Compositions and methods for controlling leptinotarsa.
Abstract
Disclosed herein are methods of controlling insect pests, in particular Leptinotarsa spp. which infest crop plants, and methods of providing plants resistant to such pests. Also disclosed are polynucleotides and recombinant DNA molecules and constructs useful in such methods, insecticidal compositions such as topical sprays containing insecticidal double-stranded RNAs, and solanaceous plants with improved resistance to infestation by Leptinotarsa spp. Further disclosed are methods of selecting target genes for RNAi-mediated silencing and control of Leptinotarsa spp.

Term
7.8 yearsleft in the term
Expires 18 July 2034.
- Priority
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37 claims: 7 independent, 30 dependent
- 1NOVEDAD DE LA INVENCIÓN REIVINDICACIONES 1. Un método para controlar una Infestación de la especie Leptinotarsa de una planta, caracterizado porque comprende:(a) poner en contacto dicha especie Leptinotarsa con un ARN de cadena doble (ARNcd) que comprende una cadena que comprende una secuencia de nucleótldos que es complementaria a al menos 22 nucleótldos contiguos de una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730;o (b) proporcionar en la dieta de dicha especie Leptinotarsa un ARNcd que comprende una cadena que comprende una secuencia de nucleótldos que es complementarla a al menos 22 nucleótidos contiguos de una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730;o (c) provocar mortalidad o debilitamiento de las larvas de dicha especie Leptinotarsa al proporcionar en la dieta de dichas larvas al menos un ARNcd que comprende al menos un elemento de sllenclamlento que comprende una cadena que comprende 22 nucleótldos contiguos que son complementarlos a una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730;o (d) aplicar tópicamente a dicha planta una composición que comprende al menos un ARNcd que comprende una cadena que comprende una secuencia de nucleótldos que es complementarla a al menos 22 nucleótidos contiguos de una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730, en donde dicho al menos un ARNcd se proporciona de una manera que permite que una especie Leptinotarsa se alimente en dicha planta para Ingerir dicho al menos un ARNcd;o (e) expresar en dicha planta al menos un ARNcd que comprende al menos un segmento que es idéntico o complementario a al menos 22 nucleótldos contiguos de una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730, en donde dicho al menos un ARNcd se proporciona de una manera que permite que una especie Leptinotarsa se alimente en dicha planta para Ingerir dicho al menos un ARNcd;o (f) proporcionar a dicha planta al menos un ARNcd que comprende al menos un segmento que es Idéntico o complementarlo a al menos 22 nucleótldos contiguos de una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730;o (g) poner en contacto dicha especie Leptinotarsa con un ARNcd que comprende al menos un segmento que es Idéntico o complementarlo a al menos 22 nucleótldos contiguos de una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730, en donde dicho ARNcd se proporciona de una manera que permite que una especie Leptinotarsa se alimente en dicha planta para Ingerir dicho al menos un ARNcd.
- 2El método de conformidad con la reivindicación 1, caracterizado además porque dicho ARN de cadena doble se sintetiza químicamente o se produce por expresión en un microorganismo o por expresión en una célula de planta. 167
- 3El método de conformidad con la reivindicaciónX .caractenzado además porque dicho ARN de cadena doble comprende una cadena que comprende una secuencia seleccionada del grupo que consiste de SEQ ID NOs:989, 988, y 1103-1105.
- 4El método de conformidad con la reivindicación 1, caracterizado además porque dicho método comprende aplicar tópicamente a dicha planta una composición que comprende al menos un ARNcd que comprende una cadena que comprende una secuencia de nucleótidos que es complementaria a al menos 22 nucleótidos contiguos de una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO:730, y en donde dicha composición comprende adicionalmente uno o más componentes seleccionados del grupo que consiste de un agente portador, un tensioactivo, un lípido catiónico, una organosilicona, un tensioactivo de organosilicona, una molécula herbicida de polinucleótido, una molécula herbicida no polinucleótido, un pesticida no polinucleótido, un protector, y un regulador del crecimiento de insectos.
- 5El método de conformidad con la reivindicación 1, caracterizado además porque dicho método comprende poner en contacto dicha especie Leptinotarsa con una cantidad eficaz de una solución que comprende un ARN de cadena doble, en donde al menos una cadena del ARN de cadena doble es complementaria a al menos 22 nucleótidos contiguos de una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO:730, en donde dicha especie Leptinotarsa es Leptinotarsa decemiineata, y en donde la interferencia del ARN se induce y ocurre la mortalidad de Leptinotarsa decemiineata.
- 6El método de conformidad con la reivindicación 5, caracterizado además porque dicha solución comprende adicionalmente uno o más componentes seleccionados del grupo que consiste de un tensioactivo de organosilicona o un lípido catiónico.
- 7El método de conformidad con la reivindicación 1, caracterizado además porque dicho método comprende aplicar tópicamente a dicha planta una composición que comprende un ARN de cadena doble que comprende una cadena que comprende una secuencia de nucleótidos que es complementaria a al menos 22 nucleótidos contiguos de una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO:730, y en donde dicha especie Leptinotarsa es Leptinotarsa decemiineata.
- 8El método de conformidad con la reivindicación 1, caracterizado además porque dicha especie Leptinotarsa se selecciona del grupo que consiste de:Leptinotarsa behrensi, Leptinotarsa coiiinsi, Leptinotarsa decemiineata (escarabajo de la patata de Colorado), Leptinotarsa defecta, Leptinotarsa haidemani (escarabajo verde de la patata de Haldeman), Leptinotarsa heydeni, Leptinotarsa juncta (falso escarabajo de la patata), Leptinotarsa iineoiata (escarabajo de la hoja de burrobrush), Leptinotarsa peninsuiaris, Leptinotarsa rubiginosa, Leptinotarsa texana, Leptinotarsa tiascaiana, Leptinotarsa tumamoca, y Leptinotarsa typographica. IMPI 168
- 9Una planta que tiene resistencia mejorada a una infestación do lo ospocisLeptinotarsa, proporcionada por el método como el que se reclama en la reivindicación 1, o un fruto, semilla, o parte propagable de dicha planta.
- 10La planta de conformidad con la reivindicación 9, caracterizada además porque dicha planta se selecciona del grupo que consiste de patata, tomate, y berenjena.
- 11Una composición insecticida para controlar una especie Leptinotarsa, caracterizada porque comprende:(a) al menos un ARN de cadena doble (ARNcd) que consiste de dos cadenas, en donde una primera cadena comprende al menos 22 nudeótidos contiguos que son complementarios a una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730, en donde dicho al menos un ARNcd provoca mortalidad o debilitamiento de crecimiento de dicha especie Leptinotarsa cuando se ingiere o pone en contacto con dicha especie Leptinotarsa·, o (b) al menos un ARNcd que consiste de al menos un elemento de silenciamiento que es complementario a al menos 22 nudeótidos contiguos de una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730, en donde dicho al menos un ARNcd provoca mortalidad o debilitamiento de crecimiento de dicha especie Leptinotarsa cuando se ingiere o pone en contacto con dicha especie Leptinotarsa-, o (c) al menos un ARNcd que consiste de dos cadenas, en donde una primera cadena comprende una secuencia que es idéntica o complementaria a al menos 22 nudeótidos contiguos de una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730, en donde dicho al menos un ARNcd provoca mortalidad o debilitamiento de crecimiento de dicha especie Leptinotarsa cuando se ingiere o pone en contacto con dicha especie Leptinotarsa] o (d) una molécula de ARNcd que provoca mortalidad o debilitamiento de crecimiento de una especie Leptinotarsa cuando se ingiere o pone en contacto con dicha especie Leptinotarsa, en donde dicha molécula de ARNcd consiste de dos cadenas, en donde una primera cadena comprende al menos 22 nudeótidos contiguos que son complementarios a una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730;o (e) una molécula de ARN de cadena doble insecticida que provoca mortalidad o debilitamiento de crecimiento de una especie Leptinotarsa cuando se ingiere o pone en contacto con dicha especie Leptinotarsa, en donde al menos una cadena de dicha molécula de ARN de cadena doble insecticida comprende 22 nudeótidos contiguos que son complementarios a una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730;o (f) al menos un ARN de cadena doble que consiste de dos cadenas, en donde la secuencia de una primera cadena se selecciona del grupo que consiste de SEQ ID NOs: 989, 988, 1103-1105, y 1116-1119, en donde dicho al menos un ARNcd provoca mortalidad o debilitamiento de crecimiento de dicha especie Leptinotarsa cuando se ingiere o pone en contacto con dicha especie Leptinotarsa.
- 12La composición insecticida de conformidad con la reivindicación 11, caracterizada además porque dicha composición insecticida está en la forma de al menos una IMPI 169 seleccionada del grupo que consiste de un sólido, líquido, polvo, suspensión, emulsión, aerosol;encapsulación, microesferas, partículas portadoras, película, matriz, tratamiento de semilla, empapado del suelo, formulación ¡mplantable, y formulación en surco.
- 13La composición insecticida de conformidad con la reivindicación 11, caracterizada además porque comprende adicionalmente al menos un componente seleccionado del grupo que consiste de un agente portador, un tensioactivo, un lípido catiónico, una organosilicona, un tensioactivo de organosilicona, una molécula herbicida de polinucleótido, una molécula herbicida no polinucleótido, un pesticida no polinucleótido, un protector, y un regulador del crecimiento de Insectos.
- 14La composición insecticida de conformidad con la reivindicación 11, caracterizada además porque dicha composición insecticida comprende una molécula de ARN de cadena doble insecticida que provoca mortalidad o debilitamiento de crecimiento de una especie Leptinotarsa cuando se ingiere o pone en contacto con dicha especie Leptinotarsa, en donde dicha molécula de ARN de cadena doble insecticida consiste de dos cadenas, en donde una primera cadena comprende una secuencia que es complementaria a 22 nucleótidos contiguos de una secuencia que corresponde a las posiciones T2J a 758 de SEQ ID NO:730, y en donde dicha molécula de ARN de cadena doble es de al menos 50 pares de bases de longitud o es de entre aproximadamente 100 a aproximadamente 500 pares de bases de longitud.
- 15Una construcción de ADN recombinante, caracterizada porque comprende un promotor heterólogo operablemente enlazado a:(a) ADN que comprende una secuencia de nucleótidos que es complementaria a al menos 22 nucleótidos contiguos de una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730;o (b) ADN que comprende 22 o más nucleótidos contiguos que tienen 100% de identidad a una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730;o (c) ADN que codifica al menos un elemento de silenciamlento que es complementarlo a al menos 22 nucleótidos contiguos de una secuencia que corresponde a las posiciones 227 a 758 de SEQ ID NO: 730;o (d) ADN que codifica un ARN que comprende al menos 22 nucleótidos contiguos que son complementarios a una secuencia de nucleótidos seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103-1105, y 11161119, o el complemento de estas, o una secuencia ortóloga de nucleótidos de una especie Leptinotarsa o una especie Triboiium, en donde la secuencia ortóloga de nucleótidos tiene al menos 98% de identidad de secuencia con una secuencia de nucleótidos seleccionada del grupo que consiste de SEQ ID NOs: 989, 988, 1103-1105, y 1116-1119, en donde el porcentaje de identidad de secuencia se calcula sobre la misma longitud;o (e) ADN que codifica un ARN que comprende al menos una región de ARN de cadena doble, al menos una cadena de la cual comprende al menos 22 nucleótidos contiguos que son complementarios a una secuencia de nucleótidos seleccionada del IMPI 170 grupo que consiste de: SEQ ID NOs: 989, 988, 1103-1105, y 111 fi-11™ »i mmpipmpntn ríe pstas. o una secuencia ortóloga de nucleótidos de una especie Leptinotarsa o una especie Triboiium, en donde la secuencia ortóloga de nucleótldos tiene al menos 98% de Identidad de secuencia con una secuencia de nucleótidos seleccionada del grupo que consiste de SEQ ID NOs: 989, 988, 1103-1105, y 1116-1119, en donde el porcentaje de identidad de secuencia se calcula sobre la misma longitud;o (f) ADN que codifica un ARN que comprende una secuencia de nucleótldos seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103-1105, y 1116-1119, o el complemento de estas.
- 16Un cromosoma o un plástido de planta o un vector de virus de planta recomblnante o un vector de baculovlrus recomblnante, caracterizado porque comprende la construcción de ADN recomblnante como la que se reclama en la reivindicación 15.
- 17Una célula de planta solanácea transgénlca, caracterizada porque tiene en su genoma la construcción de ADN recomblnante como la que se reclama en la reivindicación 15.
- 18La célula de planta solanácea transgénlca de conformidad con la reivindicación 17, caracterizada además porque dicha célula de planta solanácea transgénlca tiene adlclonalmente en su genoma ADN que codifica al menos un agente pesticida seleccionado del grupo que consiste de una patatlna, una lectlna vegetal, un fltoecdlsterolde, una proteína Insecticida de Bacillus thuringiensis, una proteína Insecticida de Xenorhabdus, una proteína insecticida de Photorhabdus, una proteína Insecticida de Bacillus iaterosporous, y una proteína Insecticida de Bacillus sphaericus.
- 19Una planta solanácea transgénlca, caracterizada porque comprende la célula de planta solanácea transgénlca como la que se reclama en la reivindicación 17, o un fruto, semilla, o, parte propagable de dicha planta solanácea transgénlca.
- 20Un método para controlar una infestación de la especie Leptinotarsa de una planta, caracterizado porque comprende:a. poner en contacto dicha especie Leptinotarsa con un ARN de cadena doble (ARNcd) que comprende una cadena que comprende una secuencia que tiene 98% de identidad a una secuencia seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105;o b. proporcionar en la dieta de dicha especie Leptinotarsa un ARNcd que comprende una cadena que comprende una secuencia que tiene al menos 98% de identidad con una secuencia seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105;o c. provocar mortalidad o debilitamiento de las larvas de dicha especie Leptinotarsa al proporcionar en la dieta de dichas larvas al menos un ARNcd que comprende al menos un elemento de silenciamiento que comprende una cadena que comprende una secuencia que tiene al menos 98% de Identidad a una secuencia seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105;o d. aplicar tópicamente a dicha planta una composición que comprende al menos un ARNcd que comprende una cadena que comprende una secuencia que tiene al menos 98% de identidad a una secuencia seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, IMPI 171 1104, y 1105, en donde dicho al menos un ARNcd se proporciona de una manera·que pormito que· una especie Leptinotarsa se alimente en dicha planta para ingerir dicho al menos un ARNcd;o e. expresar en dicha planta al menos un ARNcd que comprende una cadena que comprende una secuencia que tiene al menos 98% de identidad a una secuencia seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105, o complementaria a una secuencia que tiene al menos 98% de Identidad a una secuencia seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105, en donde dicho al menos un ARNcd se ingiere por una especie Leptinotarsa que se alimenta en dicha planta;o f. proporcionar a dicha planta al menos un ARNcd que comprende una cadena que comprende una secuencia que tiene al menos 98% de identidad a una secuencia seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105, o complementarla a una secuencia que tiene al menos 98% de identidad a una secuencia seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105, en donde dicho al menos un ARNcd se Ingiere por una especie Leptinotarsa que se alimenta en dicha planta;o g. poner en contacto dicha especie Leptinotarsa con un ARNcd que comprende una cadena que comprende una secuencia que tiene al menos 98% de identidad a una secuencia seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105, o complementaria a una secuencia que tiene al menos 98% de identidad a una secuencia seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105.
- 21El método de conformidad con la reivindicación 20, caracterizado además porque dicho ARN de cadena doble se sintetiza químicamente o se produce por expresión en un microorganismo o por expresión en una célula de planta.
- 22El método de conformidad con la reivindicación 20, caracterizado además porque dicho ARN de cadena doble comprende una cadena que comprende la secuencia seleccionada del grupo que consiste de:SEQ ID NOs: 989, 988, 1103, 1104, y 1105.
- 23El método de conformidad con la reivindicación 20, caracterizado además porque dicho método comprende aplicar tópicamente a dicha planta una composición que comprende al menos un ARNcd que comprende una cadena que comprende una secuencia que tiene ai menos 98% de identidad a una secuencia seleccionada del grupo que consiste de:SEQ ID NOs: 989, 988, 1103, 1104, y 1105;y en donde dicha composición comprende adicionalmente uno o más componentes seleccionados del grupo que consiste de un agente portador, un tensioactivo, un lípido catiónico, una organosilicona, un tensioactivo de organosilicona, una molécula herbicida de polinucleótido, una molécula herbicida no polinucleótido, un pesticida no polinucleótido, un protector, y un regulador del crecimiento de insectos.
- 24El método de conformidad con la reivindicación 20, caracterizado además porque dicho método comprende poner en contacto dicha especie Leptinotarsa con una cantidad 172 efectiva de una solución que comprende un ARN de cadena doble, en donde tri· mcROO una eodeea» del ARN de cadena doble comprende al menos 98% de Identidad a una secuencia seleccionada del grupo que consiste de:SEQ ID NOs: 989, 988, 1103, 1104, y 1105, en donde dicha especie Leptinotarsa es Leptinotarsa decemiineata, y en donde la interferencia del ARN se induce y ocurre la 5 mortalidad de Leptinotarsa decemiineata.
- 25El método de conformidad con la reivindicación 24, caracterizado además porque dicha solución comprende adicionalmente uno o más componentes seleccionados del grupo que consiste de un tensoactivo de organosilicona o un lípido catiónico.
- 26El método de conformidad con la reivindicación 20, caracterizado además 10 porque dicho método comprende aplicar tópicamente a dicha planta una composición que comprende al menos un ARNcd que comprende una cadena que comprende una secuencia que tiene al menos 98% de identidad a una secuencia seleccionada del grupo que consiste de:SEQ ID NOs: 989, 988, 1103, 1104, y 1105;y en donde dicha especie Leptinotarsa es Leptinotarsa decemiineata.
- 27El método de conformidad con la reivindicación 20, caracterizado además 15 porque dicha especie Leptinotarsa se selecciona del grupo que consiste de:Leptinotarsa behrensi, Leptinotarsa coiiinsi, Leptinotarsa decemiineata (escarabajo de la patata de Colorado), Leptinotarsa defecta, Leptinotarsa haidemani (escarabajo verde de la patata de Haldeman), Leptinotarsa heydeni, Leptinotarsa juncta (falso escarabajo de la patata), Leptinotarsa iineoiata (escarabajo de la hoja de burrobrush), Leptinotarsa peninsuiaris, Leptinotarsa rubiginosa, Leptinotarsa texana, Leptinotarsa 20 tiascaiana, Leptinotarsa tumamoca, y Leptinotarsa typographica.
- 28Una planta que tiene resistencia mejorada a una infestación de la especie Leptinotarsa, proporcionada por el método como el que se reclama en la reivindicación 20, o un fruto, semilla, o parte propagable de dicha planta, en donde el fruto, semilla, o parte propagable de la planta comprende el ARNcd. 25
- 29La planta de conformidad con la reivindicación 28, caracterizada además porque dicha planta se selecciona del grupo que consiste de patata, tomate, y berenjena.
- 30Una composición insecticida para controlar un especie Leptinotarsa, caracterizada porque comprende:a. al menos un ARNcd que consiste de dos cadenas, en donde una primera cadena comprende una secuencia que tiene al menos 98% de identidad a una secuencia 30 seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105, en donde dicho al menos un ARNcd provoca mortalidad o debilitamiento de crecimiento de dicha especie Leptinotarsa cuando se ingiere o pone en contacto con dicha especie Leptinotarsa - , o b. al menos un ARNcd que consiste de al menos un elemento de silenciamiento que comprende una cadena que comprende una secuencia que tiene al menos 98% de identidad a una secuencia seleccionada del 35 grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105, en donde dicho al menos un IMPI»· INSTITUTO MEXICANO a'TaÍÍí.-CÍ DE LA PROPIEDAD ΛβΣ_ζ2£Γ 173 INDUSTRIAL ARNcd provoca mortalidad o debilitamiento de crecimiento de dicha especie Leptinotarsa cuando se Ingiere o pone en contacto con dicha especie Leptinotarsa] o c. al menos un ARNcd que consiste de dos cadenas, en donde una primera cadena comprende una secuencia que tiene al menos 98% de Identidad a una secuencia seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105, o complementaria a una secuencia que tiene al menos 98% de Identidad a una secuencia seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105, en donde dicho al menos un ARNcd provoca mortalidad o debilitamiento de crecimiento de dicha especie Leptinotarsa cuando se Ingiere o pone en contacto con dicha especie Leptinotarsa] o d. una molécula de ARN que provoca mortalidad o debilitamiento de crecimiento de una especie Leptinotarsa cuando se ingiere o pone en contacto con dicha especie Leptinotarsa, en donde dicha molécula de ARNcd consiste de dos cadenas, en donde una primera cadena es al menos 98% Idéntica a una secuencia seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105;o e. una molécula de ARN de cadena doble Insecticida que provoca mortalidad o debilitamiento de crecimiento de una especie Leptinotarsa cuando se Ingiere o pone en contacto con dicha especie Leptinotarsa, en donde al menos una cadena de dicha molécula de ARN de cadena doble Insecticida consiste de una secuencia que comprende al menos 98% de identidad a una secuencia seleccionada del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105;o f. al menos un ARN de cadena doble que consiste de dos cadenas, en donde la secuencia de una primera cadena se selecciona del grupo que consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105, en donde dicho al menos un ARN de cadena doble provoca mortalidad o debilitamiento de crecimiento de dicha especie Leptinotarsa cuando se Ingiere o pone en contacto con dicha especie Leptinotarsa.
- 31La composición insecticida de conformidad con la reivindicación 30, caracterizada además porque dicha composición Insecticida está en la forma de al menos una seleccionada del grupo que consiste de un sólido, líquido, polvo, suspensión, emulsión, aerosol, encapsulaclón, mlcroesferas, partículas portadoras, película, matriz, tratamiento de semilla, empapado del suelo, formulación implantable, y formulación en surco.
- 32La composición Insecticida de conformidad con la reivindicación 30, caracterizada además porque comprende adlcionalmente al menos un componente seleccionado del grupo que consiste de un agente portador, un tensloactlvo, un lípido catiónlco, una organoslllcona, un tensloactlvo de organoslllcona, una molécula herbicida de pollnucleótldo, una molécula herbicida no polinucleótido, un pesticida no polinucleótido, un protector, y un regulador del crecimiento de insectos.
- 33Una construcción de ADN recomblnante, caracterizada porque comprende un promotor heterólogo operablemente enlazado a:(a) ADN que codifica un ARN que comprende al 174 IMPI INSTITUTO MfXICANO W LA MONEDA» iNnurrwiA». menos 98% de identidad a una secuencia de nucleótidos seleccionada del yiupo que camijte de SEQ ID NOs: 989, 988, 1103, 1104, y 1105, o el complemento de estas;o (b) ADN que codifica un ARN que comprende al menos una región de ARN de cadena doble, al menos una cadena de la cual comprende al menos 98% de identidad a la secuencia de nucleótidos seleccionada del grupo que 5 consiste de: SEQ ID NOs: 989, 988, 1103, 1104, y 1105, o el complemento de estas;o (c) ADN que codifica un ARN que comprende la secuencia de nucleótidos seleccionada del grupo que consiste de SEQ ID NOs: 989, 988, 1103,1104, y 1105, o el complemento de estas.
- 34Un cromosoma o un plástido de planta o un vector de virus de planta recombinante o un vector de baculovirus recombinante, caracterizado porque comprende la 10 construcción de ADN recombinante como la que se reclama en la reivindicación 33.
- 35Una célula de planta solanácea transgénica, caracterizada porque tiene en su genoma la construcción de ADN recombinante como la que se reclama en la reivindicación 33.
- 36La célula de planta solanácea transgénica de conformidad con la reivindicación 35, caracterizada además porque dicha célula de planta solanácea transgénica tiene adicionalmente 15 en su genoma ADN que codifica al menos un agente pesticida seleccionado del grupo que consiste de una patatina, una lectina vegetal, un fitoecdisteroide, una proteína insecticida de Bacillus thuringiensis, una proteína insecticida de Xenorhabdus, una proteína insecticida de Photorhabdus, una proteína insecticida de Bacillus iaterosporous, y una proteína insecticida de Bacillus sphaericus.
- 37Una planta solanácea transgénica, caracterizada porque comprende la célula de 20 planta solanácea transgénica como la que se reclama en la reivindicación 35, o un fruto, semilla, o parte propagable de dicha planta solanácea transgénica, en donde el fruto, semilla, o parte propagable comprende el ARNcd. 175 ΙΜΡΓ
Independent claims37
1,121 paragraphs in 170 sections, as filed
(54) Title: COMPOSITIONS AND METHODS TO CONTROL LEPTINOTARSA. (54) Title: COMPOSITIONS AND METHODS FOR CONTROLLING LEPTINOTARSA.
(57) Summary
The present invention relates to methods for controlling insect pests; in particular Leptinotarsa spp. that infests crop plants, and methods of providing plants resistant to such pests; Recombinant DNA polynucleotides and molecules and constructs useful in such methods are also described; insecticidal compositions, such as topical sprays, containing insecticidal double-stranded RNA and nightshade plants with improved resistance to Leptinotarsa spp. infestation; Methods for selecting target genes for RNAi mediated silencing and Leptinotarsa spp control are also described.
(57) Abstract
Disclosed herein are methods of controlling insect pests, in particular Leptinotarsa spp. which infest crop plants, and methods of providing plants resistant to such pests. Also disclosed are polynucleotides and recombinant DNA molecules and constructs useful in such methods, insecticidal compositions such as topical sprays containing insecticidal doublestranded RNAs, and solanaceous plants with improved resistance to infestation by Leptinotarsa spp. Further disclosed are methods of selecting target genes for RNAi-mediated silencing and control of Leptinotarsa spp.
IM Ρ ί ί ΐ
Γ—,
PATENT TITLE No. 359191
Owner (s): MONSANTO TECHNOLOGY LLC
Address: 800 North Lindbergh Boulevard, Mail Zone E1NA, St. Louis, Missouri, 63167, USA
Name: COMPOSITIONS AND METHODS FOR CONTROLLING LEPTINOTARSA.
Classification: CIP: C12N15 / 82; A01H5 / 10; C07K14 / 435; C12N15 / 113
CPC: C12N15 / 82; Α01Ν57ΛΙ6; C07K14 / 435; C07K14 / 43563; C12N15 / 113;
C12N15 / 8218: C12N15 / 8286
Inventors): JODI LYNN BEATTIE; MICHAELJOHN CRAWFORD; BRIAN DONOVAN EADS; LEX
EVAN FLAGEL: MAHAK KAPOOR; CHFHSTINA MARfE TAYLOR
REQUEST
Number: '' Date of International Pfeetion:
MX / a / 2016/000741; Jullode 18, 2014
<td colspan="3">PRIORITY</td>
<td>Country:</td><td></td><td>Number:</td>
<td>US</td><td>July 19, $ 201</td><td> 61/856,137</td>
<td>US</td><td>November 1st of 2013</td><td> 61/899,000</td>
<td>US</td><td>April 17, 2014</td><td> 61/980,800</td>
Validity: Twenty years
Expiration Date: July 18, 2034
Issue Date: September 18 p »2018
The reference patent is granted based on articles 1. 2nd fraction V. 6th fraction flt, and 59 of the Industrial Property Law.
In accordance with article 23 of the Industrial Property Law, this patent has a non-extendable term of twenty years, counted from the date of filing of the international application and will be subject to the payment rate to maintain the rights in force.
Who subscribes to this title does so based on the provisions of articles 6 sections lil and 7 bis 2 of the Industrial Property Law (Official Gazette of the Federation (DOF,) 06/27/1991, amended on 02 / 08/1994, 10/25/1906, 12/26/1907, 05/17/1999. 01/26/2004, 06/16/2005, 01/25/2006, 05/06/2009, 01/06/2010, 06/18/2010, 08/28/20) 0.27 / 01/2012, 04/09 / 2012,01 / 06/2016 and 03/13/2018); Articles 1, 3 'section V subsection a), 4 and 12 sections I and lil of the Regulations of the Mexican Institute of Industrial Property (DOF 14/12/4999, Reformed on 07/01/2002, 07/15/19 2004, 07/28/2004 and 09/07/2007); 1st articles. 3rd, 4th. 6th section V ínciso a), 16, Tracciones I yttty 30 of the Organic Statute of the Mexican Institute of Industrial Property (DOF 12/27/1999, amended on 10/10/2002,20/07 / ^ 004. Θ4 / Ο8 / 2Ο04 V 13 / Q8 / 2OO7)<sup>,!</sup>1, 3 and 5 paragraph a) of the Agreement that delegates powers to the Deputy Directors General, CooeUnaóar, Directors ISvieioneles'. WuíMBs'dfi the Regional Offices, Divisional Deputy Directors. Departmental Coordinators and other subordinates of the Instituto MextcánodelaPropiedatilrtdústnal · (DOF 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).
This letter is signed with an advanced electronic signature (FIEL), based on articles 7 SIS 2 of the Industrial Property Law; 3 of its Regulations, and 1 section III, 2 section V, 26 BIS and 26 TER of the Agreement establishing the guidelines for the use of the Electronic Payment and Services Portal (PASE) of the Mexican Institute of Industrial Property, in the procedures indicated.
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DIVISIONAL DIRECTOR OF PATENTS NAHANNY CANAL REYES
Original string:
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MX / 2018/78983
IMPI
MEXICAN INSTITUTE Df THE PROPERTY INDUSTRY!
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COMPOSITIONS AND METHODS TO CONTROL LEPTINOTARSA_
CROSS REFERENCE WITH RELATED REQUESTS AND INCORPORATION OF
SEQUENCE LISTINGS
This application claims priority to U.S. Provisional Patent Application No. 61 / 856,137 filed on July 19, 2013, U.S. Provisional Patent Application No. 61 / 899,000 filed on November 1, 2013, and U.S. Provisional Patent Application No. 61 / 980,800 filed on April 17, 2014, which are incorporated herein in their entirety by this reference. The sequence listings contained in files 40-21_60191_A.t (2,291 kilobytes, created on July 19, 2013, filed with U.S. Provisional Patent Application No. 61 / 856,137 on July 19, 2013), 4021_60191_0001US_ST25.txt ( 2,322 kilobytes, created on October 30, 2013, filed with U.S. Provisional Patent Application No. 61 / 899,000 on November 1, 2013), and 4021_60191_0002_US_ST25.t (2,338 kilobytes, created on April 17, 2014, filed with U.S. Provisional Patent Application No. 61 / 980,800 on April 17, 2014) are incorporated into the present in its entirety by this reference. Sequence listings comprised in file 40-21_60191_0003_ST25_new.t bytes, created Thursday, July 17, 2014) are presented as attachments and are hereby incorporated in their entirety by this reference.
FIELD OF THE INVENTION
Methods for controlling infections by Invertebrate pests, particularly in plants, as well as compositions, pollinucleotides, and recombinant DNA constructs useful in such methods are described. More specifically, this Invention relates to polynucleotides and methods for their use to modify gene expression in an Insect pest, particularly through RNA Interference. Pest species of Interest include the Leptinotarsa species, especially those that Infest cultivated plants.
BACKGROUND OF THE INVENTION
Cash crops are generally the targets of attack by invertebrate pests such as insects. Compositions for controlling insect infestations in plants have typically been found in the form of chemical insecticides. However, there are several disadvantages to the use of chemical insecticides. For example, chemical insecticides
<img file="MX359191B_D0004.tif" />
IMPI _ INSTITUTO MEXICANO ¿oe the amount * ·
INDUSTRIAL are generally not selective and the applications of the ingprt-iHHac gvímic ^ cg<sub>M9</sub> dffben controlling insect pests in plant crops can exert their effects on non-target insects, as well as other invertebrates. Chemical insecticides often remain in the environment and can have a slow degradation, potentially accumulating in the food chain. Furthermore, the use of chemical insecticides on a consistent basis can result in target Insect species developing resistance. Therefore, there has been a need for more environmentally friendly methods to control or eradicate insect pests on or within plants, that is, methods that are selective with the species, Inert in the environment, not constant. and blodegradable and that correctly fulfill the projects of treatment of the resistance of the plagues.
RNA interference (RNA and RNA mediated gene deletion) is another approach used for pest control. In vertebrates, iRNA-based gene deletion was first demonstrated in nematodes (Fire et al., (1998) Nature, 391: 806-811; Tlmmons & Fire (1998) Nature, 395: 854). Subsequently, iRNA-based deletion of invertebrate genes using recombinant nucleic acid techniques has been reported in several species including agriculturally or economically important pests of various insects and nematode taxa.
Leptinotarsa spp. it forms a gene that includes a number of species that infest commercially important plants, that include many nightshade plants (eg, potato, tomato, eggplant, bell pepper, tobacco, and petunia). For example, Leptinotarsa decemiineata / Colorado potato beetle, CPB), is a low to medium-season pest that affects solonaceous plants such as potatoes. Colorado potato beetles feed mainly on above-ground portions of the plant and defoliation leads to lower tuber yields. Methods and compositions are desired to control insect pests, in particular Leptinotarsa spp., That infest crop plants.
BRIEF DESCRIPTION OF THE INVENTION
The present modalities refer to controlling the Leptinotarsa species, especially those that are important pests from the economic or agricultural point of view. In various modalities, the Leptinotarsa species is at least one selected from the group consisting of Leptinotarsa behrensi, Leptinotarsa collinsi, Leptinotarsa decemiineata (potato beetle), Leptinotarsa detects, Leptinotarsa haidemani (Haldeman leaf beetle), Leptinotarsa heyden false potato beetle), Leptinotarsa iineoiata (leaf beetle
IMPÍ
<img file="MX359191B_D0005.tif" />
INDUSTRIAL burrobrush), Leptinotarsa peninsularis, Leptinotarsa rubiginosa, LeptinotarsaTiexana, LepttiYóiarsa tiascaiana, Leptinotarsa tumamoca, and Leptinotarsa typographica. In specific modalities, the Leptinotarsa species is at least one selected from the group consisting of Leptinotarsa decemiineata (Colorado potato beetle), Leptinotarsa juncta (false potato beetle), Leptinotarsa haidemani (green potato beetle of Haldeman ) and Leptinotarsa // neo / ate (burrobrush leaf beetle).
The compositions and methods described herein include recombinant polynudeotide molecules, such as recombinant DNA constructs for making Infestation resistant transgenic plants with the Leptinotarsa species, and single or double stranded DNA or RNA molecules, referred to herein as triggers. that are useful to control or prevent the Infestation of a plant by that Leptinotarsa species. In some embodiments, polynucleotide triggers are provided as topically applied agents to control or prevent infestation of a plant by a Leptinotarsa species. In some embodiments, nightshade plants with improved resistance to infestation with the Leptinotarsa species are provided, such as transgenic nightshade plants (which include seeds or propagating parts such as tubers) that express a polynucleotide trigger. In some embodiments, nightshade plants (including seeds or propagating parts such as tubers) are provided that have been topically treated with a composition comprising a polynucleotide trigger (eg nightshade plants that were sprayed with a solution of dsRNA molecules) . Also provided are compositions containing polynudeotides that are applied topically to a Leptinotarsa species or to a plant, part of a plant, or seed to be protected from infestation with a Leptinotarsa species.
Several modalities refer to the deletion of a target gene in a Leptinotarsa species by a polynudeotide trigger. Some modalities relate to methods for selecting Leptinotarse target genes that are likely effective targets for RNA mediated control of a Leptinotarse species. In some embodiments, the target genes selected for RNA-mediated deletion are genes that are not repetitive and are not redundant in a genome of the Leptinotarsa species, or that have little diversity of nuncleotides, or that are considered from the point of view evolutionary or functional having more synonymous nucleotide changes (K<sub>s</sub>) not synonyms (K<sub>to</sub>). Nucleotide sequences referred to in this Gen Diand 'Gene Sequence Group are provided herein, consisting of SEQ ID NO: 1-725 and SEQ ID NO: 726-830 and SEQ ID NO: 1087-1094. Also provided herein are nucleotide sequences referred to in this Trigger Sequence Group, consisting of SEQ ID NO: 831, 842, 849, 898, 910, 925, 928, 931, 932, 937, 938, 940 , 941, 942, 943, 944, 945, 947, 948, 949, 950, 951, 952, 955, 956, 957, 958, 960, 961, 964,
<img file="MX359191B_D0006.tif" />
966, 967, 968, 969, 970, 971, 973, 976, 978, 979, 982, 983, 985, 987, 988, 98 ^, 991, J'J2, 'J'J I,' J'JL · , 996, 997, 999, 1006, 1007, 1008, 1009, 1010, 1013, 1018, 1019, 1020, 1022, 1025, 1029, 1030,
1033, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1049, 1050, 1053,
1054, 1058, 1060, 1061, 1064, 1065, 1066, 1067, 1068, 1070, 1073, 1074, 1075, 1077, 1078, 1080,
1081, 1082, 1084, 1085, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1110, lili, 1112, 1113 and 1114.
In one aspect, a method of controlling an infestation with the Leptinotarsa species of a plant comprising contacting the Leptinotarsa species with a pollnucleotide comprising at least a segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% Identity (for example, a segment of 21 contiguous nucleotides with a sequence of 100% Identity) with a corresponding fragment of a DNA having a sequence that is selected from the group consisting of: the Gen Diana Sequence Group, or the DNA complement thereof . In one embodiment, the method of controlling an Infestation with the Leptinotarsa species of a plant comprises contacting the Leptinotarsa species with a pollnucleotide comprising a nucleotide sequence that is complementing at least 21 contiguous nucleotides of a target gene having a sequence of nucleotides selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1-725, SEQ ID NO: 726-729, SEQ ID NO: 731-806, SEQ ID NO : 808 - 830, and SEQ ID NO: 1087 - 1094, or an RNA transcribed from the target gene. In some embodiments, the pollnucleotide is a double-stranded RNA. In some embodiments, the pollnucleotide comprises one or more nucleotide sequences that are selected from the Trigger Sequence Group. In some embodiments, contact with a pollnucleotide is accomplished by topical application of the pollnucleotide or a composition or solution containing the pollnucleotide (eg, spraying or dusting or soaking), directly to the Leptinotarsa species or to a surface or matrix (eg , a plant or soil) in contact with the Leptinotarsa species. In some embodiments, contact with a pollnucleotide is accomplished by providing a pollnucleotide that is ingested by the Leptinotarsa species. In some embodiments, contact with a polynucleotide is accomplished by providing a transgenic plant that is expressed to the Leptinotarsa species.
Various modalities refer to a method of controlling an Infestation with the Leptinotarsa species of a plant by providing in the diet of a Leptinotarsa species an agent comprising a pollnucleotide having at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% Identity (for example, a segment of 21 contiguous nucleotides with a sequence of 100% Identity) with a corresponding fragment of a DNA having a sequence that is selected from the group consisting of: the Gen Diana Sequence Group, or the DNA complement thereof , and where the
<img file="MX359191B_D0007.tif" />
Industrial agent works after ingestion by the ¿epl / HiWJcJ pdld liilnbii species, a biological function within the Leptinotarsa species, thus controlling the infestation with the Leptinotarsa species. In one embodiment, the method of controlling an infestation with the Leptinotarsa species of a plant comprises providing in the diet of the Leptinotarsa species a polynucleotide comprising a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO: 1087 - 1094, or an RNA transcribed from the target gene. In some embodiments, the polynucleotide comprises one or more nucleotide sequences that are selected from the Trigger Sequence Group. In some embodiments, the polynucleotide is a double-stranded RNA. In some embodiments, the polynucleotide-containing agent is formulated for application to crop plant fields, for example, in emulsions or sprayable solutions, tank mixes, or powders. In some embodiments, the agent is produced biologically, for example, in the form of a microbial fermentation product or expressed in a transgenic plant cell.
In another aspect, a method is provided for causing mortality or weakening in the larvae of the Leptinotarsa species. In some embodiments, at least one RNA is provided which comprises at least one silencing element in the diet of Leptinotarsa species larvae where ingestion of the RNA by Leptinotarsa species larvae results in mortality or weakening in the larvae of the species Leptinotarsa. In some embodiments, the silencing element is essentially identical or essentially complementary to a fragment of a target gene sequence from Leptinotarsa larvae, where the target gene is selected from the group consisting of genes in the Sequence Group of Gen Diana. In one embodiment, the method of causing mortality or weakening in the larvae of the waiting & Leptinotarsa comprises providing in the diet of the larvae at least one polynucleotide comprising at least one silencing element comprising 21 contiguous nucleotides that are complementary to a target gene that it has a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO: 1087 - 1094, or an RNA transcribed from the target gene. In some embodiments, the silencing element comprises one or more nucleotide sequences that are selected from the Trigger Sequence Group. In some embodiments, the polynucleotide is a double-stranded RNA. Some embodiments relate to a method of causing mortality or lower fertility in the Leptinotarsa species which comprises providing at least one RNA in the diet of the Leptinotarsa species comprising at least one essentially identical silencing element or
<img file="MX359191B_D0008.tif" />
IMPI, MEXICAN INSTITUTE
O FROM PROPERTY
INDUSTRIAL essentially complementary to a fragment of a sequence ^ le- ^ eo-diaoajieJasJaüía ^ deJa. Leptinotarsa species where ingestion of RNA by the Leptinotarsa species results in mortality or lower fertility in the Leptinotarsa species. In some embodiments, the target gene is selected from the group consisting of genes in the Target Gene Sequence Group. In some embodiments, the method causes a reduction in the rate of metamorphosis or a reduction in feeding activity. In some embodiments, the method is useful in providing plants that have increased resistance to infestation with the Leptinotarsa species.
Various embodiments relate to a method of providing a plant having improved resistance to an Infestation with the Leptinotarsa species comprising applying topically to the plant a composition comprising at least one polynucleotide having at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity (for example, a segment of 21 contiguous nucleotides with a sequence of 100% Identity) with a corresponding fragment of a DNA having a sequence that is selected from the group consisting of: the Gen Diana Sequence Group, or the DNA complement thereof . In one embodiment, the method of providing a plant having improved resistance to an infestation with the Leptinotarsa species comprising applying topically to the plant a composition comprising at least one polynucleotide comprising a nucleotide sequence that is complementary to at least 21 nucleotides contiguous of a target gene having a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO: 1087 - 1094, or an RNA transcribed from the target gene. In one embodiment, the method of providing a plant having improved resistance to an infestation with the Leptinotarsa species comprises applying topically to the plant a composition comprising at least one polynucleotide such that an effective amount of the polynucleotide is ingested by the Leptinotarse species. that feeds on the plant, the polynucleotide comprises at least 21 contiguous nucleotides that are complementary to a target gene having a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1-725 , SEQ ID NO: 726-729, SEQ ID NO: 731-806, SEQ ID NO: 808-830, and SEQ ID NO: 1087-1094, or a transcribed RNA of the target gene. In some embodiments, the polynucleotide comprises one or more nucleotide sequences that are selected from the Trigger Sequence Group. In some embodiments, the polynucleotide is a double-stranded RNA. Various embodiments relate to compositions comprising the polynucleotide, formulated for application to crop plant fields, for example, in emulsions or sprayable solutions, tank mixes, or powders.
Various modalities refer to an insecticidal composition to control a
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IMPI
MEXICAN INSTITUTE 'K LA MONEDAD
INDUSTRIAL Leptinotarsa species comprising an insecticide effective amount of at least one pollenudeotide molecule comprising at least a segment of 18 or more contiguous nucleotides that are essentially Identical or complementary (eg, a segment of 21 contiguous nucleotides with a sequence of 100 % identity) to a corresponding fragment of a DNA having a sequence that is selected from the group consisting of: the Gene Target Sequence Group, or its DNA complement. In some embodiments, the polynucleotide molecule comprises at least 21 contiguous nucleotides that are complementary to a target gene that has a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1-725, SEQ ID NO: 726-729, SEQ ID NO: 731-806, SEQ ID NO: 808-830, and SEQ ID NO: 1087-1094, or a transcribed RNA of the target gene. In some embodiments, the polynucleotide comprises one or more nucleotide sequences that are selected from the Trigger Sequence Group. In some embodiments, the polynucleotide molecule is a recombinant polynucleotide. In some embodiments, the polynucleotide molecule is RNA. In some embodiments, the polynucleotide molecule is double-stranded RNA. Related embodiments include insecticidal compositions comprising the polynucleotide molecule formulated for application to crop plant fields, for example, in emulsions or sprayable solutions, tank mixes, or powders, and optionally comprising one or more additional components, such as a carrier agent, a surfactant, a cationic lipid, an organosllicone, an organosilicone surfactant, a herbicidal polynucleotide molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide pesticide, a protector, and an insect growth regulator.
Various embodiments relate to a method of providing a plant having improved resistance to infestation with the Leptinotarsa species comprising expressing at least one polynucleotide in the plant comprising at least a segment of 18 or more contiguous nucleotides that are essentially Identical or complementary (for example, a segment of 21 contiguous nucleotides with a sequence of 100% identity or complementarity) to the corresponding fragment of a DNA having a sequence that is selected from the group consisting of: the Gen Diana Sequence Group, or the DNA complement of this. In some embodiments, the polynucleotide comprises one or more nucleotide sequences that are selected from the Trigger Sequence Group. In some embodiments, the polynucleotide is a double-stranded RNA.
Various embodiments relate to a recombinant DNA construct comprising a heterologous promoter operably linked to a DNA element comprising at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity ( for example, a segment of 21 contiguous nucleotides with a sequence of 100% identity) with the corresponding fragment of a DNA that has a
IMPI
<img file="MX359191B_D0010.tif" />
sequence selected from the group consisting of: the Grupd 'dS'SéCUSn'CiaS dé GSn'UISña Ό fel DNA complement of this. In some embodiments, the DNA element encodes a double-stranded RNA. In some embodiments, the double-stranded RNA comprises one or more nucleotide sequences that are selected from the Trigger Sequence Group. Related modalities include a plant chromosome or plastid or a recombinant plant virus vector or a recombinant baculovlrus vector that comprises the recombinant DNA construct, or that comprises the DNA element without the heterologous promoter.
Several modalities refer to transgenic solanaceous plant cells that have in their genome an RNA that encodes recombinant DNA that suppresses the expression of a target gene in a Leptinotarsa species that contacts or ingests the RNA, where the RNA comprises at least one An elemental element that has at least one segment of 18 or more contiguous nucleotides complementing it to a fragment of a target gene. In some embodiments, the target gene is selected from the Target Gene Sequence Group. A specific embodiment is a transgenic solanaceous plant cell having in its genome an RNA that encodes recombinant DNA to silence one or more target genes that are selected from the group consisting of exoqulste genes, rlbosomal protein genes, and proteosome genes. In some embodiments, the RNA comprises one or more nucleotide sequences that are selected from the Trigger Sequence Group.
Various embodiments relate to an isolated recombinant RNA molecule that causes mortality or growth failure in a Leptinotarse species when ingested or in contact with the Leptinotarse species, where the recombinant RNA molecule comprises at least one segment of 18 or more nucleotides. contiguous that are essentially complementary (for example, a segment of 21 contiguous nucleotides with a sequence of 100% complementarity) to the corresponding fragment of a DNA that has a sequence that is selected from the group consisting of: the Gen Diana Sequence Group, or its DNA complement. In some embodiments, the recombinant RNA molecule is double-stranded RNA. Specific modalities include an isolated recombinant RNA molecule to suppress the expression of a ribosomai protein such as a rlbosomal protein L7 or a protein encoded by SEQ ID NO: 730, and an isolated recombinant double-stranded RNA molecule that has a sequence that is selected from the group consisting of SEQ ID NO: 989, 988, 1104, or 1105.
Various embodiments relate to a method of providing a plant having improved resistance to infestation with the Leptinotarsa species comprising providing the plant with at least one pollnucleotide comprising at least a segment of 18 or more contiguous nucleotides that are essentially identical or complementary (for example, a segment of 21 contiguous nucleotides with a sequence of 100% identity or complementarity) to the corresponding fragment of a target gene that is selected from the Target Gene Sequence Group. In a
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modality, the method of providing a plant that has jsysteadaJB ^ ada ^ uaajüfestación with the Leptlnotarsa species comprising providing the plant at least one polynucleotide comprising at least one segment that is Identical or complementary to at least 21 contiguous nucleotides of a target gene or a RNA transcribed from the target gene, where the target gene is selected from the group consisting of: the genes identified in the Target Gene Sequence Group. In some embodiments, the polynucleotide comprises one or more nucleotide sequences that are selected from the Trigger Sequence Group. In some embodiments, the polynucleotide is a double-stranded RNA.
Various embodiments relate to a method of controlling an infestation with the Leptinotarsa species of a plant comprising contacting the Leptinotarsa species with a polynucleotide comprising at least a segment of 18 or more contiguous nucleotides that are essentially identical or complementary (eg. , a segment of 21 contiguous nucleotides with a sequence of 100% identity or complementarity) to the corresponding fragment of equivalent length of a DNA of a target gene that is selected from the Target Gene Sequence Group. In some embodiments, the polynucleotide is a double-stranded RNA. In one embodiment, the method of controlling an infestation with the Leptinotarsa species of a plant comprises contacting the Leptinotarsa species with an effective amount of a double-stranded RNA, a strand of which is complementary to at least 21 contiguous nucleotides of a gene encoding a ribosomal protein, where RNA interference is induced and mortality occurs. In some embodiments, the double-stranded RNA comprises one or more nucleotide sequences that are selected from the Trigger Sequence Group.
Several modalities relate to a method of selecting target genes for RNAI mediated silencing of a plant genome or an animal genome. In several modalities, the method provides a subset of target genes that are present in single or low copy number (non-repeating and non-redundant) in a particular genome, or that have little nucleotide diversity, or that have a ratio of changes nucleotide synonym (K<sub>s</sub>) to no synonyms (K<sub>to</sub>) where K<sub>s</sub> >> K<sub>to</sub>.
Various embodiments relate to man-made compositions comprising at least one polynucleotide as described herein. In some embodiments, useful formulations are provided for topical application to a plant or substance that needs protection from an infestation with the Leptinotarsa species. In some embodiments, useful recombinant constructs and vectors are provided for making cells from transgenic nightshade plants and transgenic nightshade plants. In some embodiments, formulations and coatings useful for treating nightshade plants, nightshade plant seeds, or propagating parts such as tubers are provided. In some modalities,
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INSTITUTO MEXICANO Dt LA PROPIEDAD | Q INDUSTRIAL provide basic products and edible products produced from such nightshade plants, seeds or propagating parts treated with or containing a polynucleotide as described herein (especially basic products and edible products that have a detectable amount of a polynucleotide as described herein). Various modalities refer to polyclonal or monoclonal antibodies that bind to a protein encoded by a sequence or a fragment of a sequence that is selected from the Gen Diana Sequence Group. Another aspect relates to polyclonal or monoclonal antibodies that bind to a protein encoded by a sequence or a fragment of a sequence that is selected from the Trigger Sequence Group or the complement thereof. Such antibodies are made by routine methods known to those skilled in the art.
In the various embodiments described herein, the plant can be any plant subject to an infestation with a Leptinotarsa species. Modalities where the plant is a nightshade plant (family Solanaceae) are of particular interest. Examples include a plant that is selected from the group consisting of potato, tomato, and eggplant. Modalities include those where the plant is an ungerminated nightshade plant seed, a nightshade plant in the vegetative stage, or a nightshade plant in the reproductive stage. Modalities include those where the plant is a seed potato, meaning a potato tuber or part of a potato tuber that can propagate into new potato plants.
Other specific aspects and embodiments of this invention are described in the following detailed description.
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise indicated, all technical and scientific terms used have the meaning commonly understood by one skilled in the art to which the present invention belongs. When providing a singular term the inventors also contemplate aspects of the invention described by the plural of that term. When there are discrepancies in the terms and definitions used in references that are incorporated by reference, the terms used in this application will have the definitions given herein. Other technical terms used have their common meaning in the art in which they are used, as exemplified in several art-specific dictionaries, for example, The American Heritage® Science Dictionary (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), the McGraw-Hill Dictionary of Scientific and Technical Terms (6<sup>to</sup> edition, 2002, McGraw-Hill, New York) or the Oxford Dictionary of Biology (6<sup>to</sup> edition, 2008, Oxford University Press, Oxford and New York). The inventors do not intend to limit themselves to one
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mechanism or mode of action. References herein are provided for illustrative purposes only.
Unless otherwise indicated, nucleic acid sequences in the text of this specification are provided, when read from Left to right, in the 5 'to 3' direction. One of skill in the art will be aware that a DNA sequence is understood to define a corresponding RNA sequence that is identical to the DNA sequence, except for the replacement of the tlmina (T) nucleotides of the DNA with uracil nucleotides. (OR). Therefore, the input of a specific DNA sequence is understood to define the exact RNA equivalent. A given first chromosomal sequence, either DNA or RNA, further defines the sequence of its exact complement (which may be DNA or RNA), a second polynucleotide that perfectly hybridizes the first chromosomal nucleotide to Watson-Crick base pairs. For DNA: DNA duplexes (hybridized chains), the base pairs are adenine: tlmlna or guan¡na: cltoslna; for DNA: RNA duplexes, the base pairs are adenlna: uracllo or guanine: cltoslna. Therefore, the nucleotide sequence of a perfectly double-stranded blunt-ended polynucleotide (where there is 100% complementarity between the chains or where the chains are complementary) is unequivocally defined by providing the nucleotide sequence of a chain, either as DNA or RNA. Essentially Identical to or essentially complementary to a target gene or a fragment of a target gene means that a chain of polynucleotides (or at least one chain of a double-chain polynucleotide) is designed to hybridize (usually under physiological conditions such as those found in a living animal or plant cell) with a target gene or a fragment of a target gene or the transcription of a target gene or a fragment of a target gene; The person skilled in the art will understand that such hybridization does not necessarily require 100% sequence identity or complementarity. A first nucleic acid sequence is operably linked or linked to a second nucleic acid sequence when the first nucleic acid sequence is in functional relationship with the second nucleic acid sequence. For example, a promoter sequence is operably linked to a DNA if the promoter provides for the transcription or expression of the DNA. Typically, the operably linked DNA sequences are contiguous.
The term polynucleotide commonly refers to a DNA or RNA molecule that contains multiple nucleotides and generally refers to both oligonucleotides (a molecule of 18-25 nucleotide-long polynucleotides) and longer polynucleotides of 26 or more nucleotides. Pollnucleotides also Include molecules that contain multiple nucleotides, Including non-canonical nucleotides or chemically modified nucleotides as is customary in the art; see, for example, the chemical modifications described in the RNA Interference (RNAI) technical manual to (Integrated DNA Technologies
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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY
Coralville, IA). Generally, polynucleotides such as Η ^ ίΓΓ ίΠ 11 μι ι__liiíc, i are ΛΡΜ or RNA or both single and double stranded, include at least a segment of 18 or more contiguous nucleotides (or, in the case of polynucleotides double stranded (at least 18 contiguous base pairs) that are essentially Identical to or complement an equivalent size fragment of the DNA of a target gene or RNA transcription of the target gene. Throughout this description, at least 18 contiguous refers to from about 18 to about 10,000, including each integer between them. Therefore, modalities of this invention include ollgonucleotides that are 18-25 nucleotides in length (18-mers, 19-mers, 20-mers, 21mers, 22-mers, 23-mers, 24-mers, or 25- mers), or half-length polynucleotides that are 26 or more nucleotides in length (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, around 65, around 70, around 75, around 80, around 85, around 90, around 95, around 100, around 110, around 120, around 130, around 140, around 150, around 160, around 170, around 180, around 190, around 200, around 210, around 220, around 230, around 240, around 250, around 260, around 270, around 280, around 290, or around 300 nucleotides), or long polynucleotides that are greater than about 300 nucleotides in length (eg, polynucleotides of between about 300 and about 400 nucleotides, between about 400 and about 500 nucleotides, between about 500 and about 600 nucleotides, between around 600 and around 700 nucleotides, between around 700 and around 800 nucleotides, between around 800 and around 900 nucleotides, between around 900 and around 1000 nucleotides, between around 300 and around 500 nucleotides, between around 300 and around 600 nucleotides, between around 300 and around 700 nucleotides, between around 300 and around 800 nucleotides, between about 300 and about 900 nucleotides, or about 1000 nucleotides in length, or even greater than about 1000 nucleotides in length, for example, by the total length of a target gene that includes coding or non-coding parts or both coding and non-coding for the target gene). When a polynucleotide is double-stranded, its length can be similarly described in terms of base pairs.
The polynucleotides described herein can be single chain (single chain) or double chain (single chain). Double-stranded refers to base pairs that occur between sufficiently parallel antiparallel nucleic acid chains to form a double-stranded nucleic acid structure, generally under physiologically relevant conditions. Modalities include those where the polynucleotide is selected from the group consisting of sense single-stranded DNA (cDNA), RNA
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sense single-stranded (cRNAs), double-stranded RNA (udeiiu dublé dsRNA (dsDNA), a double-stranded DNA / RNA hybrid, antisense cDNAs, or antisense cDNAs; a mixture of polynucleotides of any of these types can be used. In some embodiments, the polynucleotide is double-stranded RNA of a length greater than that typical of naturally occurring small regulatory RNAs (such as endogenously produced siRNAs and mature mlRNAs). In some embodiments, the polynucleotide is double-stranded RNA of at least about 30 contiguous base pairs in length. In some embodiments, the polynucleotide is double-stranded RNA with a length of between about 50 and about 500 base pairs. In some embodiments, the polynucleotide may include different components than standard rlbonucleotides, for example, one embodiment is an RNA comprising terminal deoxyrrbonucleotides.
In various embodiments, the polynucleotide described herein comprises naturally occurring nucleotides, such as those occurring in DNA and RNA. In certain embodiments, the polynucleotide is a combination of rlbonucleotides and deoxlrrlbonucleotides, for example, synthetic polynucleotides consisting mainly of terminal rlbonucleotides but with one or more terminal deoxyrrbonbonucleotides or one or more end-deoxyrrbonnucleotides with or more than one nucleotide quenucleotides with synthetic or polynucleotides containing primarily plus terminal dideoxyribonucleotides. In certain embodiments, the polynucleotide comprises non-canonical nucleotides such as inosine, tlouridine, or pseudouridine. In certain embodiments, the polynucleotide comprises chemically modified nucleotides. Examples of chemically modified ollgonucleotides or polynucleotides are known in the art; see, for example, US Patent Publication 2011/0171287, US Patent Publication 2011/0171176, US Patent Publication 2011/0152353, US Patent Publication 2011/0152346, and US Patent Publication 2011/0160082, which are incorporated herein by this reference. Illustrative Examples Include, but are not limited to, the naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide that may be partially or completely modified with internucleotide linkage modifications of phosphorotloate, phosphorodlthioate, or methylphosphonate, modified nucleoside bases may be used. or modified sugars in the synthesis of ollgonucleotides or pollnucleotides, and ollgonucleotides or pollnucleotides can be labeled with a fluorescent moiety (eg, fluorescein or rhodamna) or another label (eg, blotin).
Various modalities refer to a polynucleotide comprising at least a segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with a fragment of equivalent length of a target DNA or gene having a sequence that is selected from the Gene Target Sequence Group or the DNA complement of
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this. In some embodiments, contiguous nucleotides number 18, for example, between 18-24, or 18-28, or 20-30, or 20-50, or 20-100, or 50-100. , or between 50 - 500, or between 100 - 250, or between 100 - 500, or between 200 - 1000, or between 500 - 2000, or even more. In some embodiments, contiguous nucleotides list more than 18, for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, for example, about 35 , around 40, around 45, around 50, around 55, around 60, around 65, around 70, around 75, around 80, around 85, around 90, around 95, around 100, around 110, around 120, around 130, around 140, around 150, around 160, around 170, around 180, around 190, around 200, around 210, around 220, around 230, around 240, around 250, around 260, around 270, around 280, about 290, about 300, about 350, about 400, about 450, about 500, or more than 500 contiguous nucleotides. In some embodiments, the pollnucleotide comprises at least one segment of at least 21 contiguous nucleotides with a sequence of about 100% identity with a fragment of equivalent length of a target DNA or gene that has a sequence that is selected from the Sequence Group Diana gene or DNA complement of this. In some embodiments, the polynucleotide is a double-stranded nucleic acid (eg, dsRNA) with a strand that comprises at least one segment of at least 21 contiguous nucleotides with 100% identity with a fragment of equivalent length of a DNA or gene target having a sequence that is selected from the group consisting of the Target Gene Sequence Set or the DNA complement thereof; expressed as base pairs, such a double-stranded nucleic acid comprises at least one segment of at least 21 contiguous base pairs, which match perfectly corresponding to a fragment of equivalent length of a target DNA or gene having a sequence that is selected from the Diana Gene Sequence Group or its DNA complement. In some embodiments, each segment contained in the polynucleotide is longer than typical of naturally occurring small regulatory RNAs, for example, each segment is at least about 30 contiguous nucleotides (or base pairs) in length. In some embodiments, the total length of the polynucleotide, or the length of each segment contained in the polynucleotide, is less than the total length of the target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group. In some embodiments, the total length of the polynucleotide is between about 50 and about 500 nucleotides (for single chain polynucleotides) or base pairs (for double chain polynucleotides). In some embodiments, the polynucleotide is a dsRNA of between about 100 and about 500 base pairs, such as a dsRNA the length of any of the dsRNA triggers described in Tables 3, 5, 8, 9, and 10. Modalities include those where the polynucleotide expressed in the plant is an RNA
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M SXJCA INSTITUTE NO
FROM THE PftONEPAD
INDUSTRIAL comprising a segment that has a sequence that is selected from the sequence consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or is an RNA hairpin encoded by a sequence selected from the group consisting of SEQ ID NO: 1105 1109. In some embodiments, the polynucleotide is expressed in a plant. In some embodiments, the polynucleotide is provided topically to the surface of a plant or to the Leptinotarsa species.
Various modalities refer to polynudeotides that are designed to modulate expression by inducing regulation or deletion of a target gene of the Leptinotarsa species. In some embodiments, the polynudeotides are designed to have a nucleotide sequence essentially identical or essentially complementary to the nucleotide sequence of a target gene or cDNA of the Leptinotarsa species (eg, the Target Gene Sequence Group) or the sequence of RNA transcribed from the target gene of the Leptinotarsa species, which may be a coding sequence or a non-coding sequence. These effective polynucleotide molecules that modulate expression can be referred to herein as polynucleotide, polynucleotide trigger, trigger or triggers.
Effective polynudeotides of any size can be used, alone or in combination, in various methods and compositions described herein. In some embodiments, a single polynucleotide trigger is used to make a composition (eg, a composition for topical application or a recombinant DNA construct useful in making a transgenic plant). In other embodiments, a mixture or group of different polynudeotide triggers is used; in such cases the polynudeotide triggers can be for a single target gene or for multiple target genes.
As used herein, the term "isolated" refers to the separation of a molecule from other molecules that are normally associated with it in its natural or original state. The term isolated can therefore refer to a DNA molecule that has been separated from other DNA molecules that are normally associated with it in its natural or original state. Such a DNA molecule can be present in a recombined state, such as a recombinant DNA molecule. Therefore, DNA molecules fused to coding or regulatory sequences with which they are not normally associated, for example as a result of recombinant techniques, are considered isolated, even when they are integrated as a transgene into the chromosome of a cell or are found with other DNA molecules.
As used herein, the term 'Gen Diand' Sequence Group refers to the group of sequences consisting of SEQ ID NO: 1-725 and SEQ ID NO: 726-830 and SEQ ID NO: 1087-1094) . As used herein, the term Sequence Group of
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INSTITIITO MSXICANO
OF THE PROPERTY
INDUSTRIAL
Triggers refers to the group of sequences consisting of SEQ ID NO: 831, 842, 849, 898, 910, 925, 928, 931, 932, 937, 938, 940, 941, 942, 943, 944, 945, 947, 948, 949, 950, 951, 952, 955, 956, 957, 958, 960, 961, 964, 966, 967, 968, 969, 970, 971, 973, 976, 978, 979, 982, 983, 985, 987, 988, 989, 991, 992, 994, 995, 996, 997, 999, 1006, 1007, 1008, 1009, 1010, 1013, 1018, 1019, 1020, 1022, 1025, 1029, 1030, 1033, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1049, 1050, 1053, 1054, 1058, 1060, 1061, 1064, 1065, 1066, 1067, 1068, 1070, 1073, 1074, 1075, 1077, 1078, 1080, 1081, 1082, 1084, 1085, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1110, lili, 1112, 1113 and 1114.
In various embodiments, the Leptinotarsa species is at least one selected from the group consisting of Leptinotarsa behrensi, Leptinotarsa collinsi, Leptinotarsa decemiineata (Colorado potato beetle), Leptinotarsa defecta, Leptinotarsa haidemani (Green potato beetle of Haldeman) , Leptinotarsa heydeni, Leptinotarsa sedge (false potato beetle), Leptinotarsa iineoiata (burrobrush leaf beetle), Leptinotarsa peninsuiarís, Leptinotarsa rubiginosa, Leptinotarsa texana, Leptinotarsa tiascaiana, Leptinotarsa tumamoca and Leptinotarsa typographica. In specific modalities, the Leptinotarsa species is at least one selected from the group consisting of Leptinotarsa decemiineata (Colorado potato beetle), Leptinotarsa juncta (false potato beetle), Leptinotarsa haidemani (green potato beetle of Haldeman ) and Leptinotarsa iineoiata (burrobrush leaf beetle).
Control of Leptinotarse Infestations by Contact with a Pollenucleotide
Methods are provided herein for controlling an infestation with the Leptinotarsa species of a plant by contacting the Leptinotarsa species with a pollnucleotide comprising at least a segment of 18 or more contiguous nucleotides having about 95% to about 100% of Identity or complementarity to a corresponding fragment of a target DNA or gene that is selected from the group consisting of: the Target Gene Sequence Group, or its DNA complement. In one embodiment, the method of controlling an infestation with the Leptinotarsa species of a plant comprises contacting the Leptinotarsa species with a polynucleotide comprising at least 21 contiguous nucleotides with 100% Identity with a corresponding fragment of a target gene having a DNA sequence that is selected from the group consisting of: SEQ ID N0: 730, SEQ ID NO: 807, SEQ ID NO: 1 725, SEQ ID NO: 726-729, SEQ ID NO: 731-806, SEQ ID NO : 808 - 830, and SEQ ID NO: 1087 1094, or its DNA complement. In some embodiments, the pollnucleotide is a double-stranded RNA. In some embodiments, the pollnucleotide (eg, double-stranded RNA) is chemically synthesized or produced by expression in a microorganism or by expression in a plant cell. Modalities Include those where the polynucleotide is a dsRNA that
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MEXICAN INSTITUTE
OF THE MMW.0AO
1 / INDUSTRIAL comprises a sequence that is selected from the group consisting of SÉQ ID NO: 831 - 1ÜHS, lUyS - 1104, and 1110 - 1114, or the complement thereof, or where the polynucleotide is encoded by a sequence that is selected from the group consisting of SEQ ID NO: 1105-1109. In one embodiment, the method of controlling an infestation with the Leptinotarsa species of a plant comprises contacting the Leptinotarsa species with a pollnucleotide comprising a nucleotide sequence that is to complement it with at least 21 contiguous nucleotides of a target gene encoded by a sequence nucleotide that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 830, and SEQ ID NO: 1087 - 1094, or an RNA transcribed from the target gene. Modalities include those where the polynucleotide is a dsRNA comprising a strand having a sequence that is selected from the Trigger Sequence Group. In some embodiments, the method uses a polynucleotide comprising a segment of 127 contiguous nucleotides (SEQ ID NO: 831) which is the antisense sequence (reverse complement) of 127 contiguous nucleotides of the target gene encoded by SEQ ID NO: 825. In some embodiments, the method uses a polynucleotide comprising contiguous 409 and 403 nucleotide segments (SEQ ID NO: 937 and SEQ ID NO: 938, respectively) which is the antisense sequence (Inverse complement) of 409 and 403 contiguous nucleotides, respectively , from a target gene encoded by SEQ ID NO: 732. The polynucleotides for use in the method can be designed for multiple target genes. Related aspects of the invention include isolated polynucleotides for use in the method and plants having improved resistance to Leptinotarse provided by the method.
In some embodiments, contiguous nucleotides have a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity with a fragment of equivalent length of a DNA or target gene having a sequence selected from the group consisting of: SEQ ID NO: 1-725 or SEQ ID NO: 726-830 and SEQ ID NO: 1087-1094 and the DNA complement thereof. In some embodiments, the contiguous nucleotides are exactly (100%) identical to a fragment of equivalent length of a target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group or its DNA complement. In some embodiments, the polynucleotide has an overall sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity with a fragment of equivalent length of a target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group or the DNA complement thereof. In one embodiment, the polynucleotide comprises at least one segment of 21 contiguous nucleotides with 100% identity to the corresponding fragment of a target gene having a DNA sequence that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID N0: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ
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iMPrr lKíCTmn '/ Λ'
OF INDUSTRIAL PROPERTY
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modalities, the polynucleotide comprises a neutral sequence (sequence that has no identity or sequence complementarity to the target gene) in addition to one or more contiguous 21 nucleotide segments with 100% identity to the corresponding fragment of the target gene, and therefore the The polynucleotide as a whole has much lower General Sequence Identity with a target gene.
Various modalities refer to a polynucleotide designed to suppress one or more genes (target genes). The term gene refers to any part of a nucleic acid that provides for the expression of a transcript or encodes a transcript. A gene may include, but is not limited to, a promoter region, 5 'untranslated regions, regions encoding transcripts that may include intronic regions, 3' untranslated regions, or combinations of these regions. In some embodiments, the target genes can include coding or non-coding sequences, or both. In other embodiments, the target gene has an identical or complementary sequence to a messenger RNA, for example, in some embodiments, the target gene is a cDNA. In specific embodiments, the polynucleotide is designed to delete one or more target genes, where each target gene is encoded by a DNA sequence that is selected from the Target Gene Sequence Group. In various embodiments, the polynucleotide is designed to delete one or more target genes, where each target gene is encoded by a sequence that is selected from the Target Gene Sequence Group, and can be designed to delete multiple target genes from this group, or to target different regions of one or more of these target genes. In one embodiment, the polynucleotide comprises multiple contiguous 21 nucleotide segments with 100% Identity with an equivalent length fragment of a target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group or the DNA complement of this. In such cases, each segment may be Identical or different in size or sequence, and may be sense or antisense relative to the target gene. For example, in one embodiment, the polynucleotide comprises multiple tandem segments or repeating arrays, where each segment comprises 21 contiguous nucleotides with a sequence of 100% identity with a fragment of equivalent length of a target DNA or gene that has a sequence that it is selected from the Gen Diana Gene Sequence Group or its DNA complement. In some embodiments, the segments may be from different regions of the target gene, for example, the segments may correspond to different regions of the exon of the target gene. In some embodiments, spacer nucleotides that do not correspond to a target gene can optionally be used between or adjacent to the segments.
The total length of the polynucleotide for use in this method may be greater than 18 contiguous nucleotides, and may include nucleotides in addition to contiguous nucleotides that have the
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about 95% to about 100% sequence identity ^ cooaM4 fragment ^ e-4 equivalent length of a target DNA or gene that has a sequence that is selected from the group consisting of: the Target Gene Sequence Group, or the complement of DNA from this. In other words, the total length of the polynucleotide may be greater than the length of the section or segment of the polynucleotide designed to delete one or more target genes, where each target gene has a DNA sequence that is selected from the group consisting of Group of Gen Diana Sequences. For example, the polynucleotide may have nucleotides flanking the active segment of at least one segment of 18 or more contiguous nucleotides that suppress the target gene, or Include spacer nucleotides between the active segments, or they may have additional nucleotides at the 5 'end, either at the 3 'end, or at both the 5' and 3 'ends. In one embodiment, the polynucleotide may Include additional nucleotides that are not specifically related (that have a non-complementary or identical sequence) to the target DNA or gene that has a sequence that is selected from the group consisting of: the Group of Gene Sequences Target or its DNA complement, for example nucleotides that provide a secondary stabilizing or convenience structure for cloning or manufacturing. In one embodiment, the polynucleotide may include additional nucleotides located immediately adjacent to one or more segments of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with a fragment of equivalent length of a DNA or gene. target having a sequence that is selected from the group consisting of: the Gen Diana Gene Sequence Group, or its DNA complement. In one embodiment, the pollnucleotide comprises such a segment, with an additional 5'G or an additional 3'C, or both, adjacent to the segment. In another embodiment, the pollnucleotide is a double-stranded RNA that comprises additional nucleotides to form a surplus, for example, a dsRNA that comprises 2 deoxyrrbonucleotides to form a 3 'surplus. Therefore in various embodiments, the nucleotide sequence of the entire polynucleotide is not 100% Identical to or complementary to a contiguous nucleotide sequence in the target DNA or gene that has a sequence that is selected from the group consisting of: the Group of Diana gene sequences, or their DNA complement. For example, in some embodiments, the polynucleotide comprises at least two segments each of 21 contiguous nucleotides with a sequence of 100% Identity with a fragment of a DNA having a sequence that is selected from the group consisting of: the Diana Gene Sequence Group, or its DNA complement, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in a different order from corresponding fragments occur in DNA that has a sequence that is selected from the group consisting of: the Gen Diana Sequence Group, or its DNA complement.
The polynucleotide for use in this method is provided by suitable means.
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known to a person skilled in the art. The modalities; and, -; ||<sub>OR</sub>j dumJc cl polynucleate is chemically synthesized (eg, by in vitro transcription, such as transcription using a T7 polymerase or other polymerase), produced by expression in a microorganism or in cell culture (such as cultured plant or insect cells in culture), produced by expression in a plant cell, or produced by microbial fermentation.
In some embodiments, the polynucleotide for use in this method is provided as an isolated DNA or RNA fragment. In some embodiments, the polynucleotide for use in this method is not part of an expression construct and does not have additional elements such as a promoter or termination sequences). These polynucleotides can be relatively short, such as single or double chain polynucleotides of between about 18 and about 300 or between about 50 and about 500 nucleotides (for single chain polynucleotides) or between about 18 and about 300 or between about 50 and about 500 base pairs (for double chain polynucleotides). In some embodiments, the polynucleotide is a dsRNA of between about 100 and about 500 base pairs, such as a dsRNA the length of any of the dsRNA triggers described in Tables 3, 5, 8, 9, and 10. Modalities include those where the polynucleotide is a dsRNA comprising a segment that has a sequence that is selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or where the polynucleotide is encoded by a sequence that is selected from the group consisting of SEQ ID NO: 1105-1109. Alternatively, the polynucleotide can be provided in more complex constructs, eg, as part of a recombinant expression construct, or included in a recombinant vector, eg, in a recombinant plant virus vector or in a baculovirus vector. recombinant. In some embodiments, these recombinant expression vectors or constructs are designed to include additional elements, such as expression cassettes to express a gene of interest (eg, an insecticidal protein).
In various embodiments of the method, contacting comprises the application to a surface of the Leptinotarsa species of a suitable composition comprising the polynucleotide for use in this method; Such a composition can be provided, for example, as a solid, liquid (including homogeneous mixtures as solutions and non-homogeneous mixtures as suspensions, colloids, micelles, and emulsions), powder, suspension, emulsion, spray, encapsulated or microencapsulation formulation, within or on top of microbeads or other carrier particles, in a film or coating, or in or on a matrix, or as a seed treatment. The contact may be in the form of a seed treatment or in the form of a treatment of seed potato tubers or tuber pieces (eg, by soaking, coating, or dusting the seed potato). Binders, inert carriers, may optionally be included.
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Suitable surfactants and the like in the composition, such as pesticide Ufoxmulaúán and seed treatment. In some embodiments, contacting comprises providing the polynucleotide in a composition further comprising one or more components that are selected from the group consisting of a carrier agent, a surfactant, a cationic lipid (such as that described in Example 18 of US patent application publication 2011/0296556, incorporated herein by this reference), an organosilicone, an organosilicone surfactant, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide pesticide, a protector and a regulator of insect growth. In some embodiments, contacting comprises providing the polynucleotide in a composition further comprising at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecysteroid, an insecticidal protein Bacillus thuríngiensis, an insecticidal protein Xenorhabdus, a Photorhabdus insecticidal protein, a BaciHus taterosporous insecticidal protein and a Bacillus sphaerícus insecticidal protein. In one embodiment, contacting comprises providing the polynucleotide in a composition that can be ingested or otherwise internally absorbed by the Leptinotarse species.
It is anticipated that the combination of certain polynucleotides for use in this method (eg, the polynucleotide triggers described in the Working Examples) with one or more non-polynucleotide pesticidal agents will result in a synergistic improvement in the prevention or control of infestations of the Leptinotarsa species, when compared with the effect obtained with the polynucleotide alone or the non-polynucleotide pesticide agent alone. In one embodiment, a composition was found to contain one or more polynucleotides and one or more non-polynucleotide pesticidal agents that are selected from the group consisting of a patatin, a plant lectin, a phytoecysteroid, an insecticidal protein Bacillus thuríngiensis, a protein Xenorhabdus insecticide, a Photorhabdus insecticidal protein, a Bacillus taterosporous insecticidal protein and a Bacillus sphaerícus insecticidal protein, performs synergistically improved prevention or control of Leptinotarsa infestations.
Control of Leptinotarse infestations by providing a food polynucleotide
Another aspect of this invention provides a method of controlling an infestation with the Leptinotarsa species of a plant comprising providing in the diet of a Leptinotarsa species an agent comprising a polynucleotide having at least one segment of 18 or more contiguous nucleotides with a sequence about 95% to about 100% identity with a fragment of equivalent length of DNA that has a sequence that
<img file="MX359191B_D0025.tif" />
IMPI
MEXICAN INSTITUTE 99 of the industrial MONEDAD selects from the group consisting of: The Sequence Group of ~~ Ggñ ~ Pia ~ r¡a; ~ or its DNA complement, where the agent works after ingestion by the Leptinotarsa species to inhibit a biological function within the Leptinotarsa species thus controlling Infestation with the Leptinotarsa species. The polynucleotide may be longer than the segment or segments it contains, but each polynucleotide segment and the corresponding DNA fragment are equivalent in length. The polynucleotides for use in the method can be designed for multiple target genes. Modalities include those where the agent comprises a dsRNA comprising a segment that has a sequence selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or where the agent comprises a polynucleotide or RNA is encoded by a sequence that is selected from the group consisting of SEQ ID NO: 1105-1109. In one embodiment, a method is provided for controlling an infestation with the Leptinotarsa species of a plant comprising providing in the diet of the Leptinotarsa species a polynucleotide comprising a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a target gene it has a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO: 1087 - 1094, or an RNA transcribed from the target gene. In some embodiments, the polynucleotide is a double-stranded RNA. In some embodiments, the polynucleotide (eg, double-stranded RNA) is chemically synthesized or produced by expression in a microorganism or by expression in a plant cell. Modalities include those where the polynucleotide is a dsRNA with a strand having a sequence that is selected from the group consisting of the Trigger Sequence Group. Related aspects of the invention include isolated polynucleotides for use in the method and plants having improved resistance to Leptinotarse provided by the method.
In various embodiments, the agent comprising a polynucleotide comprises a microbial cell or is produced in a microorganism. For example, the agent can include or can be produced in bacteria or yeast cells. In other embodiments, the agent comprising a polynucleotide comprises a transgenic plant cell or is produced in a plant cell (eg, a plant cell that transiently expresses the polynucleotide); Such plant cells can be cells in a plant or cells grown in cell culture or cell suspension.
In various embodiments, the agent comprising a polynucleotide is provided for food absorption of the Leptinotarsa species in a form suitable for ingestion, for example, as a solid, liquid (including homogeneous mixtures as solutions or non-homogeneous mixtures as suspensions, colloids, mlcelas and emulsions), powder, suspension, emulsion,
<img file="MX359191B_D0026.tif" />
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY sprayer, encapsulated or microencapsule formulation, Üófliro 0 above iniuupcrtas-or other carrier particles, in a film or coating, or in or on a matrix, or as a seed treatment. The agent comprising a polynucleotide can be provided for food absorption by the Leptinotarsa species by applying the agent to a plant subject to Leptinotarsa species infestation or by applying the agent to a plant seed, for example by spraying, dusting or coating the plant , or soaking the earth, or providing an artificial diet. The agent comprising a polynucleotide can be provided for food absorption by the Leptinotarsa species in an artificial diet formulated to meet the particular nutritional requirements for maintaining the Leptinotarsa species, where the artificial diet is supplemented with a certain amount of the polynucleotide obtained from a source. separated as chemical synthesis or purified from microbial fermentation; This modality may be useful, for example, in determining the timing and amounts of effective chromosomal treatment regimens. In some embodiments, the agent comprising a polynucleotide is provided for food absorption by the Leptinotarsa species in the form of a plant cell or in components of a plant cell, or in a microorganism (such as bacteria or yeast) or a product. from microbial fermentation, or on a synthetic or man-made diet. In one embodiment, the agent comprising a polynucleotide is provided in the form of a bait that is ingested by the species Leptinotarsa. The agent comprising a polynucleotide may be provided for food absorption by the Leptinotarsa species in the form of a seed treatment or in the form of a treatment of seed potato tubers or tuber pieces (eg, by soaking, coating, or dusting of the seed potato). Binders, inert carriers, suitable surfactants and the like may be included in the agent, as is known to one skilled in the formulation of pesticides and seed treatment. In some embodiments, the agent comprising a polynucleotide further comprises one or more components that are selected from the group consisting of a carrier agent, a surfactant, a cationic lipid (such as that described in Example 18 of the application publication US Patent 2011/0296555, incorporated herein by this reference), an organosllicone, an organosllicone surfactant, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide pesticide, a protector and a regulator of insect growth. In some embodiments, the agent comprising a polynucleotide further comprises at least one pesticidal agent which is selected from the group consisting of a patatin, a plant lectin, a phytoecdlsterol, an insecticidal protein Baciiius thuringiensis, an insecticidal protein Xenorhabdus, an insecticidal protein Photorhabdus, a Bacüius iaterosporous Insecticidal Protein and a BaciHus sphaericus Insecticidal Protein. In some embodiments, the agent comprising a polynucleotide comprises at least one implantable formulation that is selected from the group consisting of a
<img file="MX359191B_D0027.tif" />
particle, sediment or capsule implanted in the plant; in such modalities the method comprises<sup>1 </sup>implant the implantable formulation in the plant. In some embodiments, the agent comprising a polynucleotide comprises at least one groove formulation selected from the group consisting of a powder, granule, pellet, capsule, spray, or potion or any other suitable form for application to a groove; In such embodiments, the method comprises a groove treatment with the groove formulation. In some embodiments, the method comprises treating a solanaceous plant seed, potato tuber, or part of a potato tuber with the agent.
It is anticipated that the combination of certain polynucleotides for use in agents for use in this method (eg, the polynucleotide triggers described in the Working Examples) with one or more non-polynucleotide pesticidal agents will result in a synergistic improvement in the prevention or control of Leptinotarsa species infestations, when compared with the effect obtained with the polynucleotide alone or the non-polynucleotide pesticide agent alone. In one embodiment, a composition was found to contain one or more polynucleotides and one or more non-polynucleotide pesticidal agents that are selected from the group consisting of a patatin, a plant lectin, a phytoecysteroid, an insecticidal protein Bacillus thuringiensis, a protein Xenorhabdus insecticide, a Photorhabdus insecticidal protein, a Bacillus iaterosporous insecticidal protein and a Bacillus sphaericus insecticidal protein, it synergistically improves prevention or control of infestations when provided to the species Leptinotarsa in a diet.
In some embodiments, the polynucleotide is a dsRNA that comprises a segment that has a sequence that is selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or where the polynucleotide is encoded by a sequence that is selected from the group consisting of SEQ ID NO: 1105-1109.
In some embodiments, contiguous nucleotides have a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity with a fragment of equivalent length of a target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group or the DNA complement thereof. In some embodiments, the contiguous nucleotides are exactly (100%) identical to a fragment of equivalent length of a target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group or its DNA complement. In some embodiments, the polynucleotide has an overall sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity with a fragment of equivalent length of a target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group or the DNA complement thereof. In a
<img file="MX359191B_D0028.tif" />
IMPI
INSTITUTO MEXICANO ftt LA ΜΟΠΙΓΆΓ)
INnUSTfclAl the modality, the polynucleotide comprises at least one segment of 21 contiguous nucleotides with a sequence of 100% identity to the corresponding fragment of a target gene having a DNA sequence that is selected from the group consisting of: SEQ ID NO: 730 , SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO: 1087 - 1094, or the DNA complement of this; In some embodiments, the pollnucleotide comprises a neutral sequence (which has no sequence identity or complementarity with the target gene) in addition to a segment of 21 contiguous nucleotides with 100% identity to the corresponding fragment of the target gene, and therefore the The entire polynucleotide has a much lower overall sequence identity with a target gene.
The polynucleotide for use in this method is generally designed to suppress one or more genes (target genes). The term gene refers to any part of a nucleic acid that provides for the expression of a transcript or encodes a transcript. A gene can include, but is not limited to, a promoter region, 5 'untranslated regions, regions that encode transcripts that can include intraregional regions, 3' untranslated regions, or combinations of these regions. In some embodiments, the target genes may include coding or non-coding sequences, or both. In other embodiments, the target gene has an identical or complementary sequence to a messenger RNA, for example, in some embodiments the target gene is a cDNA. In specific embodiments, the polynucleotide is designed to delete one or more target genes, where each target gene has a DNA sequence that is selected from the group consisting of the Target Gene Sequence Group. In various embodiments, the pollnucleotide is designed to delete one or more target genes, where each target gene has a sequence that is selected from the group consisting of the Target Gene Sequence Group, and can be designed to delete multiple target genes from this group , or to target different strands of one or more of these target genes. In one embodiment, the pollnucleotide comprises multiple contiguous 21 nucleotide segments with a sequence of 100% Identity with a fragment of equivalent length of a target DNA or gene having a sequence that is selected from the Target Gene Sequence Group or the complement of DNA from this. In such cases, each segment may be identical or different in size or in sequence, and may be sense or antisense relative to the target gene. For example, in one embodiment the polynucleotide comprises multiple tandem segments or repeating arrays, where each segment comprises 21 contiguous nucleotides with a sequence of 100% Identity with a fragment of equivalent length of a target DNA or gene that has a sequence that is select from the Target Gene Sequence Group or its DNA complement; the segments may be from different regions of the target gene, for example, t not corresponding to a target gene may optionally be used between or adjacent to the segments.
<img file="MX359191B_D0029.tif" />
MEXICAN INSTITUTE ye. W '· * MONEDAD
INDUSTRIAL
The total length of the polynucleotide in use in Μί.ΊΙΙίΙ Ιΐΐ Ιυ puidL ~ ci mo / or quo 19 contiguous nucleotides, and may include nucleotides in addition to contiguous nucleotides that have the sequence of about 95% to about 100% identity with a fragment of equivalent length of a target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group or the DNA complement thereof. In other words, the total length of the polynucleotide may be greater than the length of the section or segment of the polynucleotide designed to delete one or more target genes, where each target gene has a DNA sequence that is selected from the group consisting of the Group of Gen Diana Sequences. For example, the polynucleotide may have nucleotides flanking the active segment of at least one segment of 18 or more contiguous nucleotides that suppress the target gene, or include spacer nucleotides between the active segments, or they may have additional nucleotides at the 5 'end, either at the 3 'end, or at both the 5' and 3 'ends. In one embodiment, the polynucleotide can include additional nucleotides that are not specifically related (that have a non-complementary or Identical sequence) to the target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group or the DNA complement of this, for example, nucleotides that provide a secondary stabilizing or convenience structure for cloning or manufacturing. In one embodiment, the polynucleotide may include additional nucleotides located immediately adjacent to one or more segments of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% Identity with a fragment of equivalent length of a DNA or gene. target having a sequence that is selected from the Target Gene Sequence Group or its DNA complement. In one embodiment, the polynucleotide comprises such a segment, with an additional 5'G or an additional 3'C, or both, adjacent to the segment. In another embodiment, the polynucleotide is a double-stranded RNA that comprises additional nucleotides to form a surplus, for example, a dsRNA that comprises 2 deoxyrrbononucleotides to form a 3 'surplus. Therefore in various embodiments, the nucleotide sequence of the entire polynucleotide is not 100% identical or complementary to a contiguous nucleotide sequence in the target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group, or its DNA complement. For example, in some embodiments the pollnucleotide comprises at least two contiguous 21 nucleotide segments with a sequence of 100% identity to a fragment of DNA that has a sequence that is selected from the Gen Diana Sequence Group, or the complement of DNA thereof, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in a different order than the order in which the corresponding fragments occur in the DNA that has a sequence that is selected from the Gen Diana Sequence Group, or its DNA complement.
<img file="MX359191B_D0030.tif" />
MF.XICAN 'I INSTITUTE
Say THE PROPERTY ¿/ industrial
The polynucleotide for use in this method is provided by mSÜIUL. suitable known to one skilled in the art. Modalities include those where the polynucleotide is chemically synthesized (eg, by in vitro transcription, such as transcription using a T7 polymerase or other polymerase), produced by expression in a microorganism or in cell culture (such as plant cells or of insect grown in culture), produced by expression in a plant cell, or produced by microbial fermentation.
In some embodiments, the polynucleotide for use in this method is provided as an isolated DNA or RNA fragment. In some embodiments, the polynucleotide for use in this method is not part of an expression construct and has no additional elements such as a promoter or termination sequences). These pollnucleotides can be relatively short, such as single or double-stranded pollnucleotides of between about 18 and about 300 or between about 50 and about 500 nucleotides (for single-chain pollnucleotides) or between about 18 and about 300 or between about 50 and about 500 base pairs (for double stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of between about 100 and about 500 base pairs, such as a dsRNA the length of any of the dsRNA triggers described in Tables 3, 5, 8, 9, and 10. Alternatively, the polynucleotide can be provided in more complex constructs, eg, as part of a recombinant expression construct, or Include in a recombinant vector, eg, in a recombinant plant virus vector or in a baculovlrus vector. recombinant. In some embodiments, these recombinant expression constructs or vectors are designed to Include additional elements, such as expression cassettes to express a gene of Interest (eg, an Insecticidal protein).
Control of Leptinotarsa infestations by providing a dietary RNA
Another aspect of this invention provides a method of causing mortality or weakening of Leptinotarsa species larvae by providing in the diet of such larvae at least one polynucleotide comprising at least one silencing element comprising 21 contiguous nucleotides that are complementary to a target gene that has a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO: 1087 - 1094, or an RNA transcribed from the target gene. In some embodiments, the polynucleotide is a double-stranded RNA. In some embodiments, the polynucleotide (eg, double-stranded RNA) is chemically synthesized or produced by expression in a microorganism or by expression in a plant cell. In one embodiment, a method is provided for causing mortality or weakening in Leptinotarsa species larvae comprising providing in the
<img file="MX359191B_D0031.tif" />
MEXICAN INSTITUTE
OF INDUSTRIAL PROPERTY
<img file="MX359191B_D0032.tif" />
diet of the Leptinotarsa species at least one RNA that comprises the silencing essentially identical or essentially complementary to a fragment of a target gene sequence of the larvae of the Leptinotarsa species, where the target gene sequence is selected from the group consisting of the Gen Diana Sequence Group, and where the Ingestion of the RNA by the larvae of the Leptinotarsa species results in the mortality or weakening of the larvae of the Leptinotarsa species. A related aspect of this invention is an RNA comprising at least one silencing element, where the at least one silencing element is essentially Identical or essentially complementary to a fragment of a target gene from the larvae of the Leptinotarsa species, where the sequence The target gene is selected from the group consisting of the Target Gene Sequence Group. RNA can be longer than the silencing element or silencing elements it contains, but each silencing element and the corresponding fragment of a target gene sequence are equivalent in length. The RNAs for use in the method can be designed for multiple target genes; modalities include RNA comprising at least one silencing element comprising a sequence selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or where the silencing element is encoded by a sequence that is selected from the group consisting of SEQ ID NO: 1105-1109. Modalities include those where the RNA comprises a dsRNA with a strand having a sequence that is selected from the group consisting of the Trigger Sequence Group. In a related aspect, there is provided a method of causing mortality or lower fertility in the Leptinotarsa species comprising providing in the diet of the Leptinotarsa species at least one RNA comprising at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence from Leptinotarsa species larvae, where the target gene sequence is selected from the Target Gene Sequence Group, or the DNA complement of this, and where the Ingestion of RNA by the Leptinotarsa species results in mortality or lower fertility in the Leptinotarsa species. Related aspects of the invention include isolated RNAs for use in the method and plants having improved resistance to Leptinotarse provided by the method.
In various embodiments, the RNA-supplied diet comprises a microbial cell or is produced in a microorganism. For example, the diet that RNA provides can include or can be produced in bacteria or yeast cells. In similar embodiments, the RNA-providing diet comprises a transgenic plant cell or is produced in a plant cell (eg, a plant cell that transiently expresses the polynucleotide); Such plant cells can be cells in a plant or cells grown in cell culture or cell suspension.
IMPI
<img file="MX359191B_D0033.tif" />
In one embodiment, the diet provided by the RNA was ppurified Uii fuimu <4 «any plant that is subject to infestation with a Leptinotarsa species, where the RNA is contained in the plant. Such plants can be stably transgenic plants that express the RNA, or non-transgenic plants that transiently express the RNA or that were treated with the RNA, for example, by spraying or coating. Stably transgenic plants generally contain an integrated recombinant RNA-encoding construct in their genome. Modalities where the plant is a nightshade plant (family Solanaceae) are of particular interest. Examples Include a plant that is selected from the group consisting of potato, tomato, and eggplant. Modalities include those where the plant is a seed of a non-germinated nightshade plant, a nightshade plant in the vegetative stage, or a nightshade plant in the reproductive stage. Modalities Include those where the plant is a seed potato, meaning a potato tuber or part of a potato tuber that can propagate into new potato plants.
In various embodiments, the RNA-providing diet is provided in a form suitable for ingestion by the Leptinotarsa species, for example, as a solid, liquid (including homogeneous mixtures as solutions or inhomogeneous mixtures as suspensions, colloids, mlcels, and emulsions ), powder, suspension, emulsion, sprayer, encapsulated or microencapsulated formulation, in or on top of microbeads or other carrier particles, in a film or coating, or in or on a matrix, or as a seed treatment. The RNA-providing diet can be provided by applying the diet to a plant subject to Leptinotarsa species Infestation, for example by spraying, dusting or coating the plant, or soaking the soil, or by providing an artificial diet. In one embodiment, the diet that provides the recombinant RNA is provided in the form of a bait that is ingested by the Leptinotarsa species. The diet that provides the RNA can be an artificial diet formulated to meet the particular nutritional requirements to maintain the Leptinotarsa species, where the artificial diet is supplemented with a certain amount of the RNA obtained from a separate source as chemical synthesis or purified from microbial fermentation ; This modality may be useful, for example, in determining the timing and amounts of effective polynucleotide treatment regimens. In some embodiments, the RNA-providing diet is provided in the form of a plant cell or in components of a plant cell, or in a microorganism (such as bacteria or yeast) or a microbial fermentation product, or in a synthetic diet. In one embodiment, the RNA-providing diet is provided in the form of a bait that is ingested by the Leptinotarsa species. The RNA-providing diet can be provided in the form of a seed treatment or in the form of a treatment of seed potato tubers or tuber parts (eg, by soaking, coating, or dusting the seed potato). I know
IMPI
<img file="MX359191B_D0034.tif" />
they may include binders, inert carriers, suitable surfactants and the like in the diet, as is known to one skilled in the formulation of pesticides and seed treatment. In some embodiments, the RNA-providing diet further comprises one or more components that are selected from the group consisting of a carrier agent, a surfactant, a cationic lipid (such as that described in Example 18 of the application publication US Patent 2011/0296556, incorporated herein by this reference), an organosilicone, an organosilicone surfactant, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide pesticide, a protector and a regulator of insect growth. In some embodiments, the RNA-providing diet further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdlsteroid, an insecticidal protein Bacillus thuríngiensis, an insecticidal protein Xenorhabdus, an insecticidal protein Photorhabdus, a Bacillus iaterosporous insecticidal protein and a Bacillus sphaericus insecticidal protein. In some embodiments, the RNA-providing diet includes at least one implantable formulation that is selected from the group consisting of a particle, sediment, or capsule implanted in the plant; in such modalities the method comprises implanting the implantable formulation in the plant. In some embodiments, the RNA-providing diet includes at least one formulation in the groove that is selected from the group consisting of a powder, granule, pellet, capsule, spray, or potion, or any other suitable forms to apply to a groove; in such embodiments, the method includes a groove treatment with the groove formulation. In some embodiments, the method comprises treating a seed of a soya plant, potato tuber, or part of a potato tuber with the agent.
It is anticipated that the combination of certain RNAs for use in this method (eg, the dsRNA triggers described in the Working Examples) with one or more non-poly nucleotide pesticidal agents will result in a synergistic improvement in the prevention or control of Leptinotarsa species infestations, when compared with the effect obtained with RNA alone or the non-polynucleotide pesticide agent alone. In one embodiment, a composition containing one or more RNA and one or more non-polynucleotide pesticidal agents was found to be selected from the group consisting of a patatin, a plant lectin, a phytoecysteroid, an insecticidal protein Bacillus thuríngiensis, a protein Xenorhabdus insecticide, a Photorhabdus insecticidal protein, a Bacillus iaterosporous insecticidal protein and a Bacillus sphaericus insecticidal protein, performs synergistically improved prevention or control of Leptinotarsa species infestations.
The RNA used in this method can have single chain (ss) or double chain (ds). Modalities of the method include those where the RNA is at least one that is selected from the group consisting of antisense single-stranded, single stranded RNA (cRNAs)
<img file="MX359191B_D0035.tif" />
(CRNAs), or double-stranded (dsRNA); a mixture of cDlqUiel'd RNA of these types can be used. In one embodiment, a double-stranded DNA / RNA hybrid is used. RNA can include components other than standard ribonucleotides, for example, one embodiment is an RNA comprising terminal deoxyribonucleotides.
RNA comprises at least one silencing element, where the silencing element is essentially identical (as the RNA equivalent) or essentially complementary to a fragment of a target gene from Leptinotarsa species larvae, where the target gene sequence is select from the group consisting of the Gen Diana Sequence Group. In some embodiments, the silencing element has a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity with or complementing a fragment of equivalent length of a DNA having a sequence that is selected from the group consisting of the Gen Diana Gene Sequence Group. In some embodiments, the silencing element is exactly (100%) Identical or exactly (100%) complementary (as the RNA equivalent) to a fragment of equivalent length of DNA that has a sequence that is selected from the Sequence Group of Diana gene or its DNA complement. In some embodiments, the RNA containing the silencing element (s) has an overall sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of identity with or complementary to a DNA fragment having a sequence that is selected from the group consisting of the Gen Diana Sequence Group.
In some embodiments, the silencing element comprises at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with or complementing a fragment of length equivalent to that of the target gene. In some embodiments, the silencing element comprises at least a segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with or complementing a fragment of equivalent length of DNA that has a sequence which is selected from the group consisting of the Gen Diana Sequence Group. In some embodiments, the silencing element comprises at least one segment of 18 or more contiguous nucleotides, for example, between 18-24, or between 18-28, or between 20-30, or between 20-50, or between 20- 100, or between 50 - 100, or between 50 - 500, or between 100 - 250, or between 100 - 500, or between 200 - 1000, or between 500 - 2000, or even more. In some embodiments, the silencing element comprises more than 18 contiguous nucleotides, for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, for example, about 35, around 40, around 45, around 50, around 55, around 60, around 65, around 70, around 75, around 80, around 85, around 90, around 95 , around 100, around 110,
<img file="MX359191B_D0036.tif" />
IMPI
INSTITUTO MEXICANO DE LA ΕΚΟΡΙΕΠΑΟ JZ INDUSTRIAL around 120, around 130, around 140, around ~ de ISPj '^ TTggectol from ~ 160, around 170, around 180, around 190, around 200, around 210, around 220, around 230, around 240, around 250, around 260, around 270, around 280, around 290, around 300, around 350, around 400, around 450, around 500, or more than 500 contiguous nudeotides. In particular embodiments, the silencing element comprises at least a segment of at least 21 contiguous nudeotides with a sequence of 100% Identity with a fragment of equivalent length of a target DNA or gene having a sequence that is selected from the Sequence Group Diana gene or DNA complement of this. In particular embodiments, the RNA is a double-stranded nucleic acid (eg, dsRNA) with a strand comprising at least one segment of at least 21 contiguous nudeotides with a sequence of 100% Identity with a fragment of equivalent length of a DNA or target gene having a sequence selected from the group consisting of the Target Gene Sequence Group or the DNA complement thereof; expressed as base pairs, such a double-stranded nucleic acid comprises at least one segment of at least 21 contiguous nudeotides, perfectly matched base pairs corresponding to a fragment of equivalent length of a target DNA or gene that has a sequence that is select from the Target Gene Sequence Group or its DNA complement. In particular embodiments, each silencing element contained in the RNA is longer than typical of naturally occurring small regulatory RNAs, for example, each segment is at least about 30 contiguous nudeotides (or base pairs) in length. In some embodiments the total length of the RNA, or the length of each silencing element contained in the RNA, is less than the total length of the sequence of interest (DNA or target gene that has a sequence that is selected from the group consisting of the Gen Diana Sequence Group). In some embodiments, the total length of the RNA is between about 50 and about 500 nudeotides (for single chain polynucleotides) or base pairs (for double chain polynucleotides). In some embodiments, the RNA is a dsRNA of between about 100 and about 500 base pairs, such as a dsRNA the length of any of the dsRNA triggers described in Tables 3, 5, 8, 9, and 10. Modalities include those where the RNA is a dsRNA comprising a segment that has a sequence that is selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or where the RNA is encoded by a sequence that is selected from the group consisting of SEQ ID NO: 1105-1109.
The RNA for use in this method is generally designed to suppress one or more genes (target genes). The term gene refers to any part of a nucleic acid that provides for the expression of a transcript or encodes a transcript. A gene can include,
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INDUSTRIAL but not limitingly, a promoter region, 5 'untranslated regions, regions encoding transcripts that may include intronic regions, 3' untranslated regions, or combinations of these regions. In some embodiments, the target genes can include coding or non-coding sequences, or both. In other embodiments, the target gene has an identical or complementary sequence to a messenger RNA, for example, in some embodiments, the target gene is a cDNA. In specific embodiments, the RNA is designed to suppress one or more target genes, where each target gene has a DNA sequence that is selected from the group consisting of the Target Gene Sequence Group. In various modalities, RNA is designed to delete one or more genes, where each gene has a sequence that is selected from the group consisting of the Target Gene Sequence Group, and can be designed to delete multiple genes from this group, or to target different regions of one or more of these genes. In one embodiment, the RNA comprises multiple silencing elements each of which comprises at least one segment of 21 contiguous nucleotides with a sequence of 100% identity or 100% complementarity to a fragment of equivalent length of DNA having a sequence that is selected from the Gen Diana Gene Sequence Group or its DNA complement. In such cases, each silencing element may be identical or different in size or sequence, and may be sense or antisense relative to the target gene. For example, in one embodiment the RNA can include multiple tandem silencing elements or repeating arrays, where each silencing element comprises at least one segment of 21 contiguous nucleotides with a sequence of 100% identity or 100% complementarity to a fragment of equivalent length of a DNA having a sequence selected from the group consisting of the Gen Diana Sequence Group; the segments may be from different regions of the target gene, for example, the segments may correspond to different regions of the exon of the target gene, and the spacer nucleotides that do not correspond to a target gene may optionally be used between or adjacent to the segments.
The total length of the RNA can be greater than 18 contiguous nucleotides, and can include nucleotides in addition to the silencing element that has a sequence of about 95% to about 100% identity or complementarity to a fragment of equivalent length of DNA or target gene having a sequence that is selected from the group consisting of the Target Gene Sequence Group. In other words, the total length of the RNA may be greater than the length of the silencing element designed to suppress one or more target genes, where each target gene has a DNA sequence that is selected from the group consisting of the Group of Sequences of Gen Diana. For example, RNA can have nucleotides flanking the active silencing element of at least a segment of 18 or more contiguous nucleotides that suppress the target gene, or include spacer nucleotides between the elements of
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INSTITUTO MEXICANO r'-WMSSp DE LA PROPIEDAD Λ)
INDUSTRIAL silencing active, or may have additional nucleotides at the 5 'end, or at the 5' and 3 'ends. In one embodiment, the RNA comprises additional nucleotides that are not specifically related (that have a non-complementary or Identical sequence) to the target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group or DNA complement thereof. , eg, nucleotides that provide a stabilizing or convenience secondary structure for cloning or manufacturing. In one embodiment, the RNA comprises additional nucleotides located Immediately adjacent to one or more silencing elements of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% Identity with or complementary to a fragment of equivalent length of a target DNA or gene that has a sequence that is selected from the group consisting of the Target Gene Sequence Group. In one embodiment, the RNA comprises such a silencing element, with an additional 5'G or an additional 3'C or both, adjacent to the silencing element. In another embodiment, the RNA is a double-stranded RNA comprising additional nucleotides to form a surplus, eg, a dsRNA comprising 2 deoxyrrbonbonucleotides to form a 3 'surplus. Therefore in various embodiments, the nucleotide sequence of the entire RNA is not 100% identical or complementary to a contiguous nucleotide fragment in the target DNA or gene that has a sequence that is selected from the group consisting of the Sequence Group by Gen Diana. For example, in some embodiments the RNA comprises at least two silencing elements each of 21 contiguous nucleotides with a sequence of 100% identity to a fragment of DNA having a sequence that is selected from the group consisting of the Group of Dlana4 Gene sequences, or the DNA complement thereof, where (1) the at least two silencing elements are separated by one or more spacer nucleotides, or (2) the at least two silencing elements are arranged in a different order than the order in which the corresponding fragments occur in DNA having a sequence that is selected from the group consisting of the Gen Diana Sequence Group, or the DNA complement of this.
In some embodiments, the RNA consists of naturally occurring rlbonucleotides. In certain embodiments, the RNA comprises components other than rlbonucleotides, for example, synthetic RNAs * consist primarily of rlbonucleotides but with one or more terminal deoxyrrbonbonucleotides or one or more terminal deoxyrrbonbonucleotides. In certain embodiments, the RNA comprises non-canonical nucleotides such as Inosine, thiourldlna, or pseudouridine. In certain embodiments, the RNA comprises chemically modified nucleotides.
RNA for use in this method is provided by suitable means known to one of skill in the art. Modalities include those where RNA is chemically synthesized
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INSTITUTO MEXICANO DE LA PRORIF.OAD INOO.TRial (for example, by in vitro transcription, such as transcríDiórum¡ñnrln ..- a T7 polymerase or other polymerase), produced by expression in a microorganism or in cell culture ( such as plant or insect cells grown in culture), produced by expression in a plant cell, or produced by microbial fermentation.
In some embodiments, the RNA is provided as an isolated RNA that is not part of an expression construct and has no additional elements such as a promoter or termination sequences). Such RNAs can be relatively short, such as single- or double-stranded RNAs of between about 18 and about 300 or between about 50 and about 500 nucleotides (for single-stranded RNA) or between about 18 and about 300 or between about 50 and about 500 base pairs (for double stranded RNA). Alternatively, RNA can be provided in more complex constructs, eg, as part of a recombinant expression construct, or included in a recombinant vector, eg, in a recombinant plant virus vector or in a baculovirus vector recombinant. In some embodiments, these recombinant expression vectors or constructs are designed to include additional elements, such as including additional RNA encoding an aptamer or ribozyme or an expression cassette to express a gene of interest (eg, an insecticidal protein).
Methods of providing plants having improved resistance to infestations of the Leptinotarsa species, and the plants, plant parts, and seeds thus provided
Another aspect of this invention provides a method of providing a plant with improved resistance to an infestation with the Leptinotarsa species, comprising applying topically to the plant a composition comprising at least one polynucleotide having at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with a fragment of a target gene or DNA that has a sequence that is selected from the Gen Diana Gene Sequence Group or its DNA complement, such that the plant treated with the polynucleotide-containing composition exhibits improved resistance to an infestation with the Leptinotarsa species, relative to an untreated plant. In one embodiment, the at least one polynucleotide comprises at least a segment of 18 or more contiguous nucleotides that are essentially identical to a fragment of equivalent length of DNA that has a sequence that is selected from the Gen Diana Gene Sequence Group or the complement of DNA from this. The polynucleotide may be longer than the segment or segments it contains, but each segment and the corresponding fragment of a target gene are equivalent in length. In one embodiment, this invention provides a method of providing a plant having improved resistance to an infestation with the Leptinotarsa species comprising applying
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- Γ 'MEXICAN NSTITUTE
Μ IA INDUSTRIAL PROPERTY topically to the plant a composition comprising the ηιιηοΓΓιϊι pi iIiiiUlIlJULIlIu that complies with a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a target gene that has a nucleotide sequence selected from the group consisting of in: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO : 1087-1094, or an RNA transcribed from the target gene. In one embodiment, this invention provides a method of providing a plant having improved resistance to an infestation with the Leptinotarsa species comprising applying topically to the plant a composition comprising at least one pollnucleotide such that an effective amount of the pollnucleotide is ingested by the Leptinotarsa species that feeds on the plant, the polynucleotide comprises at least 21 contiguous nucleotides that are complementary to a target gene having a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1-725, SEQ ID NO: 726-729, SEQ ID NO: 731-806, SEQ ID NO: 808-830, and SEQ ID NO-.1087-1094, or a transcribed RNA of the target gene. In some embodiments, this invention provides a method of controlling an infestation with the Leptinotarsa species of a plant comprising applying topically to the plant a composition comprising at least one polynucleotide such that an effective amount of the polynucleotide is ingested by the Leptinotarse species. that feeds on the plant, the polynucleotide comprises a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a target gene that has a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO : 1-725, SEQ ID NO: 726-729, SEQ ID NO: 731-806, SEQ ID NO: 808-830, and SEQ ID NO: 1087-1094, or an RNA transcribed from such a target gene; where such a Leptinotarsa species is Leptinotarsa decemiineata ', and where the target gene has the sequence of SEQ ID NO: 730 or where the polynucleotide is a double stranded RNA that has a strand with a sequence selected from the group consisting of SEQ ID NO: 989, 988, 1104, or 1105. The polynucleotides for use in the method can be designed for multiple target genes. Modalities include those where the polynucleotide comprises a segment that has a sequence that is selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or where the polynucleotide it is encoded by a sequence that is selected from the group consisting of SEQ ID NO: 1105-1109. Modalities include those where the composition comprises a dsRNA with a strand having a sequence that is selected from the group consisting of the Trigger Sequence Group. Related aspects of the invention include compositions for topical application and isolated polynucleotides for use in the method, and plants with improved resistance to Leptinotarse provided by the method.
Topical application refers to the application to the surface or exterior of an object, such as the surface or exterior of a plant, such as application to the surface of a part of
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the plant such as a leaf, stem, flower, fruit, bud, root, seed, tuber, flower, antennae, or pollen, or application to an entire plant, or the above-ground or below-ground parts of a plant. Topical application can be performed on non-living surfaces such as application to soil, or to a surface or matrix through which a Leptinotarsa Insect may come in contact with the polynucleotide. In various embodiments of the method, the composition comprising at least one polynucleotide is applied topically to the plant suitably, for example, as a solid, liquid (including homogeneous mixtures such as solutions and non-homogeneous mixtures such as suspensions, colloids, micelles and emulsions), powder, suspension, emulsion, sprayer, encapsulated or microencapsulated formulation, in or on top of microbeads or other carrier particles, in a film or coating, or in or on a matrix, or as a seed treatment. In some embodiments of the method, the polynucleotide-containing composition is applied topically to the above-ground parts of the plant, for example, sprayed or dusted on leaves, stems, and flower parts of the plant. Modalities of the method include topical application of a foliar spray (eg, spraying a liquid composition containing polynucleotides onto the leaves of a nightshade plant) or a leaf powder (eg, dusting a nightshade plant with a polynucleotide-containing composition in the form of a powder or carrier particles). In other embodiments, the polynucleotide-containing composition is applied topically to below-ground parts of the plant, such as to the roots, for example, wetting the soil. In other embodiments, the polynucleotide-containing composition is applied topically to a seed grown in the plant. Topical application may be in the form of a topical treatment of nightshade plant fruits or fruit seeds of nightshade plants or in the form of topical treatment of seed potato tubers or parts of a tuber (for example, soaking, coating or sprinkling the seed potato). Binders, inert carriers, suitable surfactants and the like may optionally be included in the polynucleotide-containing composition, as is known to one skilled in the formulation of pesticides and seed treatment. In some embodiments, the polynucleotide-containing composition is at least a topically implantable formulation that is selected from the group consisting of a particle, pellet, or capsule topically implanted in the plant; in such embodiments the method comprises topically implanting the topically implantable formulation into the plant. In some embodiments, the polynucleotide-containing composition is at least one groove formulation selected from the group consisting of a powder, granule, pellet, capsule, spray, or potion or any other suitable form to be applied topically to a groove; in such modalities, the method includes a groove treatment with the groove formulation. In one embodiment, the polynucleotide-containing composition can be ingested or otherwise absorbed internally by the Leptinotarsa species. For example, the polynucleotide-containing composition may be in the form of a bait. In some
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MEXICAN INSTITUTE of [je property
INDUSTRIAL modalities, the polynucleotide-containing composition further comprises one or more components that are selected from the group consisting of a carrier agent, a surfactant, a cationic lipid (such as that described in Example 18 of the patent application publication 2011/0296556, incorporated herein by this reference), an organosllicone, an organosllicone surfactant, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide pesticide, a protector and a regulator of insect growth. In one embodiment the composition further comprises a nonionic organosllicone surfactant such as SILWET® brand surfactants, eg SILWET L-77® brand surfactant with CAS number 27306-78-1 and EPA number: CAL.REG. No. 5905-50073-AA, currently available from Momentive Performance Materials, Albany, New York. In some embodiments, the topically applied composition further comprises at least one pesticidal agent that is selected from the group consisting of a patatin, a plant lectin, a phytoecdlsterol, a BaciHus thuringiensis Insecticidal protein, a Xenorhabdus Insecticidal protein, a Photorhabdus Insecticidal protein, a BaciHus iaterosporous insecticidal protein and a BaciHus sphaericus Insecticidal protein. Alternatively, such additional components or pesticidal agents can be provided separately, for example, by separate topical application or by transgenic expression in the plant. Alternatively, the plant is treated topically with the composition containing polynucleotides as well as with a separate application (previous, next, or simultaneous) of a substance that improves the efficacy of the composition containing polynucleotides. For example, a plant can be sprayed with a first topical application of a solution containing a non-ionic organosllicone surfactant such as SILWET® brand surfactants, for example, SILWET L-77® brand surfactant, followed by a second topical application of the composition containing polynucleotides, or vice versa.
It is anticipated that the combination of certain useful polynucleotides in the polynucleotide-containing composition (eg, the polynucleotide triggers described in the Working Examples) with one or more non-polynucleotide pesticide agents will result in synergistic improvement to prevent or control infestations of the Leptinotarsa species, compared to the effect obtained with the polynucleotide alone or the non-polynucleotide pesticide agent alone. In one embodiment, the composition containing polynucleotides is provided as a transgenic plant expressing one or more polynucleotides and one or more genes encoding a non-polynucleotide pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdlsterolde , an insecticidal BaciHus thuringiensis protein, an Insecticidal protein Xenorhabdus, an Insecticidal protein Photorhabdus, an insecticidal protein BaciHus iaterosporous, and an insecticidal protein Baciiius sphaericus, where the transgenic plant is found to exhibit improved resistance to species infestations.
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7Q MEXICAN INSTITUTE
SAY THE INWSTRIAL PROPERTY
Leptinotarsa. __
The polynucleotide useful in the polynucleotide-containing composition is provided by suitable means known to those skilled in the art. Modalities include those where the polynucleotide is chemically synthesized (eg, by in vitro transcription, such as transcription using a T7 polymerase or other polymerase), produced by expression in a microorganism or in cell culture (such as plant cells or of insect grown in culture), produced by expression in a plant cell, or produced by microbial fermentation.
In many embodiments, the polynucleotide useful in the polynucleotide-containing composition is provided as an isolated DNA or RNA fragment. In some embodiments, the polynucleotide useful in the polynucleotide-containing composition is not part of an expression construct and has no additional elements such as a promoter or termination sequences). These polynucleotides can be relatively short, such as single or double-stranded polynucleotides of between about 18 and about 300 or between about 50 and about 500 nucleotides (for single chain polynucleotides) or between about 18 and about 300 or between about 50 and about 500 base pairs (for double stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of between about 100 and about 500 base pairs, such as a dsRNA the length of any of the dsRNA triggers described in Tables 3, 5, 8, 9, and 10. Alternatively, the polynucleotide can be provided in more complex constructs, eg, as part of a recombinant expression construct, or included in a recombinant vector, eg, in a recombinant plant virus vector or in a baculovirus vector. recombinant. Such recombinant expression constructs or vectors can be designed to include additional elements, such as expression cassettes to express a gene of Interest (eg, an insecticidal protein).
The polynucleotide useful in the polynucleotide-containing composition has at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with a fragment of equivalent length of DNA that has a sequence that is select from the Gen Diana Gene Sequence Group or its DNA complement. In one embodiment, the polynucleotide comprises at least one segment of 18 or more contiguous nucleotides that are essentially Identical or essentially complementary to a fragment of equivalent length of DNA that has a sequence that is selected from the group consisting of the Group of Sequences of Gen Diana. In some embodiments, contiguous nucleotides have a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a fragment of DNA that has a sequence that is selected from the group consisting of: SEQ ID NO: 1-725 or
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SEQ ID NO: 726-830 or SEQ ID ΝΟ.Ί087-1094 or the DNA complement thereof. In some embodiments, the contiguous nucleotides are exactly (100%) identical to a fragment of equivalent length of DNA that has a sequence that is selected from the Gene Target Sequence Group or its DNA complement. In some embodiments, the polynucleotide has an overall sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a fragment of DNA that it has a sequence that is selected from the Gen Diana Sequence Group or its DNA complement.
The polynucleotide useful in the polynucleotide-containing composition comprises at least a segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% Identity with a fragment of equivalent length of DNA having a sequence that is select from the Target Gene Sequence Group or its DNA complement. In some embodiments, the polynucleotide comprises at least one segment of 18 or more contiguous nucleotides, for example, between 18-24, or between 18-28, or between 20-30, or between 20-50, or between 20-100, or between 50 - 100, or between 50 - 500, or between 100 - 250, or between 100 - 500, or between 200 - 1000, or between 500 - 2000, or even more. In some embodiments, the segment comprises more than 18 contiguous nucleotides, for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, for example, about 35, around 40, around 45, around 50, around 55, around 60, around 65, around 70, around 75, around 80, around 85, around 90, around 95, around 100, around 110, around 120, around 130, around 140, around 150, around 160, around 170, around 180, around 190, around 200, around 210, around 220, around 230, around 240, around 250, around 260, around 270, around 280, about 290, about 300, about 350, about 400, about 450, about 500, or more than 500 contiguous nucleotides. In particular embodiments, the polynucleotide comprises at least a segment of at least 21 contiguous nucleotides with a sequence of about 100% identity with a fragment of equivalent length of a target DNA or gene having a sequence that is selected from the Group of
Gene Diana sequences or their DNA complement. In particular embodiments, the polynucleotide is a double-stranded nucleic acid (eg, dsRNA) with a strand comprising at least one segment of at least 21 contiguous nucleotides with a sequence of 100% identity with a fragment of equivalent length of a DNA or target gene having a sequence that is selected from the Target Gene Sequence Group or the DNA complement thereof; expressed as base pairs, this double-stranded nucleic acid comprises at least one segment of at least 21 contiguous nucleotides, base pairs that perfectly match that
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they correspond to a fragment of equivalent length of a target DNA or gene that fills a sequence that is selected from the Target Gene Sequence Group or the DNA complement of this. In particular embodiments, each segment contained in the polynucleotide is longer than typical of small, naturally occurring regulatory RNAs, for example, each segment is at least about 30 contiguous nucleotides (or base pairs) long. In some embodiments, the total length of the polynucleotide, or the length of each segment contained in the polynucleotide, is less than the total length of the sequence of interest (DNA or target gene that has a sequence that is selected from the group consisting of Group of Gen Diana Sequences).
In some embodiments, the total length of the polynucleotide is between about 50 and about 500 nucleotides (for single-chain pollnucleotides) or base pairs (for double-chain pollnucleotides). In some embodiments, the polynucleotide is a dsRNA of between about 100 and about 500 base pairs, such as a dsRNA the length of any of the dsRNA triggers described in Tables 3, 5, 8, 9, and 10. In some embodiments, the polynucleotide is a dsRNA that comprises a segment that has a sequence that is selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or the pollnucleotide is encoded by a sequence selected from the group consisting of SEQ ID NO: 1105-1109.
The topically applied polynucleotide is generally designed to suppress one or more genes (target genes). Such target genes can include coding or non-coding sequences, or both. In specific embodiments, the polynucleotide is designed to delete one or more target genes, where each target gene has a DNA sequence that is selected from the group consisting of the Target Gene Sequence Group. In various embodiments, the topically applied polynucleotide is designed to delete one or more target genes, where each gene has a sequence that is selected from the group consisting of the Target Gene Sequence Group, and can be designed to delete multiple genes from this group , or to target different strands of one or more of these genes. In one embodiment, the topically applied polynucleotide comprises multiple sections or segments each comprising at least one segment of 21 contiguous nucleotides with a sequence of 100% Identity with a fragment of equivalent length of DNA having a sequence that is select from the Target Gene Sequence Group or its DNA complement. In such cases, each section may be Identical or different in size or sequence, and may be sense or antisense relative to the target gene. For example, in one embodiment, the topically applied polynucleotide may include multiple tandem sections or repeating arrays, where each section comprises at least one segment of 21 contiguous nucleotides with a sequence of 100% identity with a fragment of equivalent length of DNA that has a sequence that is selected from the group consisting of: SEQ ID
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NO: 1-725 or SEQ ID NO: 726-830 or SEQ ID NO: 1087-1094 or the DNA complement thereof; the segments may be from different regions of the target gene, for example, the segments may correspond to different regions of the exon of the target gene, and the spacer nucleotides that do not correspond to a target gene may optionally be used between or adjacent to the segments.
The total length of the topically applied polynucleotide may be greater than 18 contiguous nucleotides, and may include nucleotides in addition to contiguous nucleotides that have the sequence of about 95% to about 100% Identity with a fragment of equivalent length of DNA that it has a sequence that is selected from the Gen Diana Sequence Group or its DNA complement. In other words, the total length of the topically applied polynucleotide may be greater than the length of the section or segment of the polynucleotide designed to suppress one or more target genes, where each target gene has a DNA sequence that is selected from the group consisting of the Gen Diana Sequence Group. For example, the topically applied polynucleotide may have nucleotides flanking the active segment of at least one segment of 18 or more contiguous nucleotides that suppress the target gene, or include spacer nucleotides between the active segments, or they may have additional nucleotides at the 5-terminus. ', or at the 3' end, or at both the 5 'and 3' ends. In one embodiment, the topically applied polynucleotide comprises additional nucleotides that are not specifically related (that have a non-complementary or identical sequence) to the target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group or DNA complement of this, for example, nucleotides that provide a stabilizing or convenience secondary structure for cloning or manufacturing. In one embodiment, the topically applied polynucleotide comprises additional nucleotides located immediately adjacent to one or more segments of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% Identity with or complementary to a fragment of equivalent length of a target DNA or gene that has a sequence that is selected from the group consisting of the Target Gene Sequence Group. In one embodiment, the topically applied polynucleotide comprises such a segment, with an additional 5'G or an additional 3'C, or both, adjacent to the segment. In another embodiment, the topically applied polynucleotide is a double-stranded RNA that comprises additional nucleotides to form a surplus, eg, a dsRNA comprising 2 deoxyribonucleotides to form a 3 'surplus. Therefore in various modalities, the nucleotide sequence of the entire topically applied pollnucleotide is not 100% identical or complementary to a contiguous nucleotide fragment in the target DNA or gene that has a sequence that is selected from the group consisting of Group of Gen Diana Sequences. For example, in some embodiments the topically applied polynucleotide comprises at least two segments each of 21 contiguous nucleotides with a sequence of 100% identity to a fragment of DNA that has a
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sequence that is selected from, or the DNA complement of, the Gen Diana Sequence Group, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in an order other than the order in which the corresponding fragments occur in DNA that has a sequence that is selected from the Gen Diana Sequence Group, or its DNA complement.
In a related aspect, this invention relates to the plant having improved resistance to an Infestation with the Leptinotarsa species, provided by this method comprising applying topically to the plant a composition comprising at least one polynucleotide having at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% Identity with a fragment of equivalent length of a DNA that has a sequence that is selected from the Gen Diana Gene Sequence Group or its DNA complement, whereby the plant treated with the polynucleotide composition exhibits improved resistance to an infestation with the Leptinotarsa species, relative to an untreated plant. One modality is a nightshade plant that has improved resistance to an Infestation with the Leptinotarsa species compared to a control plant, provided by applying topically to the plant or to a seed grown in the plant (or, when the plant is a potato plant, to a seed potato grown in the potato plant) a dsRNA trigger that has a sequence selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or a dsRNA trigger that is encoded by a sequence selected from the group consisting of SEQ ID NO: 1105-1109. In yet another aspect, this invention relates to seeds (especially transgenic progeny seed) produced by the plant that has improved resistance to an infestation with the Leptinotarsa species, as provided by this method. Also contemplated is a basic product produced by the plant that has improved resistance to an Infestation with the Leptinotarsa species, as provided by this method, and a basic product produced from the seed of transgenic progeny of such a plant.
Insecticidal compositions to control the species Leptinotarsa
Another aspect of this Invention provides an Insecticidal composition for controlling a Leptinotarsa species comprising an Insecticidal effective amount of at least one RNA comprising at least a segment of 18 or more contiguous nucleotides that is essentially Identical to or complement a fragment of a gene target or DNA having a sequence that is selected from the group consisting of the Target Gene Sequence Group. In this context, control includes an incentive for a physiological or behavioral change in a Leptinotarsa species (adult or larvae) such as, non-exhaustively, weakening of growth,
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ΡΙ
MEXICAN INSTITUTE 44 OF PROPERTY
INDUSTRIAL increased mortality, reduction in reproductive capacity, reduction in eeae of movlmlonto or feeding behavior, or reduction in or cessation of development in the metamorphosis stage. Effective as an insecticide refers to effective in inducing a physiological or behavioral change in a Leptinotarsa species (adult or larvae) such as, but not limited to, weakening of growth, increased mortality, reduction in reproductive capacity or reduced fertility, reduction in or cessation of movement or eating behavior, or reduction in or cessation of development in the metamorphosis stage; in some embodiments, applying an insecticide-effective amount of RNA to a plant improves the plant's resistance to an infestation with the Leptinotarsa species. RNA can be longer than the segment or segments it contains, but each segment and the corresponding fragment of a target gene are equivalent in length. The RNAs for use in the method can be designed for multiple target genes. Modalities include those where the Insecticide composition comprises an Insecticide effective amount of a polynucleotide comprising at least 21 contiguous nucleotides that are complementary to a target gene having a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO: 1087 - 1094, or an RNA transcribed from the target gene; or an insecticide-effective amount of at least one polynucleotide comprising at least one silencing element that is to complement it with at least 21 contiguous nucleotides of a target gene or a transcribed RNA of the target gene, where the target gene has a sequence of nuncleotlides that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO: 1087-1094; or an Insecticide effective amount of at least one RNA comprising at least one segment that is identical to or complements at least 21 contiguous nucleotides of a target gene having a nucleotide sequence that is selected from the group consisting of: SEQ ID NO : 730, SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO: 1087 - 1094, or an RNA transcribed from the target gene; or an RNA molecule that causes mortality or growth impairment in a Leptinotarse species when ingested or contacted with the Leptinotarse species, where the RNA molecule comprises at least 21 contiguous nucleotides that are complementary to a target gene that has a sequence of nuncleotides that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO: 1087 - 1094, or an RNA transcribed from the target gene; or an insecticidal double-stranded RNA molecule that causes mortality or growth failure in a Leptinotarsa species when ingested or contacted with the Leptinotarsa species, where at least one strand of the insecticidal double-stranded RNA molecule comprises 21 contiguous nucleotides that are complementary to a target gene or RNA
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transcribed from a target gene, where the target gene has a sequence that is selected from the group ήύδ “consists of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1-725, SEQ ID NO: 726-729 , SEQ ID NO: 731-806, SEQ ID NO: 808-830, and SEQ ID NO: 1087-1094, or an insecticide effective amount of at least one double-stranded RNA comprising a sequence that is selected from the Group of Trigger Sequences. In some embodiments, the polynucleotide is a double-stranded RNA. In some embodiments, the polynucleotide (eg, double-stranded RNA) is chemically synthesized or produced by expression in a microorganism or by expression in a plant cell. Modalities include insecticidal compositions comprising a dsRNA having a sequence selected from the group consisting of SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or where the insecticidal composition comprises a polynucleotide or RNA encoded by a sequence that is selected from the group consisting of SEQ ID NO: 1105-1109. Modalities include those where the insecticidal composition comprises a chain dsRNA having a sequence that is selected from the group consisting of the Trigger Sequence Group. In one embodiment, this invention provides an insecticidal composition for controlling a Leptinotarsa species that. it comprises an insecticide-effective amount of a double-stranded RNA molecule that causes mortality or growth failure in a Leptinotarsa species when ingested or contacted with the Leptinotarsa species, where the insecticidal double-stranded RNA molecule comprises at least one segment that is complementary to 21 contiguous nucleotides of a DNA that has a sequence that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO: 1087 - 1094, or an RNA transcribed from the DNA, and where the double stranded RNA molecule is at least 50 base pairs in length or is between about 100 and about 500 base pairs in length. In one embodiment, this invention provides an insecticidal composition for controlling a Leptinotarsa species comprising an insecticide-effective amount of a double-stranded RNA, where at least one strand of the double-stranded RNA is to complement it with at least 21 contiguous nucleotides of a gene that encodes a ribosomal protein or a transcribed RNA of the gene, where the Leptinotarsa species is Leptinotarsa decemiineata, and where RNA interference is induced and Leptinotarsa decemiineata mortality occurs, and where the ribosomal protein is an L7 ribosomal protein or a protein encoded by SEQ ID NO: 730 or where the double stranded RNA comprises a sequence that is selected from the group consisting of SEQ ID NO: 989, 988, 1104 or 1105. Related aspects of the invention include isolated RNAs for use in the composition and plants having improved resistance to Leptinotarse provided by treatment with the composition.
In various modalities, the insecticidal composition to control a species
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Leptinotarsa is in the form of at least one that is in solid, liquid form (including homogeneous mixtures such as solutions and inhomogeneous mixtures such as suspensions, colloids, micelles, and emulsions), powder, suspension, emulsion, spray, encapsulated or microencapsulated formulation, in or on top of microbeads or other carrier particles, in a film or coating, or in or on a matrix, or as a seed treatment. Suitable binders, inert carriers, surfactants and the like can optionally be included in the polynucleotide-containing composition, as one skilled in the formulation of insecticides and seed treatment knows. The Leptinotarsa species to be controlled is generally a species that infests a plant. In some embodiments, the insecticidal composition is at least one implantable formulation that is selected from the group consisting of a particle, pellet, or capsule implanted in the plant; in such modalities the method comprises implanting the implantable formulation in the plant. In some embodiments, the insecticidal composition is at least one groove formulation selected from the group consisting of a powder, granule, pellet, capsule, spray, or potion or any other suitable form to apply to a groove; In such embodiments, the method comprises a groove treatment with the groove formulation. In one embodiment, the insecticidal composition can be ingested or otherwise absorbed internally by the Leptinotarsa species. For example, the insecticidal composition may be in the form of a bait. In some embodiments, the insecticidal composition further comprises one or more components that are selected from the group consisting of a carrier agent, a surfactant, a cationic lipid (such as that described in Example 18 of the US patent application publication 2011/0296556, incorporated herein by this reference), an organosilicone, an organosilicone surfactant, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide pesticide, a protector and a regulator of insect growth. In one embodiment, the insecticidal composition further comprises a nonionic organosilicone surfactant such as SILWET® brand surfactants, eg, SILWET L-77® brand surfactant with CAS Number 27306-78-1 and EPA Number: CAL. REG. No. 5905-50073-AA, currently available from Momentive Performance Materials, Albany, New York. In some embodiments, the insecticidal composition further comprises at least one pesticidal agent that is selected from the group consisting of a patatin, a plant lectin, a phytoecysteroid, an insecticidal protein Bacillus thuringiensis, an insecticidal protein Xenorhabdus, an insecticidal protein Photorhabdus, a Insecticidal protein Badiius iaterosporous and an insecticidal protein Bacillus sphaericus. Alternatively, such additional components or pesticidal agents can be provided separately, for example, by separate topical application or by transgenic expression in the plant. Alternatively the plant is treated topically with the insecticidal composition as well as with a separate application (anterior, posterior or
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Simultaneous IMPI) of a substance that improves the efficacy of rnmpn ¡fiijh ¡hj<sub>l</sub> _1¡l¡i.1o. But a plant can be sprayed with a first topical application of a solution containing a non-ionic organosilicone surfactant such as SILWET® brand surfactants, for example SILWET L-77® brand surfactant, followed by a second topical application of the insecticidal composition, or vice versa.
It is anticipated that the combination of certain RNAs for use in this method (eg, the dsRNA triggers described in the Working Examples) with one or more non-polynucleotide pesticidal agents will result in a synergistic improvement in the prevention or control of infestations of the Leptinotarsa species, when compared with the effect obtained with RNA alone or the non-polynucleotide pesticide agent alone. In one embodiment, an insecticidal composition containing one or more RNA and one or more non-polynucleotide pesticidal agents was found to be selected from the group consisting of a patatin, a plant lectin, a phytoecysteroid, an insecticidal protein Bacillus thuringiensis, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus iaterosporous insecticidal protein and a Bacillus sphaericus insecticidal protein, it synergistically improves prevention or control of infestations by Leptinotarsa species.
The Leptinotarsa species to be controlled is generally a species that infests a plant. The plant can be any plant that is subject to infestation with a Leptinotarsa species. Modalities where the plant is a nightshade plant (family Solanaceae) are of particular interest. Examples include a plant that is selected from the group consisting of potato, tomato, and eggplant. Modalities include those where the plant is an ungerminated nightshade plant seed, a nightshade plant in the vegetative stage, or a nightshade plant in the reproductive stage. Modalities include those where the plant is a seed potato meaning a potato tuber or part of a potato tuber that can be propagated into new potato plants. In some modalities, the use of the insecticidal composition results in the control of the Leptinotarsa species, for example, in growth weakening, increased mortality, reduction in reproductive capacity, reduction in or cessation of movement or feeding behavior, or reduction in or cease of development in metamorphosis stage. In some embodiments, control of the Leptinotarsa species is seen as improved growth or improved yields of nightshade plants treated with the insecticidal composition, compared to plants not treated with the insecticidal composition. In some modalities, control of the Leptinotarsa species is seen as reduced amounts of eggs, larvae, or adults of the Leptinotarsa species, reduced defoliation or other damage to the plant, or increased yield of harvestable fruit (eg, tomatoes or eggplants) or tubers (for example, potatoes).
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In various modalities, the composition insecticide contains microbial or occurs in a microorganism. For example, the Insecticidal composition can Include or can be produced in bacteria or yeast cells. In similar embodiments, the Insecticidal composition comprises a transgenic plant cell or is produced in a plant cell (eg, a plant cell that transiently expresses the polynucleotide); Such plant cells may be cells in a plant or cells grown in tissue culture or in cell suspension.
The insecticidal composition can be provided for food absorption by the Leptinotarsa species by applying the composition to a plant or surface subject to Leptinotarsa species infestation, for example, by spraying, dusting or coating the plant or a plant seed or a seed potato. , or soaking the earth or applying a furrow treatment, or providing an artificial diet. The Insecticidal composition can be provided for food absorption by the Leptinotarsa species in an artificial diet formulated to meet the particular nutritional requirements for maintaining the Leptinotarsa species, where the artificial diet is supplemented with a certain amount of RNA obtained from a separate source as chemical synthesis. or purified from microbial fermentation; This modality may be useful, for example, in determining the timing and amounts of effective RNA treatment regimens. In some embodiments, the Insecticidal composition is provided for food absorption by the Leptinotarsa species in the form of a plant cell or in components of a plant cell, or in a microorganism (such as bacteria or yeast) or a microbial fermentation product. , or on a synthetic diet. In one embodiment, the insecticidal composition is provided in the form of a bait that is ingested by the species Leptinotarsa. The insecticidal composition can be provided for food absorption of the Leptinotarsa species in the form of a seed treatment (or seed potato).
In one embodiment, the Insecticidal composition is provided in the form of any plant that is subject to infestation with a Leptinotarsa species, where the RNA is contained in the plant. Such plants can be stably transgenic plants that express the RNA, or non-transgenic plants that transiently express the RNA or that were treated with the RNA, for example, by spraying or coating. Stably transgenic plants generally contain a recombinant construct encoding RNA integrated into their genome. The modalities where the plant is a nightshade plant (family Solanaceae) are of particular Interest. Examples Include a plant that is selected from the group consisting of potato, tomato, and eggplant. Modalities Include those where the plant is a seed of a non-germinated nightshade plant, a nightshade plant in the vegetative stage or a nightshade plant in the reproductive stage. Modalities Include those where the plant is a seed potato which means a potato tuber or part of a potato tuber that can be propagated in new
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potato plants.
The RNA useful in the insecticidal composition can have a single chain (ss) or a double chain (ds). Modalities include those where the RNA is at least one that is selected from the group consisting of single-stranded RNA (cRNA), single-stranded antisense (cRNA), or double-stranded (dsRNA); A mixture of RNA of any of these types can be used. In one embodiment, a double-stranded DNA / RNA hybrid is used. RNA can include components other than standard ribonucleotides, for example, one embodiment is an RNA comprising terminal deoxyribonucleotides.
The RNA in the insecticidal composition has at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with a fragment of equivalent length of a target gene or DNA that has a sequence that is select from the Target Gene Sequence Group or its DNA complement. In one embodiment, the RNA comprises at least one segment of 18 or more contiguous nucleotides that are essentially identical or complementary to a fragment of equivalent length of DNA that has a sequence that is selected from the group consisting of the Group of Gene Sequences Diana. In some embodiments, contiguous nucleotides have a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% Identity with a fragment of DNA that it has a sequence that is selected from the Gen Diana Sequence Group or its DNA complement. In some embodiments, the contiguous nucleotides are exactly (100%) identical to a fragment of equivalent length of a DNA that has a sequence that is selected from the Gene Target Sequence Group or its DNA complement. In some embodiments, the RNA has an overall sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a fragment of DNA that it has a sequence that is selected from the Gen Diana Sequence Group or its DNA complement.
The RNA in the insecticidal composition comprises at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with a fragment of equivalent length of a DNA having a sequence that is selected from the Group of Diana Gene Sequences or its DNA complement. In some embodiments, the RNA comprises at least one segment of 18 or more contiguous nucleotides, for example, between 18 24, or between 18 - 28, or between 20 - 30, or between 20 - 50, or between 20 - 100, or between 50 - 100, or between 50 - 500, or between 100 - 250, or between 100 - 500, or between 200 - 1000, or between 500 - 2000, or even more. In some embodiments, the segment comprises more than 18 contiguous nucleotides, for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, for example, about 35, around 40, around 45, around 50, around 55, around 60, around 65, around
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70, around 75, around 80, around 85, around 90, around 95, around 100, around 150, around 200, around 250, around 110, around 160, around 210 , around 260, around 120, around 170, around 220, around 270, around 130, around 180, around 230, around 280, around 140, around 190, around 240, around 290, around 300, around 350, about 400, about 450, about 500, or more than 500 contiguous nucleotides. In particular embodiments, the RNA comprises at least one segment of at least 21 contiguous nucleotides with a sequence of about 100% identity with a fragment of equivalent length of a target DNA or gene having a sequence that is selected from the Sequence Group Diana gene or DNA complement of this. In particular embodiments, the polynucleotide is a double-stranded nucleic acid (eg, dsRNA) with a strand comprising at least one segment of at least 21 contiguous nucleotides with a sequence of 100% identity with a fragment of equivalent length of a DNA or target gene having a sequence selected from the group consisting of the Target Gene Sequence Group or the DNA complement thereof; expressed as base pairs, such a double-stranded nucleic acid comprises at least one segment of at least 21 contiguous nucleotides, perfectly matched base pairs corresponding to a fragment of equivalent length of a target DNA or gene that has a sequence that is select from the Target Gene Sequence Group or its DNA complement. In particular embodiments, each segment contained in the RNA is longer than typical of naturally occurring small regulatory RNAs, for example, each segment is at least about 30 contiguous nucleotides (or base pairs) in length. In some embodiments the total length of the RNA, or the length of each segment contained in the RNA, is less than the total length of the sequence of interest (DNA or target gene that has a sequence that is selected from the group consisting of Group of Gen Diana Sequences). In some embodiments, the total length of the RNA is between about 50 and about 500 nucleotides (for single-stranded RNA) or base pairs (for double-stranded RNA). In some embodiments, the RNA comprises at least one strand of RNA between about 50 and about 500 nucleotides in length. Modalities include those where the RNA comprises at least one segment that has a sequence that is selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or where RNA is encoded by a sequence that is selected from the group consisting of
SEQ ID NO: 1105-1109.
The RNA expressed in the insecticidal composition is generally designed to suppress one or more genes (target genes). Such target genes can include coding or non-coding sequences, or both. In specific modalities, RNA is designed to suppress one or more
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industrial target genes, where each target gene has a DNA sequence that is selected ütíl 'yi upu qtte · consists of the Target Gene Sequence Group. In various modalities, RNA is designed to delete one or more genes, where each gene has a sequence that is selected from the group consisting of the Target Gene Sequence Group, and can be designed to delete multiple genes from this group, or to target different regions of one or more of these genes. In one embodiment, the RNA comprises multiple sections or segments each of which comprises at least one segment of 21 contiguous nucleotides with a sequence of 100% Identity with a fragment of equivalent length of DNA having a sequence that is selected from the Target Gene Sequence Group or its DNA complement. In such cases, each section may be Identical or different in size or sequence, and may be sense or antisense relative to the target gene. For example, in one embodiment the RNA may include multiple tandem sections or repeating arrays, where each section comprises at least one segment of 21 contiguous nucleotides with a sequence of 100% identity with a fragment of equivalent length of DNA that has a sequence that is selected from the Gene Target Sequence Group or the DNA complement thereof; the segments may be from different regions of the target gene, for example, the segments may correspond to different regions of the exon of the target gene, and the spacer nucleotides that do not correspond to a target gene may be used optionally between or adjacent to the segments.
The total length of the RNA in the insecticidal composition may be greater than 18 contiguous nucleotides, and may include nucleotides in addition to contiguous nucleotides that have the sequence of about 95% to about 100% identity with a fragment of equivalent length of one DNA that has a sequence that is selected from the Gen Diana Gene Sequence Group or its DNA complement. In other words, the total length of the RNA may be greater than the length of the section or segment of the RNA designed to suppress one or more target genes, where each target gene has a DNA sequence that is selected from the group consisting of the Group of Gen Diana Sequences. For example, RNA may have nucleotides that flank the active segment of at least one segment of 18 or more contiguous nucleotides that suppress the target gene, or include spacer nucleotides between the active segments, or they may have additional nucleotides at the 5 'end, either at the 3 'end, or at both the 5' and 3 'ends. In one embodiment, the RNA comprises additional nucleotides that are not specifically related (that have a non-complementary or Identical sequence) to the target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group or DNA complement thereof. for example, nucleotides that provide a stabilizing secondary structure or for convenience in cloning or manufacturing. In one embodiment, the RNA comprises additional nucleotides located immediately adjacent to one or more segments of 18 or more nucleotides.
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contiguous with a sequence of about 95% to about 100% LUII identity or complementary to a fragment of equivalent length of a target DNA or gene that has a sequence that is selected from the group consisting of the Target Gene Sequence Group . In one embodiment, the RNA comprises such a segment, with an additional 5'G or an additional 3'C or both, adjacent to the segment. In another embodiment, the RNA is a double-stranded RNA comprising additional nucleotides to form a surplus, eg, a dsRNA comprising 2 deoxyribonucleotides to form a 3 'surplus. Therefore in various embodiments, the nucleotide sequence of the entire RNA is not 100% identical or complementary to a contiguous nucleotide fragment in the target DNA or gene that has a sequence that is selected from the group consisting of the Sequence Group by Gen Diana. For example, in some embodiments the RNA comprises at least two segments each of 21 contiguous nucleotides with a sequence of 100% Identity with a fragment of a DNA having a sequence that is selected from the Gen Diana Sequence Group, or the DNA complement thereof, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in a different order than the corresponding fragments occur in DNA having a sequence that is selected from, or the DNA complement of, the Gen Diana Sequence Group.
In various embodiments, the RNA in the insecticidal composition consists of naturally occurring ribonucleotides. Modalities include, for example, synthetic RNAs consisting entirely of ribonucleotides or predominantly ribonucleotides but with one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides. In certain modalities, RNA comprises non-canonical nucleotides such as inosine, thiouridine, or pseudouridine. In certain embodiments, the RNA comprises chemically modified nucleotides. b) The RNA in the insecticidal composition is provided by suitable means known to one skilled in the art. Modalities include those where RNA is chemically synthesized (eg, by in vitro transcription, such as transcription using a T7 polymerase or other polymerase), produced by expression in a microorganism or in cell culture (such as plant cells or of insect grown in culture), produced by expression in a plant cell, or produced by microbial fermentation.
In some embodiments, the RNA is provided as an isolated RNA that is not part of an expression construct. In some embodiments, the RNA is provided as an isolated RNA that lacks additional elements such as a promoter or termination sequences. Such RNAs can be relatively short, such as single- or double-stranded RNAs of between about 18 and about 300 or between about 50 and about 500 nucleotides (for single-stranded RNA) or between about 18 and about 300 or between about 50 and
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MEXICANC INSTITUTE <sub>c</sub>, w. THE MflRIF.UAP □ J INDUSTRIAL about 500 base pairs (for double stranded RNA). From "rnanef-a áltén IdllVd, til RNA can be provided in more complex constructs, eg, as part of a recombinant expression construct, or included in a recombinant vector, eg, in a recombinant plant virus vector or in a recombinant baculovirus vector. In some embodiments, these recombinant expression vectors or constructs are designed to include additional elements, such as Include additional RNA encoding an aptamer or rlbozlma or an expression cassette to express a gene of interest (eg, an insecticidal protein).
Methods of providing plants having improved resistance to infestations of the Leptinotarsa species, and the plants and seeds thus provided
Another aspect of this invention relates to a method of providing a plant having improved resistance to an infestation with the Leptinotarsa species comprising expressing in the plant at least one polynucleotide comprising at least a segment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment of a target gene or DNA having a sequence that is selected from the group consisting of the Target Gene Sequence Group, whereby the resulting plant has improved resistance to a Leptinotarsa species when compared to a control plant where the polynucleotide is not expressed. In one embodiment, the method comprises expressing in the plant at least one polynucleotide comprising at least a segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with a fragment of equivalent length of one target gene or DNA that has a sequence that is selected from the Target Gene Sequence Group or its DNA complement. In one embodiment, the invention provides a method of providing a plant having improved resistance to an infestation with the Leptinotarsa species comprising expressing at least one polynucleotide in the plant comprising at least one segment that is identical or complementary to at least 21 nucleotides contiguous of a DNA having a sequence that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID N0: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO: 1087 - 1094. Expressing a polynucleotide in the plant generally means expressing an RNA transcript in the plant, for example, expressing in the plant an RNA comprising a sequence of rlbonucleotides that is antisense or essentially complementing it at least a fragment of a target gene or DNA that it has a sequence that is selected from the group consisting of the Gen Diana Sequence Group. Modalities include those where the plant expressed polynucleotide is an RNA comprising at least one segment having a sequence that is selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement of this, or where the polynucleotide expressed in the plant is an RNA hairpin
<img file="MX359191B_D0058.tif" />
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FROM INDUSTRIAL PROPERTY encoded by a sequence that is selected from the group consisting of SFQ TD NO: 11Q5-1109. Modalities include those where the plant expressed polynucleotide comprises a strand cDNA having a sequence that is selected from the group. consisting of the Trigger Sequence Group. However, the polynucleotide expressed in the plant may also be DNA (eg, DNA produced in the plant during genome replication), or the RNA encoded by such DNA. Related aspects of the invention include isolated polynucleotides for use in the method and plants having improved resistance to Leptinotarse provided by the method.
The method comprises expressing at least one polynucleotide in a plant, where the pollnucleotide comprises at least a segment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment of a target gene or DNA that has a sequence that is selected from the group. consisting of the Gen Diana Sequence Group. In some embodiments, a first polynucleotide is provided to a plant in the form of DNA (eg, in the form of an isolated DNA molecule, or as an expression construct, or as a transformation vector), and the expressed polynucleotide in the plant it is a second polynucleotide (eg, the RNA transcript of the first polynucleotide) in the plant. In one embodiment, the polynucleotide is expressed in the plant by transgenic expression, i.e., Stably integrating the polynucleotide into the plant genome from which it can be expressed in a plant cell or cells. In one embodiment, a first polynucleotide (eg, a recombinant DNA construct comprising a promoter operably linked to DNA comprising at least a segment of 18 or more contiguous nucleotides that is essentially Identical to or complementary to a fragment of a target gene or DNA having a sequence that is selected from the group that consists of the Gen Diana Gene Sequence Group) integrates stably into the plant genome from which secondary produced polynucleotides (eg, an RNA transcript that comprises the transcript of the segment of 18 or more contiguous nucleotides that is essentially Identical to or complementary to a fragment of a target gene or DNA that has a sequence that is selected from the group consisting of the Target Gene Sequence Group ) are expressed in a plant cell or cells. Methods for providing stably transformed plants are provided in the section entitled Making and Using Transgenic Plant Cells and Transgenic Plants.
In another embodiment, the expressed plant polynucleotide is expressed by transient expression (ie, expression that does not result from the stable integration of a sequence into the plant genome). In such embodiments the method may include a step for introducing a polynucleotide (eg, dsRNA or dsDNA) into the plant by routine techniques known in the art. For example, transient expression can be achieved by infiltration of a
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polynucleotide using a needleless syringe on a sheet of a planfe
In some embodiments where the polynucleotide expressed in the plant is expressed by transient expression, a first polynucleotide is provided to a plant in the form of RNA or DNA or both RNA and DNA, and a second polynucleotide produced secondary is transiently expressed in the plant . In some embodiments, the first polynucleotide is one or more that are selected from: (a) a single-stranded RNA molecule (cRNAs), (b) a single-stranded RNA molecule that self-hybridizes to form a double-stranded RNA molecule, (c) a double-stranded RNA molecule (dsRNA) , (d) a single-stranded DNA molecule (cDNA), (e) a single-stranded DNA molecule that self-hybridizes to form a double-stranded DNA molecule, (f) a single-stranded DNA molecule comprising a modified Pol III gene that is transcribed into an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule that comprises a modified Pol III gene that is transcribed to an RNA molecule, and (i) an RNA / DNA molecule hybrid double chain, or combinations thereof. In specific embodiments, a first polynucleotide is introduced into the plant by topically applying to the plant a composition containing polynucleotides in a suitable form, for example, as a solid, liquid (including homogeneous mixtures as solutions and non-homogeneous mixtures as suspensions, colloids, micelles and emulsions), powder, suspension, emulsion, sprayer, encapsulated or microencapsulated formulation, in or on top of microbeads or other carrier particles, in a film or coating, or in or on a matrix, or in the form of a solanaceous plant seed treatment or seed potato treatment. Binders, inert carriers, suitable surfactants and the like may optionally be included in the composition, as is known to one skilled in the formulation of pesticides and seed treatment. In such embodiments, the polynucleotide-containing composition may further include one or more components that are selected from the group consisting of a carrier agent, a surfactant, a cationic lipid (such as that described in Example 18 of the patent application publication 2011/0296556, incorporated by this reference herein), an organosilicone, an organosilicone surfactant, a polynucleotide herbicide molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a protector and a regulator of insect growth; in one embodiment the composition further comprises a nonionic organosilicone surfactant such as SILWET® brand surfactants, for example SILWET L-77® brand surfactant with CAS number 27306-78-1 and EPA number: CAL.REG . No. 5905-50073-AA, currently available from Momentlve Performance Materials, Albany, New York. In some embodiments, the topically applied composition further comprises at least one pesticidal agent that is selected from the group consisting of a patatin, a plant lectin, a phytoecysteroid, an insecticidal protein Bacillus thuríngiensis, an insecticidal protein Xenorhabdus,
<img file="MX359191B_D0060.tif" />
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an insecticidal protein Photorhabdus, an insecticidal protein üacil / us laterosporous and an insecticidal protein Bacillus sphaericus. Alternatively, such additional components or pesticidal agents can be provided separately, for example, by separate topical application or by transgene expression in the plant. Alternatively, the plant is treated topically with the composition containing pollnucleotides as well as with a separate application (previous, next or simultaneous) of a substance that improves the efficacy of the composition containing pollnucleotides. For example, a plant can be sprayed with a first topical application of a solution containing a nonionic organosilcone surfactant such as SILWET® brand surfactants, for example, SILWET L-77® brand surfactant, followed by a second topical application of the composition containing pollnucleotides, or vice versa.
It is anticipated that the combination of certain polynucleotides for use in this method (for example, the polynucleotide triggers described in the Working Examples) with one or more non-polynucleotide pesticide agents will result in a significant improvement in the prevention or control of infestations. of the Leptinotarsa species, when compared to the effect obtained with the pollenucleotide alone or the non-pollenucleotide pesticide agent alone. In one embodiment, a transgenic plant expressing at least one pollnucleotide comprising at least a segment of 18 or more contiguous nucleotides is found to be essentially Identical to or essentially complement a fragment of a target gene or DNA having a sequence that is select from the Gen Diana Sequence Group (for example, the polynucleotide triggers described in the Working Examples) and one or more genes encoding a non-polynucleotide pesticidal agent that is selected from the group consisting of a patatlna, a plant lectin, a fltoecdlsterolde, a Bacillus thuringiensis Insecticidal protein, a protein Xenorhabdus insecticide, a Photorhabdus insecticidal protein, a Bacillus laterosporous Insecticidal protein, and a Bacillus sphaericus insecticidal protein, exhibited an improved resistance to Leptinotarsa species infestations.
In some embodiments where the plant-expressed pollnucleotide is expressed by transient expression, a first pollnucleotide is provided to a plant in the form of RNA or DNA or both RNA and DNA, and a second secondarily produced pollnucleotide is transiently expressed in the plant ; the site of application of the first pollnucleotide need not necessarily be the same site where the second pollnucleotide is transiently expressed. For example, a first pollnucleotide can be provided to a plant by topical application to a leaf, or by injection to a stem, and the second pollnucleotide can be expressed transiently anywhere in the rest of the plant, for example, in the roots or in all plant. In some embodiments of the method, a composition comprising at least one polynucleotide is applied topically to the above-ground parts of the plant, for example, sprayed or dusted on
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leaves, stems and parts of the flower of the plant. In other mndaliriaHp ^ iin ^ rqmpo ^ irión gnp rnmprpqdp at least one polynucleotide is applied topically to the below ground parts of the plant, such as the roots, for example, soaking the soil. In other embodiments, a composition comprising at least one polynucleotide is topically applied to a seed (or, in the case of potatoes, topically applied to seed potato) that is grown on the plant that has improved resistance to a infestation with the Leptinotarsa species. In some embodiments, the polynucleotide expressed in the plant is RNA, which can be single-stranded (ss) or double-stranded (ds) RNA, or a combination of both.
In some embodiments, a first polynucleotide (DNA or RNA or both) is provided to a plant and a second polynucleotide that has a corresponding (identical or complementary) sequence to the first polynucleotide is subsequently expressed in the plant. In such modalities, the polynucleotide expressed in the plant is a transcript of RNA that can be cRNA or dsRNA or a combination of both. In some embodiments where the polynucleotide is expressed by transient expression, a first polynucleotide is provided to a plant in the form of RNA or DNA or both RNA and DNA, and a second polynucleotide produced secondary is transiently expressed in the plant; In such embodiments, the first polynucleotide is one or more selected from: (a) a single-stranded RNA molecule (cRNA), (b) a single-stranded RNA molecule that self-hybridizes to form a double-stranded RNA molecule, (c) a double-stranded RNA molecule (dsRNA) , (d) a single-stranded DNA molecule (cDNA), (e) a single-stranded DNA molecule that self-hybridizes to form a double-stranded DNA molecule, (f) a single-stranded DNA molecule comprising a modified Pol III gene that is transcribed into an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule that comprises a modified Pol III gene that is transcribed to an RNA molecule, and (i) an RNA / DNA molecule double-chain hybrid, or combinations thereof. In such modalities where the polynucleotide is expressed by transient expression the first polynucleotide may consist of naturally occurring nucleotides, such as nucleotides occurring in DNA and RNA. In such modalities where the polynucleotide is expressed by transient expression the first polynucleotide may be chemically modified, or comprise chemically modified nucleotides. The first polynucleotide is provided by suitable means known to one skilled in the art. Modalities Include those where the first polynucleotide is chemically synthesized (eg, by in vitro transcription, such as transcription using a T7 polymerase or other polymerase), is produced by expression in a microorganism or in cell culture (such as plant cells or from cultivated insects in culture), is produced by expression in a plant cell or is produced by microbial fermentation. The first polynucleotide can be provided as a fragment of RNA or DNA. Alternatively the first polynucleotide
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may be provided in more complex constructs, eg, COI 110 paito di_unuconstrucclón of recombinant expression, or included in a recombinant vector, for example in a vector of recombinant plant virus or a vector of recombinant baculovlrus; Such recombinant expression constructs or vectors can be designed to include additional elements, such as expression cassettes to express a gene of Interest (eg, an Insecticidal protein).
In some embodiments, the expressed polynucleotide in the plant is an RNA molecule and can be relatively short, such as single or double stranded RNA of between about 18 and about 300 or between about 50 and about 500 nucleotides (for RNA single stranded) or between about 18 and about 300 or between about 50 and about 500 base pairs (for double stranded RNA). Alternatively, the pollnucleotide may be provided in more complex constructs, eg, as part of a recombinant expression construct, or Include in a recombinant vector, eg, in a recombinant plant virus vector or baculovlrus vector recombinant. In some embodiments, these recombinant expression vectors or constructs are designed to Include additional elements, such as expression cassettes to express a gene of Interest (eg, an Insecticidal protein).
The expressed polynucleotide in the plant has at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with a fragment of equivalent length of DNA that has a sequence that is selected from the Group of Diana Gene Sequences or its DNA complement. In one embodiment the polynucleotide expressed in the plant comprises at least a segment of 18 or more contiguous nucleotides that are essentially Identical to or complement a fragment of equivalent length of DNA having a sequence that is selected from the group consisting of the Group of Gen Diana sequences. In some embodiments, contiguous nucleotides have a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% Identity with a DNA fragment that has a sequence that is selected from the Gen Diana Gene Sequence Group or its DNA complement. In some embodiments, the contiguous nucleotides are exactly (100%) Identical to a fragment of equivalent length of a DNA that has a sequence that is selected from the Gene Target Sequence Group or its DNA complement. In some embodiments, the plant expressed polynucleotide has an overall sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% Identity with a fragment DNA that has a sequence that is selected from the Gen Diana Gene Sequence Group or its DNA complement.
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The polynucleotide expressed in the plant is generally designed Pcrid tupi lililí unu or more genes (target genes). Such target genes can include coding or non-coding sequences, or both. In specific embodiments, the plant expressed polynucleotide is designed to suppress one or more target genes, where each target gene has a DNA sequence that is selected from the group consisting of the Target Gene Sequence Group. In various embodiments, the plant-expressed polynucleotide is designed to suppress one or more genes, where each gene has a sequence that is selected from the group consisting of the Gene Target Sequence Group, and can be designed to suppress multiple genes from it. group, or to target different regions of one or more of these genes. In one embodiment, the plant expressed polynucleotide comprises multiple sections or segments each comprising at least one segment of 21 contiguous nucleotides with a sequence of 100% Identity with a fragment of equivalent length of DNA having a sequence which is selected from the Gene Target Sequence Group or its DNA complement. In such cases, each section may be identical or different in size or sequence, and may be sense or antisense relative to the target gene. For example, in one embodiment the plant expressed polynucleotide may include multiple tandem sections or repeating arrangements, where each section comprises at least one segment of 21 contiguous nucleotides with a sequence of 100% identity with a fragment of equivalent length of one DNA having a sequence that is selected from the Gen Diana Gene Sequence Group or its DNA complement; the segments may be from different regions of the target gene, for example, the segments may correspond to different regions of the exon of the target gene, and the spacer nucleotides that do not correspond to a target gene may optionally be used between or adjacent to the segments.
The total length of the plant expressed polynucleotide may be greater than 18 contiguous nucleotides, and may include nucleotides in addition to contiguous nucleotides that have the sequence of about 95% to about 100% identity with a fragment of equivalent length of one DNA that has a sequence that is selected from the Gen Diana Gene Sequence Group or its DNA complement. In other words, the total length of the polynucleotide expressed in the plant can be greater than the length of the section or segment of the polynucleotide designed to delete one or more target genes, where each target gene has a DNA sequence that is selected from the group that It consists of the Gen Diana Sequence Group. For example, the plant expressed polynucleotide may have nucleotides flanking the active segment of at least one segment of 18 or more contiguous nucleotides that suppress the target gene, or include spacer nucleotides between the active segments, or they may have additional nucleotides in the 5 'end, or at the 3' end, or both 5 'and 3' ends. In one embodiment, the plant expressed polynucleotide comprises additional nucleotides that are not specifically related (is
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that is, they have a sequence that is not complementary or identical) to the target DNA or gene which Lltílltí UIRT sequence that is selected from the Target Gene Sequence Group or the DNA complement of this, for example, nucleotides that provide a stabilizing secondary structure or by convenience in cloning or manufacturing. In one embodiment, the plant expressed polynucleotide comprises additional nucleotides located immediately adjacent to one or more segments of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% Identity with or complementary to a fragment in length equivalent of a target DNA or gene that has a sequence that is selected from the group consisting of the Target Gene Sequence Group. In one embodiment, the plant expressed polynucleotide comprises such a segment, with an additional 5'G or an additional 3'C or both, adjacent to the segment. In another embodiment, the plant expressed polynucleotide is a double stranded RNA that comprises additional nucleotides to form a surplus, eg, a dsRNA comprising 2 deoxyrrbonbonucleotides to form a 3 'surplus. Therefore in various modalities, the nucleotide sequence of the entire polynucleotide expressed in the plant is not 100% identical or complementary to a contiguous nucleotide fragment in the target DNA or gene that has a sequence that is selected from the group consisting of the Gen Diana Sequence Group. For example, in some embodiments the plant expressed polynucleotide comprises at least two segments each of 21 contiguous nucleotides with a sequence of 100% Identity with a fragment of a DNA having a sequence that is selected from the Gene Sequence Group Diana, or its DNA complement, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in a different order than the order in which the corresponding fragments occur in the DNA that has a sequence that is selected from the Gen Diana Sequence Group, or its DNA complement.
In a related aspect, this Invention relates to the plant having improved resistance to an Infestation with the Leptinotarsa species, provided by expressing in the plant at least one polynucleotide comprising at least a segment of 18 or more contiguous nucleotides that are essentially Identical or complementary to a fragment of equivalent length of DNA having a sequence that is selected from the group consisting of the Gen Diana Gene Sequence Group, whereby the resulting plant has improved resistance to an Infestation with the Leptinotarsa species when compared to a control plant where the polynucleotide is not expressed. In a related aspect, this invention refers to the plant that has improved resistance to an infestation with the Leptinotarsa species, provided by expressing in the plant at least one polynucleotide that comprises at least a segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% Identity with a fragment in length
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DNA equivalent that has a sequence that is selected from the group ¿fé SeéUSUClys dtí Gen Diana or its DNA complement, whereby the resulting plant has improved resistance to an infestation with the Leptinotarsa species when compared to a control plant where the polynucleotide is not expressed. One modality is a nightshade plant that has improved resistance to an infestation with the Leptinotarsa species when compared to a control plant, provided by expressing in the plant an RNA that has a sequence that is selected from the group consisting of: SEQ ID NO: 831 1085, 1095-1104, and 1110-1114, or complement thereof, or expressing in the plant an RNA hairpin encoded by a sequence selected from the group consisting of SEQ ID NO: 1105-1109. In yet another aspect, this invention relates to seeds (especially transgenic progeny seed) produced by the plant that have improved resistance to an infestation with the Leptinotarsa species, as provided by this method. Also contemplated is a basic product produced by the plant that has improved resistance to an infestation with the Leptinotarsa species, as provided by this method, and a basic product produced from the transgenic progeny seed of such a plant.
Recombinant DNA constructs to control a Leptinotarsa species
Another aspect of this invention provides a recombinant DNA construct comprising a heterologous promoter operably linked to a DNA element comprising at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity. with a DNA fragment having a sequence that is selected from the Gen Diana Sequence Group or its DNA complement. In some embodiments, the recombinant DNA construct comprises a heterologous promoter operably linked to: (a) DNA comprising a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a target gene that has a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 - 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO: 1087-1094, or a transcribed RNA of the target gene; or (b) a DNA comprising 21 or more contiguous nucleotides having 100% identity to a fragment of equivalent length of a DNA having a sequence selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO : 807, SEQ ID NO: 1-725, SEQ ID NO: 726-729, SEQ ID NO: 731-806, SEQ ID NO: 808-830, and SEQ ID NO: 1087-1094, or the DNA complement of this; or (c) DNA encoding at least one silencing element that is complementary to at least 21 contiguous nucleotides of a target gene or an RNA transcribed from the target gene, where the target gene has a sequence that is selected from the group consisting of: SEQ ID NO: 730, SEQ ID NO: 807, SEQ ID NO: 1 - 725, SEQ ID NO: 726 729, SEQ ID NO: 731 - 806, SEQ ID NO: 808 - 830, and SEQ ID NO: 1087 - 1094; or (d) DNA that
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encodes at least one silencing element comprising at least 21 nuUdÚUduJi Lüiilkjuub that are complementary to a target gene that is selected from the genes in the Target Gene Sequence Group or an RNA transcribed from the target gene; or (e) DNA encoding an RNA comprising at least 21 contiguous nudeotides that are complementary to a nudeotide sequence that is selected from, or complement to, the Trigger Sequence Group, or an orthologous nudeotide sequence of a Leptinotarse species or a Tribolium species, where the orthologous nudeotide sequence has at least 95% sequence identity with a nudeotide sequence that is selected from the Trigger Sequence Group, where the sequence identity percentage is calculated on the same length; or (f) DNA encoding an RNA comprising at least one double-stranded RNA region, at least one strand of which comprises at least 21 contiguous nudeotides that are complementary to a nudeotide sequence that you select from the Trigger Sequence Group , or the complement thereof, or an orthologous nudeotide sequence of a Leptinotarsa species or a Tribolium species, wherein the orthologous nudeotide sequence has at least 95% sequence identity with a nudeotide sequence that is selected from the Trigger Sequence Group, where the percent sequence identity is calculated over the same length; or (g) RNA encoding DNA comprising a nudeotide sequence that is selected from, or complement to, the Trigger Sequence Group. Modalities include a recombinant DNA construct comprising a heterologous promoter operably linked to a DNA element encoding an RNA having a sequence that is selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or comprising a heterologous promoter operably linked to a DNA element encoding an RNA hairpin encoded by a sequence selected from the group consisting of SEQ ID NO: 1105-1109. Modalities include a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding a dsRNA with a strand having a sequence selected from the group consisting of the Trigger Sequence Group. Recombinant DNA constructs are useful in providing a plant that has improved resistance to an infestation with the Leptinotarsa species, for example, by expressing in a plant a transcript of such a recombinant DNA construct. Recombinant DNA constructs are also useful for making polynucleotides useful for making compositions that can be applied to a plant, seed, plant propagating part, soil or soil, or surface that needs to be protected from an infestation with the Leptinotarsa species. Related aspects of the invention include: compositions comprising the construction of recombinant DNA; a plant chromosome or a plastid or a recombinant plant virus vector or a recombinant baculovirus vector comprising the construction of recombinant DNA; a cell of the
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transgenic solanaceous plant that has in its genome the construction of recoi nbiiWlILtí DNA, “optionally comprising in its genome DNA that encodes at least one pesticidal agent that is selected from the group consisting of a patatin, a plant lectin, a phytoecysteroid, a insecticidal protein Bacillus thuringiensis, an insecticidal protein Xenorhabdus, an insecticidal protein Photorhabdus, an insecticidal protein Bacillus Iaterosporous, and an insecticidal protein Bacillus sphaericus, and a transgenic solanaceous plant including such a cell of a transgenic solanaceous plant, or a fruit, seed or spreadable part of the transgenic solanaceous plant; and plants having improved resistance to Leptinotarse provided by the expression of or treatment with the recombinant DNA construct or the RNA encoded therein.
The recombinant DNA construct comprises a heterologous promoter operably linked to a DNA comprising at least a segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with a fragment of equivalent length of DNA having a sequence that is selected from the Gen Diana Gene Sequence Group or its DNA complement. In some embodiments, the segment of 18 or more contiguous nucleotides has a sequence with about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity with a A fragment of DNA that has a sequence that is selected from the Gen Diana Gene Sequence Group or its DNA complement. In some embodiments, the contiguous nucleotides are exactly (100%) identical to a fragment of equivalent length of DNA that has a sequence that is selected from the Gene Target Sequence Group or its DNA complement. In some embodiments, DNA has an overall sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a DNA that has a sequence which is selected from the Gene Target Sequence Group or its DNA complement.
The recombinant DNA construct therefore comprises a heterologous promoter operably linked to DNA comprising at least a segment of 18 or more contiguous nucleotides designed to suppress the expression of a target gene having a sequence that is selected from the Gene Sequence Group Diana or her DNA complement. In some embodiments, the DNA comprises at least one segment of 18 or more contiguous nucleotides, for example, between 18-24, or between 18-28, or between 20-30, or between 20-50, or between 20-100, or between 50 - 100, or between 50 - 500, or between 100 - 250, or between 100 - 500, or between 200 - 1000, or between 500 - 2000, or even more. In some embodiments, the segment comprises more than 18 contiguous nucleotides, for example, 19, 20, 21, 22, 23, 24, 25, 26, TJ, 28, 29, 30, or more than 30, for example, about 35, around 40, around 45, around 50, around 55, around 60, around 65, around 70, around 75, around 80, around 85, around
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90, around 95, around 100, around 110, nn around 140, around 150, around 160, around 170, around 180, around 190, around 200, around 210, around 220, around 230, around 240, around 250, around 260, around 270, around 280, around 290, around 300, around 350, around 400, around 450, around 500, or more than 500 contiguous nucleotides. In particular embodiments, DNA encodes an RNA that contains at least one segment of at least 21 contiguous nucleotides with a sequence of 100% identity with a fragment of equivalent length of a target DNA or gene that has a sequence that is selected from the Group. of Diana Gene Sequences or its DNA complement. In particular embodiments, DNA encodes a double-stranded nucleic acid (eg, dsRNA) with a strand comprising at least one segment of at least 21 contiguous nucleotides with a sequence of 100% identity with a fragment of equivalent length of a DNA or target gene having a sequence selected from the group consisting of the Target Gene Sequence Group or the DNA complement thereof; expressed as base pairs, such a double-stranded nucleic acid comprises at least one segment of at least 21 contiguous nucleotides, perfectly matched base pairs corresponding to a fragment of equivalent length of a target DNA or gene that has a sequence that is select from the Target Gene Sequence Group or its DNA complement. In particular modalities, each segment contained in the DNA is longer than typical of small naturally occurring regulatory RNAs. In some embodiments, each segment is at least about 30 contiguous nucleotides (or base pairs) in length. In some embodiments, the total length of the DNA, or the length of each segment contained in the polynucleotide, is less than the total length of the sequence of interest (DNA or target gene that has a sequence that is selected from the group consisting of the Gen Diana Sequence Group). In some embodiments, the total length of the DNA is between about 50 and about 500. In some embodiments, the DNA encodes an RNA having a sequence that is selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or complement thereof. In some embodiments, the recombinant DNA construct comprises a sequence that is selected from the group consisting of SEQ ID NO: 1105-1109.
The recombinant DNA construct comprises a heterologous promoter operably linked to DNA generally designed to suppress one or more genes ("target genes"). Such target genes can include coding or non-coding sequences, or both. In specific embodiments, the recombinant DNA construct is designed to delete one or more target genes, where each target gene has a DNA sequence that is selected from the group consisting of the Target Gene Sequence Group. In various modalities, the recombinant DNA construct is
<img file="MX359191B_D0069.tif" />
It is designed to delete one or more genes, where each gene has a YJ lié and tí5UlLcciui iw sequence from the group that consists of the Gen Diana Gene Sequence Group, and can be designed to delete multiple genes from this group, or to target different regions of one or more of these genes. In one embodiment, the recombinant DNA construct comprises a heterologous promoter operably linked to multiple sections or segments each comprising at least one contiguous 21 nucleotide segment with a sequence of 100% identity with a fragment of equivalent length of a DNA having a sequence that is selected from the Gen Diana Gene Sequence Group or its DNA complement. In such cases, each section may be identical or different in size or sequence, and may be sense or antisense relative to the target gene. For example, in one embodiment the recombinant DNA construct may include a heterologous promoter operably linked to multiple tandem sections or repeating arrays, where each section comprises at least one segment of 21 contiguous nucleotides with a sequence of 100% identity to a fragment of equivalent length of a DNA having a sequence that is selected from the Gen Diana Sequence Group or the DNA complement thereof; the segments may be from different regions of the target gene, eg, the segments may correspond to different regions of the exon of the target gene, and the spacer nucleotides that do not correspond to a target gene may be used optionally between or adjacent to the segments.
The recombinant DNA construct comprises a heterologous promoter operably linked to DNA that can have a total length that is greater than 18 contiguous nucleotides, and may include nucleotides in addition to the segment of at least one segment of 18 or more contiguous nucleotides that has the sequence of about 95% to about 100% Identity with a fragment of equivalent length of a DNA that has a sequence that is selected from the Diana Gene Sequence Group or its DNA complement. In other words, the total length of the DNA may be greater than the length of the DNA segment designed to delete one or more target genes, where each target gene has a DNA sequence that is selected from the group consisting of the Group of Sequences of Gen Diana. For example, DNA may have nucleotides flanking the active segment of at least one segment of 18 or more contiguous nucleotides that suppress the target gene, or include spacer nucleotides between the active segments, or may have additional nucleotides at the 5 'end, either at the 3 'end, or at both the 5' and 3 'ends. In one embodiment, the heterologous promoter is operably linked to DNA that comprises additional nucleotides that are not specifically related (that have a non-complementary or identical sequence) to the target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group or the DNA complement of this, eg nucleotides that provide a stabilizing secondary structure or for convenience in cloning or
<img file="MX359191B_D0070.tif" />
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INSTITUTO MEXICANO OE EA RROHFDAO 66 industrial manufacturing. In one embodiment, the heterologous promoter is operably linked to AUN ¢) 115 comprising additional nucleotides located immediately adjacent to one or more segments of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with or complementarity to a fragment of equivalent length of a target DNA or gene that has a sequence that is selected from the group consisting of the Target Gene Sequence Group. In one embodiment, the heterologous promoter is operably linked to DNA comprising such a segment, with an additional 5'G or an additional 3'C or both, adjacent to the segment. In another embodiment, the heterologous promoter is operably linked to DNA encoding a double-stranded RNA that comprises additional nucleotides to form a surplus. Therefore in various embodiments, the nucleotide sequence of all DNA operably linked to the heterologous promoter is not 100% identical or complementary to a contiguous nucleotide fragment in the target DNA or gene that has a sequence that is selected from the group consisting of in the Gen Diana Sequence Group. For example, in some embodiments the heterologous promoter is operably linked to DNA comprising at least two segments each of 21 contiguous nucleotides with a sequence of 100% identity to a fragment of DNA having a sequence that is selected from the Group of Diana gene sequences, or the DNA complement thereof, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in a different order than the corresponding fragments occur in DNA having a sequence that is selected from, or the DNA complement of, the Gen Diana Sequence Group.
In recombinant DNA constructs, the heterologous promoter is operably linked to DNA encoding a transcript that can be single-stranded (ss) or double-stranded (ds) or a combination of both. Modalities of the method include those where the DNA encodes a transcript comprising sense single-stranded RNA (cRNA), antisense cRNA, or double-stranded RNA (dsRNA), or a combination thereof.
The recombinant DNA construct is provided by suitable means known to one of skill in the art. Modalities include those where the recombinant DNA construct is synthesized in vitro, produced by expression in a microorganism, or in cell culture (such as plant or insect cells grown in culture), produced by expression in a cell of plant or is produced by microbial fermentation.
The heterologous promoter for use in recombinant DNA constructs is selected from the group consisting of a functional promoter in the plant, a functional promoter in a prokaryote, a functional promoter in a fungal cell, and a baculovirus promoter. Non-exhaustive examples of promoters are described in the section entitled Promoters.
In some embodiments, the recombinant DNA construct comprises a
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second promoter also operably linked to DNA. For ΓφΙυ, lI ACM qut. compnnri-ií<sup>to |</sup>_ minus a segment of 18 or more contiguous nucleotides may be flanked by two promoters arranged so that the promoters are transcribed in opposite directions and convergently, providing opposite-strand transcripts of DNA that are complementary to and able to hybridize with each other to form RNA double chain. In one embodiment, the DNA is located between two root-specific promoters, which allow DNA transcription in opposite directions, resulting in the formation of dsRNA.
In some embodiments, the recombinant DNA construct comprises other DNA elements in addition to the heterologous promoter operably linked to a DNA comprising at least a segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% Identity with a fragment of equivalent length of DNA that has a sequence that is selected from the Gen Diana Gene Sequence Group or its DNA complement. Such DNA elements are known in the art, and include, but are not limited to, standards, recombinase recognition sites, aptamers, or rhoboles, additional expression cassettes to express coding sequences (eg, to express a transgene such as an insecticidal protein or selectable marker) or non-coding sequences (eg, to express additional deletion elements). The inclusion of one or more recognition sites for binding and excision by a small RNA (for example, by a mIRNA or a pRNA that is expressed only in a particular cell or tissue) allows for more accurate expression patterns in a plant, where the expression of the recombinant DNA construct is suppressed when the small RNA is expressed.
In some embodiments, the recombinant DNA construct is provided in a recombinant vector. Recombinant vector means a recombinant polynucleotide molecule that is used to transfer genetic information from one cell to another. Suitable modalities for this invention include, but are not limited to, recombinant plasmids, recombinant plasmids, artificial chromosomes, and recombinant viral vectors such as recombinant plant virus vectors and recombinant baculovlrus vectors. Alternative modalities Include recombinant plasmids, recombinant cosmids, artificial chromosomes, and recombinant viral vectors such as recombinant plant virus vectors and recombinant baculovlrus vectors comprising the DNA element without the heterologous promoter.
In some embodiments, the recombinant DNA construct is provided on a chromosome or plastid of a plant, for example, on a transgenic plant cell or a transgenic plant. Therefore, this invention also encompasses a transgenic plant cell having the recombinant DNA construct in its genome, as well as a transgenic plant or partially transgenic plant including such a transgenic plant cell. The plants
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partially transgenic include, for example, a non-<-ra'n? 5 stem<sup>fi</sup>ri¡''n-tnjn4ftflo in a r¡7<sup>n</sup>mq. transgenic including the transgenic plant cell. The modalities include a transgenic tomato rhizome that includes the transgenic plant cell. The plant can be any plant that is subject to Infestation with a Leptinotarsa species. Modalities where the plant is a nightshade plant (family Solanaceae) are of particular interest. Examples Include a plant that is selected from the group consisting of potato, tomato, and eggplant. Modalities include those where the plant is a seed of a non-germinated nightshade plant, a nightshade plant in the vegetative stage, or a nightshade plant in the reproductive stage. Modalities include those where the plant is a seed potato, meaning a potato tuber or part of a potato tuber that can propagate into new potato plants. In yet another aspect, this invention relates to seeds (especially transgenic progeny seed) produced by the transgenic plant having in its genome a recombinant DNA construct as described herein. The modalities also comprise a transgenic seed potato having a recombinant DNA construct in its genome as described herein. Also contemplated is a basic product produced by such a transgenic plant, and a basic product produced from the seed of transgenic progeny of such a transgenic plant.
The recombinant DNA construct can be provided in a composition for topical application to a surface of a plant or plant seed, or for topical application to any substrate that needs protection from an infestation with the Leptinotarsa species. Similarly, the recombinant DNA construct can be provided in a composition for topical application to a Leptinotarse species, or in a composition for ingestion by a Leptinotarse species. In various embodiments, such compositions containing the recombinant DNA construct are provided in the form of at least one that is selected from the group consisting of a solid, liquid (including homogeneous mixtures as solutions and non-homogeneous mixtures as suspensions, colloids, micelles, and emulsions), powder, suspension, emulsion, sprayer, encapsulated or microencapsulated formulation, in or on top of microbeads or other carrier particles, in a film or coating, or in or on a matrix, or as a seed treatment. Topical application may be in the form of a topical treatment of nightshade plant fruits or fruit seeds of nightshade plants or in the form of topical treatment of seed potato tubers or parts of a tuber (for example, soaking, coating or sprinkling the seed potato). Binders, inert carriers, suitable surfactants, and the like may be included in the composition containing the recombinant DNA construct, as is known to one skilled in the formulation of pesticides and seed treatment. In some embodiments, the composition for topical application containing the recombinant DNA construct is at least one topically implantable formulation that is selected from the group that
<img file="MX359191B_D0073.tif" />
ΙΜΡΪ consists of a particle, sediment, or capsule topically implanted<sup>l</sup>düS><sup>,</sup>5n ”IS '* planta; in such modalities the method comprises implanting the topically implantable formulation topically in the plant. In some embodiments, the composition for topical application containing the recombinant DNA construct is at least one groove formulation selected from the group consisting of a powder, granule, pellet, capsule, spray, or potion, or any other forms suitable for topical application to a groove; in such embodiments, the method includes a groove treatment with the groove formulation. In one embodiment the composition for topical application containing the recombinant DNA construct can be ingested or otherwise internally absorbed by the Leptinotarse species. For example, the composition for topical application containing the recombinant DNA construct may be in the form of a bait. In some embodiments, the composition containing the recombinant DNA construct further comprises one or more components that are selected from the group consisting of a carrier agent, a surfactant, a catholic lipid (such as that described in Example 18 of US Patent Application Publication 2011/0296556, incorporated herein by this reference), an organosylcone, an organosllicone surfactant, a polynucleotide herbicidal molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide pesticide, a protector and a regulator of Insect growth. In one embodiment the composition containing the recombinant DNA construct further comprises a nonionic organosilicone surfactant such as SILWET® brand surfactants, for example SILWET L-77® brand surfactant CAS number 27306-78-1 and EPA number: REG.CAL. No. 5905-50073-AA, currently available from Momentlve Performance Materials, Albany, New York. In some embodiments, the composition containing the recombinant DNA construct further comprises at least one pesticidal agent that is selected from the group consisting of a potato, a plant lectin, a fltoecdlsterolde, a fltoecdlsterolde, a Bacillus thuringiensis Insecticidal protein, a protein Xenorhabdus insecticide, a Photorhabdus insecticidal protein, a Bacillus iaterosporous Insecticidal protein, and a Bacillus sphaericus Insecticidal protein.
It is anticipated that the combination of certain recombinant DNA constructs as described herein (eg, recombinant DNA constructs including the polynucleotide triggers described in the Working Examples), either transgenically expressed or topically applied, with one or more non-polynucleotide pesticidal agents, either transgenically expressed or topically applied, will result in a surgical improvement in the prevention or control of Leptinotarsa species infestations, when compared to the effect obtained with the recombinant DNA constructs alone or the non-polynucleotide pesticide agent alone. In one embodiment, a recombinant DNA construct was found to express one or more pollnucleotides as well as one or more genes that
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they encode a non-polynucleotide pesticide agent that is selected from the group consisting of a potato, a plant lectin, a phytoecdlsteroid, a Bacillus thuringiensis Insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, and a Bacillus laterosporous protein Bacillus sphaericus insecticide, provides a highly improved resistance to Leptinotarsa species infestations in plants expressing the recombinant DNA construct. One embodiment refers to a recombinant DNA construct for expressing an RNA comprising a segment having a sequence that is selected from the group consisting of SEQ ID NO: 831-1085 and 1095 as well as one or more genes encoding an agent non-polynucleotide pesticide that is selected from the group consisting of a potato, a plant lectin, a fltoecdlsterolde, a Bacillus thuringiensis Insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous Insecticidal protein, and a Bacillus sphaericus Insecticidal protein.
The composition containing the recombinant DNA construct can be provided for food absorption by a Leptinotarsa species by applying the composition to a plant or surface subject to infestation with the Leptinotarsa species, for example by spraying, dusting or coating the plant, or soaking the soil, or providing an artificial diet. The composition containing the recombinant DNA construct can be provided for food absorption by a Leptinotarse species in an artificial diet formulated to meet the particular nutritional requirements for maintaining the Leptinotarse species, where the artificial diet is supplemented with an amount of the DNA construct. recombinant obtained from a separate source such as in vitro synthesis or purified from a microbial fermentation or other biological source; This modality may be useful, for example, in determining the times and amounts of effective treatment regimens. In some embodiments, the composition containing the recombinant DNA construct is provided for food absorption by the Leptinotarsa species in the form of a plant cell or in plant cell components, or in a microorganism (such as a bacterium or yeast) or a microbial fermentation product, or in a synthetic diet. In one embodiment, the composition containing the recombinant DNA construct is provided in the form of a bait that is ingested by the species Leptinotarsa. The composition containing the recombinant DNA construct can be provided for food absorption by the Leptinotarsa species in the form of a seed treatment.
In various embodiments, the composition containing the recombinant DNA construct comprises a microbial cell or is produced in a microorganism. For example, the composition containing the recombinant DNA construct may include or may be produced in yeast or bacterial cells. In similar embodiments, the composition containing the recombinant DNA construct comprises a transgenic plant cell or is produced in a
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plant (for example a plant cell that expresses transItonaTOIILé Id CüHíl »ULLIÜII Lio recombinant DNA); Such plant cells may be cells in a plant or cells grown in tissue culture or in cell suspension.
Transqenlcal nightshade plant cells
Various modalities relate to transgenic solanaceous plant cells expressing a polynucleotide useful in the methods described herein to suppress the expression of a target gene in a Leptinotarse species or to control an infestation with Leptinotarse. In one aspect this invention provides a transgenic soianaceae plant cell having in its genome an RNA encoding recombinant DNA comprising at least a segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% Identity with a fragment of a DNA that has a sequence that is selected from the Gen Diana Gene Sequence Group or its DNA complement. In one aspect this invention provides a transgenic soianaceae plant cell having in its genome an RNA encoding recombinant DNA comprising at least one gene element essentially identical or essentially complementary to a fragment of a target gene sequence of the species larvae Leptinotarse, where the target gene sequence is selected from the Target Gene Sequence Group, or its DNA complement. In one aspect this invention provides a transgenic soianaceae plant cell having in its genome an RNA encoding recombinant DNA that suppresses the expression of a target gene in a Leptinotarsa species that contacts or ingests the RNA, where the RNA comprises at least a silencing element having at least one segment of 18 or more contiguous nucleotides complementary to a fragment of the target gene, and where the target gene is selected from the group consisting of genes from the Target Gene Sequence Group. A specific modality is a transgenic soian plant cell that has in its genome an RNA that encodes recombinant DNA that suppresses the expression of a target gene in a Leptinotarsa species that contacts or ingests the RNA, where the RNA comprises at least one element. knockout having at least one segment of 18 or more contiguous nucleotides complementary to a fragment of one or more exocyst target genes; suitable exocyst target genes include the Leptinotarsa exocyst genes provided in Table 4 or identified homologous sequences from other insect species. In one aspect this invention provides a transgenic soianaceae plant cell having in its genome a recombinant DNA encoding an RNA having a sequence selected from the group consisting of: SEQ ID NO: 831 1085, 1095-1104, and 1110 1114, or the complement thereof, or a recombinant DNA that is selected from the group consisting of SEQ ID NO: 1105-1109; the modalities include a transgenic soianaceous plant cell having in its genome a recombinant DNA encoding a
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DsRNA with a string that has a sequence that is selected from the group that is cFUüülti arel · Trigger sequence group. Such transgenic solanaceous plant cells are useful in providing a transgenic solanaceous plant that has improved resistance to an infestation with the Leptinotarsa species when compared to a control plant that lacks such plant cells. The transgenic solanaceous plant cell may be an isolated transgenic solanaceous plant cell, or a cultured transgenic solanaceous plant cell, or a transgenic cell of any transgenic solanaceous plant that is subjected to infestation by a Leptinotarse species. Examples Include a transgenic nightshade plant that is selected from the group consisting of potato, tomato, and eggplant. Modalities include those where the transgenic nightshade plant is an ungerminated transgenic nightshade plant seed, a vegetative-stage transgenic nightshade plant, or a reproductive-stage transgenic nightshade plant. Modalities include those where the transgenic nightshade plant is a potato tuber or part of the potato tuber (seed potato) that can be propagated in new transgenic potato plants.
In one embodiment, the recombinant DNA is stably integrated into the genome of the transgenic solanaceous plant from which it can be expressed in a cell or cells of the transgenic solanaceous plant. Methods for providing stably transformed plants are provided in the section entitled Making and Using Transgenic Plant Cells and Transgenic Plants.
Several modalities refer to a transgenic solanaceous plant cell that has in its genome an RNA that encodes recombinant DNA that suppresses the expression of a target gene in a Leptinotarsa species that contacts or ingests the RNA, where the RNA comprises at least one silencing element complementary to the target gene and where the sequence of the target gene is selected from or complement the Target Gene Sequence Group. In some embodiments, the silencing element comprises at least 18 or more contiguous nucleotides with a sequence of about 95% to about 100% complementary to a fragment of equivalent length of DNA that has a sequence that is selected from the group consisting of in the Gen Diana Sequence Group. In some embodiments, the silencing element comprises at least 18 or more contiguous nucleotides capable of hybridizing in vivo or hybridizing under physiological conditions (eg, such as physiological conditions normally found in cells of a Leptinotarse species) to a fragment of length DNA equivalent that has a sequence that is selected from the group consisting of the Gen Diana Gene Sequence Group. Contiguous segment nucleotides number at least 18, for example, between 18 - 24, or between 18 - 28, or between 20 - 30, or between 20 - 50, or between 20 - 100, or between 50 - 100, or between 50 500, or between 100 - 250, or between 100 - 500, or between 200 - 1000, or between 500 - 2000, or even more.
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In some embodiments, contiguous nucleotides list more than -10, for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, for example, about 35, around 40, around 45, around 50, around 55, around 60, around 65, around 70, around 75, around 80, around 85, around 90, around 95, around 100, around 110, around 120, around 130, around 140, around 150, around 160, around 170, around 180, around 190, around 200, around 210, around 220, around 230, around 240, around 250, around 260, around 270, around 280, about 290, about 300, about 350, about 400, about 450, about 500, or more than 500 contiguous nucleotides. In particular embodiments, the gene element comprises at least one segment of at least 21 contiguous nucleotides with a sequence of 100% identity with a fragment of equivalent length of a target DNA or gene having a sequence that is selected from the Sequence Group Diana gene or DNA complement of this. In particular embodiments, the polynucleotide is a double-stranded nucleic acid (eg, dsRNA) with a strand comprising at least one segment of at least 21 contiguous nucleotides with a sequence of 100% identity with a fragment of equivalent length of a DNA or target gene having a sequence selected from the group consisting of the Target Gene Sequence Group or the DNA complement thereof; expressed as base pairs, such a double-stranded nucleic acid comprises at least one segment of at least 21 contiguous nucleotides, perfectly matched base pairs corresponding to a fragment of equivalent length of a target DNA or gene that has a sequence that is select from the Target Gene Sequence Group or its DNA complement. In particular embodiments, each linkage element contained in the RNA is longer than the typical length of naturally occurring small regulatory RNAs. In some embodiments, each segment is at least about 30 contiguous nucleotides (or base pairs) in length. In particular embodiments, the RNA is between about 50 and about 500 nucleotides in length. In particular embodiments, the RNA has a sequence that is selected from the group consisting of: SEQ ID NO: 831 1085, 1095-1104, and 1110-1114, or the complement thereof, or the RNA is encoded by a sequence that is selected from the group consisting of SEQ ID NO: 1105-1109.
In some embodiments, the transgenic nightshade plant cell is also capable of expressing additional heterologous DNA sequences. In one embodiment, the transgenic solanaceous plant cell has a genome further comprising recombinant DNA encoding at least one pesticidal agent that is selected from the group consisting of a patatin, a plant lectin, a phytoecdlsteroid, an insecticidal protein BaciHus thuringiensis, an insecticidal protein Xenorhabdus, an insecticidal protein Photorhabdus, an insecticidal protein
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Bacillus laterosporous, and an insecticidal protein BaciHus sphaerícus. In particular embodiments, the transgenic solanaceous plant cell stably integrated into its genome (i) recombinant DNA encoding at least one RNA with a sequence selected from the group consisting of SEQ ID NO: 831-1085 and 1095 and ( I) DNA encoding at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecysteroid, a phytoecysteroid, an insecticidal protein BaciHus thuringiensis, an insecticidal protein Xenorhabdus, an insecticidal protein Photorhabdus, an insecticidal protein BaciHus laterosporous, and an insecticidal protein BaciHus sphaerícus.
In a related aspect, this invention relates to a transgenic solanaceous plant including the transgenic solanaceous plant cell, a commodity produced from the transgenic solanaceous plant, and the transgenic progeny-derived solanaceous plant seed or transgenic part of the plant transgenic nightshade. Modalities include a transgenic tomato plant, a transgenic tomato rhizome, a transgenic eggplant, or a transgenic potato plant that has improved resistance to an infestation with the Leptinotarsa species. Also contemplated is a basic product produced by the transgenic solanaceous plant, and a basic product produced from the transgenic progeny seed of such a transgenic solanaceous plant.
Insecticidal compositions to control the species Leptinotarsa
Another aspect of this invention provides an insecticidal composition for controlling the Leptinotarsa species, where the insecticidal composition consists essentially of an RNA molecule that causes mortality or growth impairment in a Leptinotarsa species when ingested or contacted by the Leptinotarsa species, and wherein the RNA molecule comprises at least a segment of 18 or more contiguous nucleotides that is essentially complementary to a fragment of a DNA that has a sequence that is selected from, or complements, the DNA Gene Sequence Group. In this context, controlling a Leptinotarsa species includes the induction of a physiological or behavioral change in a Leptinotarsa species (adult or larva) such as, without limitation, weakening of growth or increased mortality. In some modalities, controlling a Leptinotarsa species is achieved by a decrease in reproductive capacity, decrease or cessation of movement or feeding behavior, or decrease or cessation of the development of the metamorphosis stage in a Leptinotarsa species. Generally, the RNA molecule was isolated, ie, purified substantially from a mixture such as a fermentation or an in vitro synthesis mixture. In one embodiment, the RNA molecule comprises at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% complementary to a fragment of equivalent length of DNA that has a
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INSTITUTO MEXICANO DE IA TMOPIEOAO sequence that is selected from the Gen Diana Sequence Group or the complement Ütí EVEN this. In some embodiments, the RNA molecule comprises at least one segment of 18 or more contiguous nucleotides that is essentially complementary to a fragment of DNA that has a sequence that is selected from the group consisting of: SEQ ID NO: 831-1085, 1095 1104, and 1110-1114, or the complement thereof, or where the RNA molecule is encoded by a sequence selected from the group consisting of SEQ ID NO: 1105-1109. In some embodiments, the RNA molecule is double-stranded, and the at least one segment is about 50 to about 500 base pairs in length. In some embodiments, the RNA molecule is a dsRNA with a strand that has a sequence that is selected from the group consisting of the Trigger Sequence Group. In some embodiments, an Insecticidal composition is provided to control a Leptinotarsa species, where the Insecticidal composition comprises a double-stranded RNA, where at least one strand of the double-stranded RNA is complementary to at least 21 contiguous nucleotides of a gene encoding a rlbosomal protein or a transcribed RNA of the gene, where the Leptinotarsa species is Leptinotarsa decem / ineata, and where RNA Interference is Induced and Leptinotarsa decem / ineata mortality occurs, and where the rlbosomal protein is a L7 rlbosomal protein or a protein encoded by SEQ ID NO: 730 or where the double stranded RNA comprises a sequence that select from the group consisting of SEQ ID NO: 989, 988, 1104, or 1105.
Molecules of the RNA molecule Include those where the segment of 18 or more contiguous nucleotides has a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% Complementarity to a DNA fragment that has a sequence that is selected from the Gen Diana Sequence Group or its DNA complement. In some embodiments, the contiguous nucleotides are exactly (100%) complementary to a fragment of equivalent length of a DNA that has a sequence that is selected from the Gene Target Sequence Group or its DNA complement. In some embodiments, the RNA molecule has an overall sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% complementary to a DNA that has a sequence that is selected from the Gen Diana Gene Sequence Group or its DNA complement.
Molecules of the RNA molecule Include at least one segment of 18 or more contiguous nucleotides designed to suppress the expression of a target gene that has a sequence that is selected from the Target Gene Sequence Group or its DNA complement. Contiguous segment nucleotides number at least 18, for example, between 18 - 24, or between 18 28, or between 20 - 30, or between 20 - 50, or between 20 - 100, or between 50 - 100, or between 50 - 500, or between 100 - 250, or between 100 - 500, or between 200 - 1000, or between 500 - 2000, or even more. In some
<img file="MX359191B_D0080.tif" />
ΙΜΡΪ modalities, contiguous nucleotides list more than 18, for example 19) 20t ^ t227-23> 24, 25, 26, 27, 28, 29, 30, or more than 30, for example, about 35, about 40, around 45, around 50, around 55, around 60, around 65, around 70, around 75, around 80, around 85, around 90, around 95, around 100 , around 110, around 120, around 130, around 140, around 150, around 160, around 170, around 180, around 190, around 200, around 210, around 220, around 230, around 240, around 250, around 260, around 270, around 280, around 290, about 300, about 350, about 400, about 450, about 500, or more than 500 contiguous nucleotides. In particular embodiments, the RNA molecule comprises at least one segment of at least 21 contiguous nucleotides with a sequence of 100% identity with a fragment of equivalent length of a target DNA or gene having a sequence that is selected from the Sequence Group Diana gene or DNA complement of this. In particular embodiments, the polynucleotide is a double-stranded nucleic acid (eg, dsRNA) with a strand comprising at least one segment of at least 21 contiguous nucleotides with a sequence of 100% identity with a fragment of equivalent length of a DNA or target gene having a sequence selected from the group consisting of the Target Gene Sequence Group or the DNA complement thereof; expressed as base pairs, such a double-stranded nucleic acid comprises at least one segment of at least 21 contiguous nucleotides, perfectly matched base pairs corresponding to a fragment of equivalent length of a target DNA or gene that has a sequence that is select from the Target Gene Sequence Group or its DNA complement. In particular embodiments, each segment contained in the RNA molecule is longer than typical for small, naturally occurring regulatory RNAs. In some embodiments, each segment is at least about 30 contiguous nucleotides (or base pairs) in length. In some embodiments, the total length of the RNA molecule, or the length of each segment contained in the RNA molecule, is less than the total length of the sequence of Interest (DNA or target gene that has a sequence that is selected from the group consisting of the Gen Diana Sequence Group). In some embodiments, the total length of the RNA molecule is from about 50 to about 500 nucleotides (for single chain polynucleotides) or base pairs (for double chain polynucleotides). In some embodiments, the RNA molecule is a dsRNA of between about 100 and about 500 base pairs, such as a dsRNA the length of any of the dsRNA triggers described in Tables 3, 5, 8, 9 and 10. In some embodiments, the insecticidal composition consists essentially of an insecticidal effective amount of a double-stranded RNA molecule with a strand having a sequence selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104 , and 1110 - 1114, or the
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complement of this, or consists essentially of a <sup>rar</sup>t * Hnd c-flcar ionw ingartrid<sup>3</sup> · _ · Η RNA hairpin encoded by a sequence selected from the group consisting of SEQ ID NO: 1105-1109. In some embodiments, the insecticidal composition consists essentially of an insecticide-effective amount of a double-stranded RNA molecule with a string having a sequence that is selected from the group consisting of the Trigger Sequence Group.
The RNA molecule is generally designed to suppress one or more genes (target genes). Such target genes can include coding or non-coding sequences, or both. In specific embodiments, the RNA molecule is designed to suppress one or more target genes, where each target gene has a DNA sequence that is selected from the group consisting of the Target Gene Sequence Group. In various modalities, the RNA molecule is designed to delete one or more genes, where each gene has a sequence that is selected from the group consisting of the Target Gene Sequence Group, and can be designed to delete multiple genes from this group, or to target different regions of one or more of these genes. Modalities of the RNA molecule include at least a segment of 18 or more contiguous nucleotides that have a sequence designed to suppress one or more genes, where each gene has a sequence that is selected from the group consisting of the Group of Gene Sequences Diana. In one embodiment, the RNA molecule comprises multiple sections or segments each of which comprises at least one segment of 21 contiguous nucleotides with a sequence 100% complementary to a fragment of equivalent length of DNA that has a sequence that is select from the Target Gene Sequence Group or its DNA complement. In such cases, each section can be identical or different in size or in sequence. For example, in one embodiment the RNA molecule comprises multiple tandem sections or repeating arrangements, where each section comprises at least one segment of 21 contiguous nucleotides with a sequence 100% complementary to a fragment of equivalent length of DNA that has a sequence that is selected from the Gene Target Sequence Group or the DNA complement thereof; the segments may be from different regions of the target gene, eg, the segments may correspond to different regions of the target gene exon, and the spacer nucleotides that do not correspond to a target gene may optionally be used between or adjacent to the segments.
The RNA molecule can have a total length that is greater than 18 contiguous nucleotides, and can include nucleotides in addition to the segment of at least one segment of 18 or more contiguous nucleotides that has the sequence of from about 95% to about 100% of Complementarity to a fragment of equivalent length of DNA that has a sequence that is selected from the Gen Diana Sequence Group or the DNA complement thereof. In other words, the total length of the RNA molecule may be greater than the length of the segment designed to suppress one or more target genes, where each target gene has a DNA sequence
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which is selected from the group consisting of the Sequence Group<sup>1</sup> by Gen Diana. Pui cjunpk »; the RNA molecule may have nucleotides that flank the active segment of at least one segment of 18 or more contiguous nucleotides that suppress the target gene, or include spacer nucleotides between the active segments, or may have additional nucleotides at the 5 'end, or at the 3 'end, or at both the 5' and 3 'ends. In one embodiment, the RNA molecule comprises additional nucleotides that are not specifically related (that have a non-complementary or Identical sequence) to the target DNA or gene that has a sequence that is selected from the Target Gene Sequence Group or DNA complement of this, for example, nucleotides that provide a stabilizing secondary structure or for convenience in cloning or manufacturing. In one embodiment, the RNA molecule comprises additional nucleotides located immediately adjacent to one or more contiguous 18 or more nucleotide segments with a sequence of about 95% to about 100% complementarity to a fragment of equivalent length of DNA or target gene having a sequence that is selected from the group consisting of the Target Gene Sequence Group. In one embodiment, the RNA molecule comprises such a segment, with an additional 5'G or an additional 3'C or both, adjacent to the segment. In another embodiment, the RNA molecule is a double-stranded RNA that comprises additional nucleotides to form a surplus, eg, a dsRNA comprising 2 deoxyrrbononucleotides to form a 3 'surplus. Therefore in various embodiments, the nucleotide sequence of the entire RNA molecule is not 100% identical or complementary to a contiguous nucleotide fragment in the target DNA or gene that has a sequence that is selected from the group consisting of Group of Gen Diana Sequences. For example, in some embodiments the RNA molecule comprises at least two contiguous 21 nucleotide segments with a sequence of 100% Identity with a fragment of DNA having a sequence that is selected from the Gen Diana Sequence Group, or the DNA complement thereof, where (1) the at least two segments are separated by one or more spade nucleotides, or (2) the at least two segments are arranged in a different order than the order in which the corresponding fragments occur in the DNA that has a sequence that is selected from the Gen Diana Sequence Group, or its DNA complement.
The RNA molecule can be single chain (s) or double chain (cd) or a combination of both. Modalities of the RNA molecule Include sense single-stranded RNAs (cRNAs), antisense cRNAs, or double-stranded RNAs (dsRNA), or a combination of these. RNA can Include components other than standard ribonucleotides, for example, one embodiment is an RNA comprising terminal deoxyribonucleotides. In several embodiments, the RNA molecule consists of naturally occurring ribonucleotides. In certain embodiments, the RNA molecule is a combination of ribonucleotides and deoxyribonucleotides, for example, the
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Synthetic RNA molecule consisting mainly of rlbonucleotides but with one or more terminal deoxyrrbonucleotides or one or more terminals. In certain embodiments, the RNA molecule comprises non-canonical nucleotides such as inosine, thiouridine, or pseudouridine. In certain embodiments, the RNA molecule comprises chemically modified nucleotides.
The RNA molecule is provided by suitable means known to one of skill in the art. Modalities include those where the RNA molecule is synthesized in vitro, is produced by expression in a microorganism or in cell culture (such as cultured plant or insect cells), is produced by expression in a plant cell or it is produced by microbial fermentation.
In some embodiments, the RNA molecule comprises other RNA elements, such as RNA aptamers or ribozymes, additional non-coding RNA (eg, additional deletion elements), or one or more recognition sites for RNA binding and cleavage small (for example, by a miRNA or a siRNA that is expressed only in a particular cell or tissue).
The insecticidal composition can be provided for topical application to a surface of a plant or a plant seed, or for topical application to any substrate that needs protection from an infestation with the Leptinotarsa species. Of! Similarly, the insecticidal composition can be provided for topical application to a Leptinotarsa species, or in a composition for ingestion by a Leptinotarsa species. In various embodiments, the insecticidal composition is provided in the form of at least one that is selected from the group consisting of a solid, liquid (including homogeneous mixtures as solutions and non-homogeneous mixtures as suspensions, colloids, micelles, and emulsions), powder, suspension , emulsion, sprayer, encapsulated or microencapsulation formulation, in or on top of microbeads or other carrier particles, in a film or coating, or in or on a matrix. Binders, inert carriers, suitable surfactants and the like can be included in the insecticidal composition, as one skilled in the formulation of pesticides and seed treatment knows. While the insecticidal composition consists essentially of an RNA molecule, in some embodiments the insecticidal composition further comprises at least one non-insecticidal agent which is selected from the group consisting of a carrier agent, a salt, a surfactant, a cationic lipid (such such as described in Example 18 of US Patent Application Publication 2011/0296556, incorporated herein by this reference), an organosilicone, an organosilicone surfactant, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule and a protector. In one embodiment the composition containing the recombinant RNA molecule further comprises a nonionic organosilicone surfactant such as surfactants
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SILWET® brand, for example SILWET L-77® brand surfactant with CAS number 27306-78-1 and EPA number: CAL.REG. No. 5905-50073-AA, currently available from Momentive Performance Materials, Albany, New York. Also, the insecticidal composition can be used, before or after, in combination with a treatment with a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide (for example, at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecysteroid, an insecticidal protein Bacillus thuringiensis, an insecticidal protein Xenorhabdus, an insecticidal protein Photorhabdus, an insecticidal protein Bacillus iaterosporous, and an insecticidal protein Bacillus sphaericus). Related compositions include combinations of the RNA molecule with a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, and a non-polynucleotide pesticide.
The insecticidal composition can be provided for food absorption by a Leptinotarsa species by applying the composition to a plant or surface subject to Leptinotarsa species infestation, for example by spraying, dusting or coating the plant, or soaking the soil, or by providing an artificial diet. The insecticidal composition may be provided for food absorption by a Leptinotarsa species in an artificial diet formulated to meet the particular nutritional requirements for maintaining the Leptinotarsa species, where the artificial diet is supplemented with an amount of the recombinant RNA molecule obtained from such a separate source. as an in vitro or purified synthesis of a microbial fermentation or other biological source; This modality may be useful, for example, in determining the times and amounts of effective treatment regimens. The insecticidal composition can be provided for food absorption by the Leptinotarsa species in the form of a seed treatment.
Methods of providing plants having improved resistance to infestations of the Leptinotarsa species, and the plants, plant parts, and seeds thus provided
Various embodiments relate to a method of providing a plant that has improved resistance to an infestation with the Leptinotarsa species comprising providing the plant with at least one polynucleotide comprising at least one segment of 18 or more contiguous nudeotides that is essentially identical or complementary to a fragment of a target gene that is selected from the group consisting of the genes identified in the Target Gene Sequence Group. In one embodiment, this invention provides a method of providing a plant having improved resistance to an infestation with the Leptinotarsa species which comprises providing the plant with at least one polynucleotide comprising at least one segment that is identical or complementary to at least 21 nudeotides. contiguous of a target gene or a transcribed RNA
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of the target gene, where the target gene is selected from the genes identified in the Target Gene Sequence Group or an RNA transcribed from the target gene. The modalities of these target genes are identified by name in Tables 1, 2, and 4 and include genes that have a sequence that is selected from the group consisting of the Target Gene Sequence Group, as well as related genes that include orthologs from related insect species, for example related genes from other Leptinotarsa species, Triboiium species, or other related genera. Examples of such related target genes include the Triboiium castaneum genes listed in Table 1. In some embodiments, the polynucleotide is a double-stranded RNA. In some embodiments, the polynucleotide (eg, double-stranded RNA) is chemically synthesized or produced by expression in a microorganism or by expression in a plant cell. In some embodiments, the polynucleotide comprises at least one segment of 18 or more contiguous nucleotides that is essentially Identical to or complementary to a sequence that is selected from the group consisting of the Gen Diana Sequence Group. In some embodiments the polynucleotide is a dsRNA with a strand that has a sequence that is selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or where the polynucleotide is encoded by a sequence that is selected from the group consisting of SEQ ID NO: 1105-1109. In some embodiments, the polynucleotide comprises a one-strand dsRNA that has a sequence that is selected from the Trigger Sequence Group.
In one embodiment the method comprises applying topically to the plant a composition comprising at least one polynucleotide comprising at least a segment of 18 or more contiguous nucleotides that are essentially identical or complementary to a fragment of equivalent length of a DNA of a target gene which is selected from the group consisting of the genes identified in the Target Gene Sequence Group, whereby the plant treated with the polynucleotide composition exhibits improved resistance to an infestation with the Leptinotarsa species, relative to an untreated plant. Topical application refers to application to the surface or exterior of an object, such as the surface or exterior of a plant, such as application to the surface of a part of the plant such as a leaf, stem, flower, fruit, sprout, root, seed, tuber, flower, anther or pollen, or application to an entire plant, or parts above ground or below ground of a plant. Topical application can be performed on non-living surfaces such as application to soil, or to a surface or matrix whereby a Leptinotarsa insect may come in contact with the polynucleotide. In various embodiments of the method, the polynucleotide-containing composition is applied topically to the plant suitably, for example, as a solid, liquid (including homogeneous mixtures as solutions and non-homogeneous mixtures as suspensions, colloids, micelles, and emulsions), powder , suspension, emulsion, spray, encapsulated or microencapsulation formulation, in or on top of microbeads u
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MEXICAN INSTITUTE OF LA MOHEDA ·
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seed treatment. In some embodiments of the method, the polynucleotide-containing composition is applied topically to the above-ground parts of the plant, for example, sprayed or dusted on leaves, stems, and flower parts of the plant. Modalities of the method include topical application of a foliar spray (eg, spraying a liquid composition containing polynucleotides onto the leaves of a nightshade plant) or a leaf powder (eg, dusting a nightshade plant with a polynucleotide-containing composition in the form of a powder or carrier particles). In other embodiments, the polynucleotide-containing composition is applied topically to below-ground parts of the plant, such as to the roots, for example, wetting the soil. In other embodiments, the polynucleotide-containing composition is applied topically to a seed grown in the plant. Topical application may be in the form of a topical treatment of nightshade plant fruits or fruit seeds of nightshade plants or in the form of topical treatment of seed potato tubers or parts of a tuber (for example, soaking, coating or sprinkling the seed potato). Binders, inert carriers, suitable surfactants and the like may optionally be included in the polynucleotide-containing composition, as is known to one skilled in the formulation of pesticides and seed treatment. In some embodiments, the polynucleotide-containing composition is at least a topically implantable formulation that is selected from the group consisting of a particle, pellet, or capsule topically implanted in the plant; in such modalities the method comprises topically implanting the topically implantable formulation in the plant. In some embodiments, the polynucleotide-containing composition is at least one groove formulation selected from the group consisting of a powder, granule, pellet, capsule, spray, or potion or any other suitable form to be applied topically to a groove; in such modalities, the method includes a groove treatment with the groove formulation. In one embodiment, the polynucleotide-containing composition can be ingested or otherwise absorbed internally by the Leptinotarsa species. For example, the polynucleotide-containing composition may be in the form of a bait. In some embodiments, the polynucleotide-containing composition further comprises one or more components that are selected from the group consisting of a carrier agent, a surfactant, a cationic lipid (such as that described in Example 18 of the US Patent 2011/0296556, incorporated herein by this reference), an organosilicone, an organosilicone surfactant, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide pesticide, a protector and a regulator of Insect growth. In one embodiment the composition further comprises a nonionic organosilicone surfactant such as SILWET® brand surfactants, eg, surfactant of
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SILWET L-77® brand with CAS number 27306-78-1 and EPA number: CAL.REG. No. 5905-50073-AA, currently available from Momentive Performance Materials, Albany, New York. In some embodiments, the topically applied composition further comprises at least one pesticidal agent that is selected from the group consisting of a patatlna, a plant lectin, a fltoecdlsteroid, a Bacillus thuringiensis Insecticidal protein, a Xenorhabdus Insecticidal protein, a Photorhabdus Insecticidal protein, a Bacillus laterosporous Insecticidal protein and a Bacillus sphaericus Insecticidal protein. Alternatively, such additional components or pesticidal agents can be provided separately, for example, by separate topical application or by transgene expression in the plant. Alternatively, the plant is treated topically with the composition containing pollnucleotides as well as with a separate application (previous, next or simultaneous) of a substance that improves the efficacy of the composition containing pollnucleotides. For example, a plant can be sprayed with a first topical application of a solution containing a non-ionic organosylcone surfactant such as SILWET® brand surfactants, for example, SILWET L-77® brand surfactant, followed by a second topical application of the composition containing pollnucleotides, or vice versa.
It is anticipated that the combination of certain pollnucleotides (eg, the pollnucleotide triggers described in the Working Examples) with one or more non-pollnucleotide pesticidal agents will result in a significant improvement in the prevention or control of Leptinotarse species infestations, when compared with the effect obtained with the polynucleotide alone or the non-polynucleotide pesticide agent alone. In one embodiment, a composition was found to contain one or more pollenucleotides and one or more non-pollenucleotide pesticidal agents that are selected from the group consisting of a potato, a plant lectin, a fltoecdlsteroid, an insecticidal protein Bacillus thuringiensis, a protein Xenorhabdus insecticide, a Photorhabdus insecticidal protein, a Bacillus laterosporous Insecticidal protein, and a Bacillus sphaericus Insecticidal protein, It performs a highly improved prevention or control of Leptinotarsa species infestations when applied topically to a plant.
In some embodiments, the method comprises topically applying to the plant a composition comprising at least one polynucleotide comprising at least a segment of 18 or more contiguous nucleotides that are essentially identical or complementary to a fragment of equivalent length of DNA from a gene target that is selected from the group consisting of genes identified in the Target Gene Sequence Group. The pollnucleotide applied topically to the plant can be single chain (ss) or double chain (ds).
The polynucleotide applied topically to the plant is provided by suitable means known to one skilled in the art. Modalities Include those where the polynucleotide is chemically synthesized (eg, by in vitro transcription, such as
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transcription using a T7 polymerase or other polymerase), expression in a microorganism or in cell culture (such as plant or insect cells grown in culture), produced by expression in a plant cell, or produced by microbial fermentation.
In various embodiments, the polynucleotide applied topically to the plant is provided as an isolated DNA or RNA. In some embodiments, the polynucleotide applied topically to the plant is not part of an expression construct and has no additional elements such as a promoter or termination sequences. These polynucleotides can be relatively short, such as single or double stranded polynucleotides of between about 18 and about 300 or between about 50 and about 500 nucleotides (for single chain pollenucleotides) or between about 18 and about 300 or between about 50 and about 500 base pairs (for double stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of between about 100 and about 500 base pairs, such as a dsRNA the length of any of the dsRNA triggers described in Tables 3, 5, 8, 9, and 10. Alternatively, the polynucleotide may be provided in more complex constructs, eg, as part of a recombinant expression construct, or Include in a recombinant vector, eg, in a recombinant plant virus vector or baculovlrus vector recombinant. Such recombinant expression constructs or vectors can be designed to Include additional elements, such as expression cassettes to express a gene of Interest (eg, an Insecticidal protein).
The polynucleotide applied topically to the plant has at least one segment of 18 or more contiguous nucleotides that are essentially identical to or complement a fragment of equivalent length of a DNA for a target gene that is selected from the group consisting of the genes identified in the Gen Diana Sequence Group, or that have a sequence of about 95% to about 100% Identity with or complement a fragment of equivalent length of a DNA of a target gene that is selected from the group consisting of the genes Identified in the Sequence Group of Gen Diana. In one embodiment the polynucleotide applied topically to the plant comprises at least a segment of 18 or more contiguous nucleotides that are essentially Identical or complement a fragment of equivalent length of a DNA of a target gene that is selected from the group consisting of genes identified in the Gen Diana Sequence Group. In some embodiments, contiguous nucleotides have a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity with or complementing the fragment of equivalent length of a DNA from a target gene that is selected from the group consisting of the genes Identified in the Target Gene Sequence Group. In some
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Modalities the contiguous nucleotides are exactly (100%) Identical or complementary to a fragment of equivalent length of a DNA of a target gene that is selected from the group consisting of genes identified in the Target Gene Sequence Group. In some embodiments, the pollnucleotide has an overall sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% Identity with or complementary to a fragment of a DNA from a target gene that is selected from the group consisting of the genes Identified in the Target Gene Sequence Group.
The polynucleotide applied topically to the plant is generally designed to suppress one or more genes (target genes). In specific embodiments, the polynucleotide is designed to delete one or more target genes that are selected from the group consisting of the genes identified in the Target Gene Sequence Group. Modalities of genes identified in the Gene Target Sequence Group include, but are not limited to, cDNA sequences that are selected from the group consisting of the Target Gene Sequence Group. In various embodiments, the polynucleotide applied topically to the plant is designed to suppress one or more genes, where each gene is selected from the group consisting of the genes identified in the Diana Gene Sequence Group, and can be designed to suppress multiple genes from this group, or to target different regions of one or more of these genes. In one embodiment, the polynucleotide topically applied to the plant comprises multiple sections or segments each of which comprises at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with or complementarity. with a fragment of equivalent length of a DNA from a target gene that is selected from the group consisting of genes identified in the Target Gene Sequence Group. In such cases, each section may be identical or different in size or sequence, and may be sense or antisense relative to the target gene. For example, in one embodiment the polynucleotide topically applied to the plant may include multiple tandem sections or repeating arrangements, where each section comprises at least one segment of 21 contiguous nucleotides with a sequence of 100% identity or 100% complementarity to a fragment of equivalent length of DNA for a target gene that is selected from the group consisting of genes identified in the Target Gene Sequence Group; the segments may be from different regions of the target gene, for example, the segments may correspond to different regions of the exon of a cDNA with a sequence selected from the group consisting of the Target Gene Sequence Group, and the spacer nucleotides that they do not correspond to a target gene and can optionally be used between or adjacent to the segments.
The total length of the pollnucleotide applied topically to the plant may be greater than 18 contiguous nucleotides, and may include nucleotides in addition to at least one segment of
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contiguous nucleotides having the sequence essentially IdérillüH or Luiiipteiniintail or · mi fragment of equivalent length of a DNA of a target gene that is selected from the group consisting of genes Identified in the Target Gene Sequence Group. In other words, the total length of the pollnucleotide applied topically to the plant may be greater than the length of the section or segment of the pollnucleotide designed to suppress one or more target genes, where each target gene is selected from the group consisting of the Identified genes in the Gen Diana Sequence Group. For example, the pollnucleotide applied topically to the plant may have nucleotides flanking the active segment of at least one segment of 18 or more contiguous nucleotides that suppress the target gene, or Include spacer nucleotides between the active segments, or may have additional nucleotides in the 5 'end, or the 3' end, or both 5 'and 3' ends. In one embodiment, the pollnucleotide applied topically to the plant comprises additional nucleotides that are not specifically related (having a non-complementary or Identical sequence) to the target gene that is selected from the group consisting of the genes Identified in the Gene Sequence Group Target, for example, nucleotides that provide a stabilizing secondary structure or for convenience in cloning or manufacturing. In one embodiment, the pollnucleotide topically applied to the plant comprises additional nucleotides located immediately adjacent to one or more segments of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% Identity with or complementary to the target gene that is selected from the group consisting of the genes Identified in the Target Gene Sequence Group. In one embodiment, the pollnucleotide topically applied to the plant comprises such a segment, with an additional 5'G or an additional 3'C, or both, adjacent to the segment. In another embodiment, the polynucleotide topically applied to the plant is a double-stranded RNA that comprises additional nucleotides to form a surplus, eg, a dsRNA comprising 2 deoxyrrbonucleotides to form a 3 'surplus. Therefore in various modalities, the nucleotide sequence of the entire pollnucleotide applied topically to the plant is not 100% identical or complementary to a contiguous nucleotide fragment in the target gene that is selected from the group consisting of the genes identified in the Gen Diana Sequence Group. For example, in some embodiments, the pollnucleotide applied topically to the plant comprises at least two segments each of 21 contiguous nucleotides with a sequence of 100% Identity with a fragment of a target gene selected from the group consisting of the Identified genes. in the Gen Diana Sequence Group, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in a different order than the corresponding fragments occur in the target gene that is selected from the group consisting of the genes Identified in the Target Gene Sequence Group.
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In a related aspect, this invention was related to an improved rosistcncia to an infestation with the Leptinotarsa species, provided by this method comprising applying topically to the plant a composition comprising at least one polynucleotide comprising at least a segment of 18 or more contiguous nucleotides that are essentially identical or complementary to a fragment of equivalent length of a DNA of a target gene which is selected from the group consisting of the genes identified in the Target Gene Sequence Group, whereby the plant treated with the polynucleotide composition exhibits improved resistance to an infestation with the Leptinotarsa species, relative to an untreated plant. In yet another aspect, this invention relates to seeds (especially transgenic progeny seed) produced by the plant that have improved resistance to an infestation with the Leptinotarsa species, as provided by this method. Also contemplated is a basic product produced by the plant that has improved resistance to an infestation with the Leptinotarsa species, as provided by this method, and a basic product produced from the transgenic progeny seed of such a plant.
In another embodiment the method comprises expressing in the plant at least one polynucleotide comprising at least a segment of 18 or more contiguous nucleotides that are essentially identical or complementary to a fragment of equivalent length of a target gene that is selected from the group consisting of genes identified in the Diana Gene Sequence Group, whereby the plant expressing the polynucleotide exhibits improved resistance to an infestation with the Leptinotarsa species, in relation to a plant that does not express the polynucleotide. In one embodiment the method comprises expressing in the plant at least one polynucleotide comprising at least a segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with or complementarity to a fragment of equivalent length from a target gene DNA that is selected from the group consisting of genes identified in the Target Gene Sequence Group. The modalities of these target genes are identified by name in Tables 1, 2, and 4 and include genes that have a sequence that is selected from the group consisting of the Target Gene Sequence Group, as well as related genes that include orthologs from related insect species, for example related genes from other Leptinotarsa species, Tríboiium species, or other related genera. Examples of such related target genes include the Triboiium castaneum genes listed in Table 1. "Expressing a polynucleotide in the plant" generally means that it expresses a transcription of RNA in the plant. However, the polynucleotide expressed in the plant can also be DNA, for example, DNA produced in the plant during genome replication.
The method comprises expressing at least one polynucleotide in a plant, where the
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polynucleotide comprises at least a segment of 18 or more contiguous nucleotides jUS er essentially identical or complementary to a fragment of a target gene that is selected from the group consisting of genes identified in the Target Gene Sequence Group. In some embodiments, a first polynucleotide is provided to a plant in the form of DNA (eg, in the form of an isolated DNA molecule, or as an expression construct, or as a transformation vector), and the expressed polynucleotide in the plant it is a second polynucleotide (eg, the RNA transcript of the first polynucleotide) in the plant. In one embodiment, the polynucleotide is expressed in the plant by transgenic expression, i.e., Stably integrating the polynucleotide into the plant genome from which it can be expressed in a plant cell or cells. In one embodiment, a first polynucleotide (eg, a recombinant DNA construct comprising a promoter operably linked to DNA comprising at least a segment of 18 or more contiguous nucleotides that is essentially identical or complementary to a target gene fragment that is selected from the group consisting of the genes identified in the Target Gene Sequence Group) integrates stably into the plant genome from which secondary polynucleotides are produced (eg, an RNA transcript comprising the transcript of the segment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment of a target gene that is selected from the group consisting of genes Identified in the Target Gene Sequence Group) is expressed in a plant cell or cells. Methods for providing stably transformed plants are provided in the section entitled Making and lllso of Transgenic Plant Cells and Transgenic Plants.
In another embodiment, the polynucleotide expressed in the plant is expressed by transient expression (ie, expression that does not result from the stable integration of a sequence into the plant genome). In such embodiments, the method may include a step to introduce a polynucleotide (eg, dsRNA or dsDNA) into the plant by routine techniques known in the art. For example, transient expression can be achieved by infiltrating a polynucleotide solution using a needleless syringe on a leaf of a plant.
In some embodiments where the polynucleotide expressed in the plant is expressed by transient expression, a first polynucleotide is provided to a plant in the form of RNA or DNA or both RNA and DNA, and a second polynucleotide produced secondary is transiently expressed in the plant . In some embodiments, the first polynucleotide is one or more that are selected from: (a) a single-stranded RNA molecule (cRNA), (b) a single-stranded RNA molecule that self-hybridizes to form a single-stranded RNA molecule. double-stranded, (c) a double-stranded RNA molecule (dsRNA), (d) a single-stranded DNA molecule (cDNA), (e) a single-stranded DNA molecule that self-inhibits to form a DNA molecule of
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double-stranded, (f) a single-stranded DNA molecule that comprises a modified Pol III gene that is transcribed into an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a DNA molecule double-stranded comprising a modified Pol III gene that is transcribed to an RNA molecule, and (i) a double-stranded hybridized RNA / DNA molecule, or combinations thereof. In specific embodiments, a first polynucleotide is introduced into the plant by topically applying to the plant a composition containing polynudeotides in a suitable form, for example, as a solid, liquid (including homogeneous mixtures as solutions and non-homogeneous mixtures as suspensions, colloids, micelles and emulsions), powder, suspension, emulsion, sprayer, encapsulated or microencapsulated formulation, in or on top of microbeads or other carrier particles, in a film or coating, or in or on a matrix, or in the form of a solanaceous plant seed treatment or seed potato treatment. Binders, inert carriers, suitable surfactants and the like may optionally be included in the composition, as is known to one skilled in the formulation of pesticides and seed treatment. In such embodiments, the polynudeotide-containing composition may further include one or more components that are selected from the group consisting of a carrier agent, a surfactant, a cationic lipid (such as that described in Example 18 of the patent application publication 2011/0296556, incorporated by this reference herein), an organosilicone, an organosilicone surfactant, a polynucleotide herbicide molecule, a non-polynucleotide herbicide molecule, a non-polynucleotide pesticide, a protector and a regulator of insect growth; in one embodiment the composition further comprises a nonionic organosilicone surfactant such as SILWET® brand surfactants, for example SILWET L-77® brand surfactant with CAS number 27306-78-1 and EPA number: CAL.REG . No. 5905-50073-AA, currently available from Momentive Performance Materials, Albany, New York. In some embodiments, the topically applied composition further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecysteroid, an insecticidal protein Bacillus thuringiensis, an insecticidal protein Xenorhabdus, an insecticidal protein Photorhabdus, an insecticidal protein Bacillus iaterosporous and an insecticidal protein Bacillus sphaericus. Alternatively, such additional components or pesticidal agents can be provided separately, for example, by separate topical application or by transgenic expression in the plant. Alternatively, the plant is treated topically with the polynudeotide-containing composition as well as with a separate (previous, next, or simultaneous) application of a substance that improves the effectiveness of the polynudeotide-containing composition. For example, a plant can be sprayed with a first topical application of a solution containing a nonionic organosilicone surfactant such as SILWET® brand surfactants, for example, SILWET L-77® brand surfactant, followed by A second
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topical application of the composition containing polynucleotides, or vice versa; ---—__
It is anticipated that the combination of certain polynucleotides for use in this method (eg, the polynucleotide triggers described in the Working Examples) with one or more non-polynucleotide pesticidal agents will result in a synergistic improvement in the prevention or control of infestations of the Leptinotarsa species, when compared with the effect obtained with the pollenucleotide alone or the non-pollenucleotide pesticide agent alone. In one embodiment, a transgenic plant expressing at least one polynucleotide comprising at least a segment of 18 or more contiguous nucleotides that is essentially Identical or complementary to a fragment of a target gene that is selected from the group consisting of the genes identified in Table 1 (for example, the polynucleotide triggers described in the Working Examples) and one or more genes encoding a non-polynucleotide pesticide agent that is selected from the group consisting of a potato, a plant lectin, a phytoecdisterolde, an Insecticidal protein Bacillus thuríngiensis, a protein Xenorhabdus insecticide, a Photorhabdus insecticidal protein, a Bacillus laterosporous Insecticidal protein, and a Bacillus sphaericus Insecticidal protein, exhibited an improved resistance to Leptinotarsa species infestations.
In some embodiments where the plant-expressed polynucleotide is expressed by transient expression, a first polynucleotide is provided to a plant in the form of RNA or DNA or both RNA and DNA, and a second secondarily produced polynucleotide is transiently expressed in the plant ; the site of application of the first pollnucleotide does not necessarily have to be the same site where the second pollnucleotide is transiently expressed. For example, a first polynucleotide can be provided to a plant by topical application to a leaf, or by injection to a stem, and the second polynucleotide can be expressed transiently anywhere in the rest of the plant, for example, in the roots or throughout plant. In some embodiments of the method, a composition comprising at least one polynucleotide is applied topically to the above-ground parts of the plant, eg, sprayed or dusted on leaves, stems, and flower parts of the plant. In other embodiments, a composition comprising at least one polynucleotide is applied topically to the below-ground parts of the plant, such as the roots, for example, soaking the soil. In other embodiments, a composition comprising at least one polynucleotide is topically applied to a seed (or, in the case of potatoes, topically applied to seed potato) that is grown on the plant that has improved resistance to a infestation with the Leptinotarsa species.
In some embodiments, the polynucleotide expressed in the plant is RNA, which can be single-stranded (ss) or double-stranded (ds) RNA or a combination of both.
In some embodiments, a first polynucleotide is provided (DNA or RNA or "ffj ·
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both) to a plant and a second polynucleotide having a 55CU5nCia C0rP65pülldldllle (Identical or complementary) to the first polynucleotide is subsequently expressed in the plant. In such modalities the polynucleotide expressed in the plant is a transcript of RNA that can be cRNA or dsRNA or a combination of both. In some embodiments where the polynucleotide is expressed by transient expression, a first polynucleotide is provided to a plant in the form of RNA or DNA or both RNA and DNA, and a second polynucleotide produced secondary is transiently expressed in the plant; In such embodiments, the first polynucleotide is one or more is selected from: (a) a single-stranded RNA molecule (cRNAs), (b) a single-stranded RNA molecule that self-hybridizes to form a double-stranded RNA molecule, (c) a double-stranded RNA molecule (dsRNA) , (d) a single-stranded DNA molecule (cDNA), (e) a single-stranded DNA molecule that self-hybridizes to form a double-stranded DNA molecule, (f) a single-stranded DNA molecule comprising a modified Pol III gene that is transcribed into an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule that comprises a modified Pol III gene that is transcribed to an RNA molecule, and (i) an RNA / DNA molecule hybrid double chain, or combinations thereof. In such modalities where the polynucleotide is expressed by transient expression the first polynucleotide may consist of naturally occurring nucleotides, such as nucleotides occurring in DNA and RNA. In such modalities where the polynucleotide is expressed by transient expression the first polynucleotide can be chemically modified, or comprises chemically modified nucleotides. The first polynucleotide is provided by suitable means known to one of skill in the art. Modalities include those where the first polynucleotide is chemically synthesized (eg, by in vitro transcription, such as transcription using a T7 polymerase or other polymerase), is produced by expression in a microorganism or in cell culture (such as plant cells or from cultivated insects in culture), is produced by expression in a plant cell or is produced by microbial fermentation. The first polynucleotide can be provided as a fragment of RNA or DNA. Alternatively, the first polynucleotide may be provided in more complex constructs, eg, as part of a recombinant expression construct, or included in a recombinant vector, eg, in a recombinant plant virus vector or in a recombinant baculovirus vector; Such recombinant expression constructs or vectors can be designed to include additional elements, such as expression cassettes to express a gene of interest (eg, an insecticidal protein).
In some embodiments the plant expressed polynucleotide is an RNA molecule and can be relatively short, such as single or double stranded RNA of between about 18 and about 300 or between about 50 and about 500 nucleotides (for RNA chain
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INSTITUTO MEXICANO BE LA PROPIEDAD 92 INDUSTRIAL simple) or between around 18 and around 300 or between around 50 and around 500 base pairs (for double-stranded RNA). Alternatively, the polynucleotide can be provided in more complex constructs, eg, as part of a recombinant expression construct, or included in a recombinant vector, eg, in a recombinant plant virus vector or in a baculovirus vector. recombinant. In some embodiments, these recombinant expression vectors or constructs are designed to include additional elements, such as expression cassettes to express a gene of interest (eg, an insecticidal protein).
The plant expressed polynucleotide has at least one segment of 18 or more contiguous nudeotides with a sequence of about 95% to about 100% identity with or complementary to a fragment of equivalent length of a target gene that is selected from the group consisting of the genes identified in the Diana gene sequence group. In one embodiment the plant expressed polynucleotide comprises at least a segment of 18 or more contiguous nudeotides that are essentially identical or complementary to a fragment of equivalent length of a target gene that is selected from the group consisting of the genes identified in the Group of Gen Diana Sequences. In some embodiments, contiguous nudeotides have a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity with or complementary to a fragment length equivalent of a target gene that is selected from the group consisting of genes identified in the Target Gene Sequence Group. In some embodiments, contiguous nudeotides are exactly (100%) identical or complementary to a fragment of equivalent length of a target gene that is selected from the group consisting of the genes identified in the Target Gene Sequence Group. In some embodiments, the plant expressed polynucleotide has an overall sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity with or complementarity to a fragment of a target gene that is selected from the group consisting of the genes identified in the Target Gene Sequence Group.
c) The polynucleotide expressed in the plant is generally designed to suppress one or more genes (target genes). Such target genes can include coding or non-coding sequences, or both. In specific embodiments, the plant expressed polynucleotide is designed to suppress one or more target genes that are selected from the group consisting of the genes identified in the Target Gene Sequence Group. In various embodiments, the plant-expressed polynucleotide is designed to delete one or more target genes that are selected from the group consisting of the genes identified in the Target Gene Sequence Group, and can be designed to delete multiple genes from this group, or to target different regions of one or more of
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MEXICAN INSTITUTE __ M THE PROPERTY
INDUSTRIAL these genes. In one embodiment, the polynucleotide expressed in<sup>1</sup> the 'plaritácompréndé mÜlEipl®' sections or segments each comprising at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with or complementarity to a fragment of a target gene which is selected from the group consisting of the genes identified in the Diana Gene Sequence Group. In such cases, each section may be identical or different in size or sequence, and may be sense or antisense relative to the target gene. For example, in one embodiment the plant expressed polynucleotide may include multiple tandem sections or repeating arrangements, where each section comprises at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% of identity with or complementarity to a fragment of a target gene that is selected from the group consisting of genes identified in the Target Gene Sequence Group; the segments may be from different regions of the target gene, for example, the segments may correspond to different regions of the exon of the target gene, and the spacer nucleotides that do not correspond to a target gene may optionally be used between or adjacent to the segments.
The total length of the plant expressed polynucleotide may be greater than 18 contiguous nucleotides, and may include nucleotides in addition to contiguous nucleotides that have the sequence of about 95% to about 100% identity with or complementarity to a fragment of a target gene that is selected from the group consisting of genes identified in the Target Gene Sequence Group. In other words, the total length of the polynucleotide expressed in the plant may be greater than the length of the section or segment of the polynucleotide designed to delete one or more target genes that are selected from the group consisting of the genes identified in the Sequence Group. by Gen Diana. For example, the plant expressed polynucleotide may have nucleotides flanking the active segment of at least one segment of 18 or more contiguous nucleotides that suppress the target gene, or include spacer nucleotides between the active segments, or may have additional nucleotides in the 5 'end, or at the 3' end, or both 5 'and 3' ends. In one embodiment, the plant expressed polynucleotide comprises additional nucleotides that are not specifically related (having a non-complementary or identical sequence) to the target gene that is selected from the group consisting of genes identified in the Target Gene Sequence Group eg nucleotides that provide a stabilizing secondary structure or for convenience in cloning or manufacturing. In one embodiment, the plant expressed polynucleotide comprises additional nucleotides located immediately adjacent to one or more contiguous 18 or more nucleotide segments with a sequence of about 95% to about 100% identity with or complementarity to a fragment in length equivalent of a target gene that is selected from the group consisting of the
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genes identified in the Diana Gene Sequence Group. In one embodiment, the plant expressed polynucleotide comprises such a segment, with an additional 5'G or an additional 3'C or both, adjacent to the segment. In another embodiment, the polynucleotide expressed in the plant is a double-stranded RNA that comprises additional nucleotides to form a surplus, eg, a dsRNA comprising 2 deoxyribonucleotides to form a 3 'surplus. Therefore in various modalities, the nucleotide sequence of the entire polynucleotide expressed in the plant is not 100% identical or complementary to a contiguous nucleotide fragment in the target gene that is selected from the group consisting of the genes identified in the Group. of Gen Diana Sequences. For example, in some embodiments the plant expressed polynucleotide comprises at least two contiguous 21 nucleotide segments with a sequence of 100% identity with or 100% complementarity to a fragment of a target gene that is selected from the group consisting of genes identified in the Diana Gene Sequence Group, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in a different order than the order in which the corresponding fragments occur in the target gene that is selected from the group consisting of the genes identified in the Target Gene Sequence Group.
In a related aspect, this invention relates to the plant having improved resistance to an infestation with the Leptinotarsa species, provided by expressing in the plant at least one polynucleotide comprising at least a segment of 18 or more contiguous nucleotides that are essentially identical or complementary to a fragment of equivalent length of a target gene that is selected from the group consisting of genes identified in the Target Gene Sequence Group, whereby the resulting plant has improved resistance to an infestation with the Leptinotarsa species when compared to a control plant where the polynucleotide is not expressed. In a related aspect, this invention relates to the plant having improved resistance to an infestation with the Leptinotarsa species, provided by expressing in the plant at least one polynucleotide comprising at least a segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with or complementary to a fragment of a target gene that is selected from the group consisting of the genes identified in the Diana Gene Sequence Group, whereby the resulting plant has improved resistance to an infestation with the Leptinotarsa species when compared to a control plant where the polynucleotide is not expressed. One modality is a nightshade plant that has improved resistance to an infestation and the Leptinotarsa species when compared to a control plant, provided by expressing in the plant an RNA that has a sequence that is selected from the group consisting of SEQ ID NO: 831 - 1085 and 1095. In yet another aspect, this invention relates to seeds or spreadable parts (especially transgenic progeny seed
TO
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or spreadable parts) produced by the plant that has improved resistance to an infestation with the Leptinotarsa species, as provided by this method. Also contemplated is a commodity produced by the plant that has improved resistance to an infestation with the Leptinotarsa species, as provided by this method, and a commodity produced from the seed of transgenic progeny or propagible parts of such a plant.
Methods to control infestations of the Leptinotarsa species of a plant
Various embodiments relate to a method of controlling an infestation with the Leptinotarsa species of a plant comprising contacting the Leptinotarsa species with a polynucleotide comprising at least one segment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment. of equivalent length of a DNA of a target gene that is selected from the group consisting of the genes identified in the Target Gene Sequence Group. In this context, control includes an incentive for a physiological or behavioral change in a Leptinotarsa species (adult or larvae) such as, but not limited to, growth weakness, increased mortality, reduction in reproductive capacity, reduction in or cessation of movement, or feeding behavior, or reduction in or cessation of development in the metamorphosis stage. In one embodiment, the method of controlling an infestation with the Leptinotarsa species of a plant comprises contacting the Leptinotarsa species with a polynucleotide comprising at least one segment that is identical or complementary to at least 21 contiguous nucleotides of a target gene that is select from genes identified in the Target Gene Sequence Group or an RNA transcribed from the target gene. In some embodiments, the polynucleotide is a double-stranded RNA. In some embodiments, the polynucleotide (eg, double-stranded RNA) is chemically synthesized or produced by expression in a microorganism or by expression in a plant cell. In some embodiments, the polynucleotide is a double-stranded RNA that comprises a strand that comprises a sequence that is selected from the Trigger Sequence Group. In one embodiment, the method of controlling an infestation with the Leptinotarsa species of a plant comprises contacting the Leptinotarsa species with an effective amount of a double-stranded RNA, a strand of which is complementary to at least 21 contiguous nucleotides of a gene encoding a ribosomal protein, where RNA interference is induced and mortality occurs. Target gene modalities are identified by name in Tables 1, 2, and 4 and include genes that have a sequence that is selected from the group consisting of the Target Gene Sequence Group, as well as related genes that include orthologs from related insect species, eg, related genes from other Leptinotarsa species, Triboiium species, or other related coleopteran genera. Examples of such related genes include the Triboiium genes
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castaneum listed in Table 1. In some embodiments the polynucleotide comprises at least a segment of 18 or more contiguous nucleotides that is essentially identical to or complements a fragment of a target gene that has a sequence that is selected from the group consisting of Group of Gen Diana Sequences. In some embodiments the polynucleotide comprises RNA having a sequence that is selected from the group consisting of SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or is an RNA hairpin encoded by a sequence that is selected from the group consisting of SEQ ID NO: 1105 1109. In some embodiments, the polynucleotide comprises a strand dsRNA having a sequence that is selected from the group consisting of the Trigger Sequence Group. In some embodiments, this invention provides a method of controlling an infestation with the Leptinotarsa species of a plant comprising contacting the Leptinotarsa species with an effective amount of a solution comprising a double stranded RNA, where at least one strand of the RNA double stranded is complementing at least 21 contiguous nucleotides of a gene encoding a ribosomal protein or a transcribed RNA of the gene, where the Leptinotarsa species is Leptinotarsa decemiineata, and where RNA interference is induced and Leptinotarsa decemiineata mortality occurs, and where the ribosomal protein is an L7 ribosomal protein or a protein encoded by SEQ ID NO: 730 or where strand RNA double comprises a sequence that is selected from the group consisting of SEQ ID NO: 989, 988, 1104, or 1105; In some embodiments, the solution further comprises one or more components that are selected from the group consisting of an organosilicone surfactant or a cationic lipid.
In some embodiments, contiguous nucleotides have a sequence of about 95%, about 96%, to about 97%, about 98%, about 99%, or about 100% identity with or complementary to a fragment of equivalent length of a target gene that is selected from the group consisting of genes identified in the Target Gene Sequence Group. In some embodiments, the contiguous nucleotides are exactly (100%) Identical or complementary to a fragment of equivalent length of a target gene that is selected from the group consisting of the genes identified in the Target Gene Sequence Group. In some embodiments, the polynucleotide has an overall sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% Identity with or complementary to a fragment length equivalent of a target gene that is selected from the group consisting of genes identified in the Target Gene Sequence Group. In one embodiment, the polynucleotide comprises at least one segment of 21 contiguous nucleotides with a sequence of 100% identity or complementarity to the corresponding fragment of a target gene that is selected from the group consisting of the genes identified in the Sequence Group of Gen Diana; in some embodiments, the polynucleotide comprises f
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MEXICAN INSTITUTE OF THE ttOiWAÍ; INDUSTRIAL neutral sequence (that does not have Identity or complementary to the target gene) in addition to a segment of 21 contiguous nucleotides with 100% Identity with the corresponding fragment of the target gene, and therefore the polynucleotide as a whole has much less sequence general Identity with a target gene.
The polynucleotide for use in this method is generally designed to delete one or more genes (target genes). The term gene refers to any part of a nucleic acid that provides for the expression of a transcript or encodes a transcript. A gene may include, but is not limited to, a promoter region, 5 'untranslated regions, regions that encode transcripts that can include intronic regions, 3' untranslated regions, or combinations of these regions. In some embodiments, the target genes can include coding or non-coding sequences, or both. In other embodiments, the target gene has an identical or complementary sequence to a messenger RNA, for example, in some embodiments the target gene is a cDNA. In specific embodiments, the polynucleotide is designed to delete one or more target genes that are selected from the group consisting of the genes Identified in the Target Gene Sequence Group. In various embodiments, the polynucleotide is designed to delete one or more target genes that are selected from the group consisting of the genes identified in the Target Gene Sequence Group, and can be designed to delete multiple target genes from this group, or to target to different strands of one or more of these target genes. In one embodiment, the polynucleotide comprises multiple contiguous 21 nucleotide segments with a sequence of 100% Identity with a fragment of equivalent length of a target gene or DNA that has a sequence that is selected from the Target Gene Sequence Group or the complement of DNA from this. In such cases, each segment may be Identical or different in size or sequence, and may be sense or antisense relative to the target gene. For example, in one embodiment the polynucleotide comprises multiple tandem segments or repeating arrangements, where each segment comprises 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with or complementary to a fragment of equivalent length from a target gene that is selected from the group consisting of genes Identified in the Target Gene Sequence Group; the segments may be from different regions of the target gene, for example, the segments may correspond to different regions of the exon of the target gene, and the spacer nucleotides that do not correspond to a target gene may be used optionally between or adjacent to the segments.
The total length of the polynucleotide for use in this method may be greater than 18 contiguous nucleotides, and may Include nucleotides in addition to contiguous nucleotides that have the sequence of about 95% to about 100% Identity with or complement a fragment of equivalent length of a target gene that is selected from the group consisting of
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genes identified in the Diana Gene Sequence Group. In other words, the total length of the polynucleotide may be greater than the length of the section or segment of the polynucleotide designed to delete one or more target genes that are selected from the group consisting of the genes Identified in the Target Gene Sequence Group. For example, the polynucleotide may have nucleotides flanking the active segment of at least one segment of 18 or more contiguous nucleotides that suppress the target gene, or include spacer nucleotides between the active segments, or they may have additional nucleotides at the 5 'end, either at the 3 'end, or at both the 5' and 3 'ends. In one embodiment, the polynucleotide may include additional nucleotides that are not specifically related (having a non-complementary or identical sequence) to the target gene that is selected from the group consisting of the genes Identified in the Target Gene Sequence Group, for example nucleotides that provide a secondary stabilizing structure or for convenience in cloning or manufacturing. In one embodiment, the polynucleotide may include additional nucleotides located immediately adjacent to one or more segments of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% Identity with or complementary to a fragment of equivalent length of a target gene that is selected from the group consisting of the genes Identified in the Target Gene Sequence Group. In one embodiment, the polynucleotide comprises such a segment, with an additional 5'G or an additional 3'C, or both, adjacent to the segment. In another embodiment, the polynucleotide is a double-stranded RNA that comprises additional nucleotides to form a surplus, for example, a dsRNA comprising 2 deoxyribonucleotides for for. Therefore in various modalities, the nucleotide sequence of the entire polynucleotide is not 100% identical or complementary to a contiguous nucleotide sequence in the target gene that is selected from the group consisting of the genes identified in the Gene Sequence Group Diana. For example, in some embodiments the polynucleotide comprises at least two segments each of 21 contiguous nucleotides with a sequence of 100% identity with a fragment of equivalent length of the target gene, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in a different order than the corresponding fragments occur in DNA having a sequence that is selected from, or the DNA complement of, the Gen Diana Sequence Group.
The polynucleotide for use in this method is provided by suitable means known to one of skill in the art. Modalities include those where the polynucleotide is chemically synthesized (eg, by in vitro transcription, such as transcription using a T7 polymerase or other polymerase), produced by expression in a microorganism or in cell culture (such as plant cells or Insect grown in culture), produced by expression in a plant cell, or produced by microbial fermentation.
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In some embodiments, the polynucleotide for use in this "se-jJFüpdrdUlia method as an isolated DNA or RNA fragment. In some embodiments, the polynucleotide for use in this method is not part of an expression construct and does not have additional elements such as a promoter or termination sequences). These polynucleotides can be relatively short, such as single or double chain polynucleotides of between about 18 and about 300 or between about 50 and about 500 nucleotides (for single chain polynucleotides) or between about 18 and about 300 or between about 50 and about 500 base pairs (for double chain polynucleotides). In some embodiments, the polynucleotide is a dsRNA of between about 100 and about 500 base pairs, such as a dsRNA the length of any of the dsRNA triggers described in Tables 3, 5, 8, 9, and 10. Modalities Include those where the polynucleotide is a dsRNA comprising a segment that has a sequence that is selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof, or where the polynucleotide is an RNA hairpin encoded by a sequence selected from the group consisting of SEQ ID NO: 1105-1109. Alternatively, the polynucleotide can be provided in more complex constructs, eg, as part of a recombinant expression construct, or included in a recombinant vector, eg, in a recombinant plant virus vector or in a baculovirus vector. recombinant. In some embodiments, these recombinant expression vectors or constructs are designed to include additional elements, such as expression cassettes to express a gene of interest (eg, an insecticidal protein).
In various embodiments of the method, contacting comprises the application to a surface of the Leptinotarsa species of a suitable composition comprising the polynucleotide for use in this method; Such a composition can be provided, for example, as a solid, liquid (including homogeneous mixtures as solutions and non-homogeneous mixtures as suspensions, colloids, micelles and emulsions), powder, suspension, emulsion, spray, encapsulated or microencapsulation formulation, within or on top of microbeads or other carrier particles, in a film or coating, or in or on a matrix, or as a seed treatment. The contact may be in the form of a seed treatment or in the form of a treatment of seed potato tubers or pieces of tuber (eg, by soaking, coating, or dusting the seed potato). Binders, inert carriers, suitable surfactants, and the like may optionally be included in the composition, as is known to one skilled in pesticide formulation and seed treatment. In some embodiments, contacting comprises providing the polynucleotide in a composition further comprising one or more components that are selected from the group consisting of a carrier agent, a surfactant, a cationic lipid (such as that described in Example 18 of the patent application publication
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100 US 2011/0296556, incorporated herein rrrririULI 'L.LJ iducnda), an organosilicone, an organosilicone surfactant, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, protector and regulator . In embodiments, contacting comprises providing the polynucleotide in a composition further comprising at least one pesticidal agent that is selected from the group consisting of a patatlin, a plant lectin, a fltoecdlsteroid, an Insecticidal protein Bacillus thuringiensis, an Insecticidal protein Xenorhabdus , a Photorhabdus Insecticidal Protein, a Bacillus laterosporous Insecticidal Protein and a Bacillus sphaericus Insecticidal Protein. In one embodiment, contacting comprises providing the polynucleotide in a composition that can be ingested or otherwise internally absorbed by the Leptinotarsa species.
The combination of certain polynucleotides for use in this method (for example, the polynucleotide triggers described in the Working Examples) with one or more non-polynucleotide pesticidal agents is anticipated to result in a smarter improvement in the prevention or control of infestations of the Leptinotarsa species, when compared with the effect obtained with the polynucleotide alone or the non-polynucleotide pesticide agent alone. In one embodiment, it was found that a composition containing one or more polynucleotides and one or more non-polynucleotide pesticidal agents that are selected from the group consisting of a potato ,. a plant lectlna, a fltoecdlsteroide, an insecticidal protein Bacillus thuringiensis, an insecticidal protein Xenorhabdus, an insecticidal protein Photorhabdus, an insecticidal protein Bacillus laterosporous, and an insecticidal protein Bacillus sphaericus, effect a prevention or control of lesser species of Leesta.
Methods for selecting target genes
Another aspect of the present invention provides a method for the non-random selection of target genes for RNA-mediated spleen. In one embodiment, the method provides a subset of the target genes that is present in a single or low couplet number (non-repeating and non-redundant) in a particular genome. These target genes can be genes from a plant genome or genes from an animal genome. In some embodiments, the target genes are genes from an Invertebrate pest, for example, an Invertebrate pest from a plant or an Invertebrate pest from a vertebrate. In some embodiments, the target genes are genes from an insect pest or a plant nematode pest. In some embodiments, the target genes are genes of a Leptinotarsa species. Other aspects include making a polynucleotide (eg, a cDNA or dsRNA trigger, such as the cDNA triggers described in the Working Examples or a recombinant DNA construct useful for making transgenic plants) based on
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101 INDUSTRIAL target genes for RNA-mediated targeting selected by any of the methods described herein.
In one embodiment, the method comprises the step of Identifying low or single copy number genes in the chosen genome or alternatively identifying low or single copy number genes in an orthologous database of related organisms to predict which genes will be copla only / low in the chosen organism. Low-coupled genes, and in particular single-coupled genes, are selected as targets for RNA-mediated targeting. In one embodiment, low or simple couplet number gene identification is performed by sequence comparison between a set of genes from a first species and a set of genes from a second species, where the set of genes from a second species is Has identified as low or simple copy number in the second species. In one embodiment, the identification of low or simple couplet number genes is performed by applying a computer-made algorithm to a set of genes from a first species to identify a subset of low or single couplet number genes in the set of genes of the first species, then comparing a set of genes from a second species to the subset of low or single couplet number genes from the first species to identify corresponding low or single couplet number genes from the second species. The low or single couplet number genes of the second species are useful as target genes for RNA-mediated splenation; the sequences of these target genes are used to design polynucleotides (eg, a cDNA or dsRNA trigger, such as the dsRNA triggers described in the Working Examples, or recombinant DNA constructs to make transgenic plants) and methods for use in the prevention or control of infestations of the second species.
Method modalities include an additional step of estimating nucleotide diversity for single / low-coupled genes in a population of the chosen organism and selecting those low / single-coupled genes that also have the least nucleotide diversity. Low / single copy genes that also have low nucleotide diversity are selected as targets for RNA-mediated targeting.
Method Modalities Include an additional step of comparing the ratio of synonymous nucleotide changes (K<sub>s</sub>) to no synonyms (K<sub>to</sub>) as an estimate of functional restriction or evolution. In one embodiment, the method comprises the step of selecting genes where K<sub>s</sub>is at least equal to or greater than K<sub>to</sub>. In one embodiment, the method comprises the step of selecting genes where K<sub>s</sub> >> K<sub>to</sub>.
A related aspect of the present invention is a set of target genes for identified RNA-mediated spleen identified from a genome by any of the gene selection methods described herein. One modality refers to a set of target genes
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MEXICAN INSTITUTE 1 m FROM THE PROPERTY <sup>iUZ</sup> INDUSTRIAL for RNA-mediated silencing that is selected from a llití Id Idtíi no ^ ύύι iüe single or low couplet number target geneset of a larger set of genes from that genome. One embodiment refers to a set of target genes for RNA mediated silencing that is selected from an invertebrate genome by identifying single or low copy number target genes from a larger set of genes from that invertebrate genome. A specific modality refers to a set of target genes for RNA-mediated targeting in a Leptinotarse species that is selected from a Leptinotarse genome by identifying single or low couplet number target genes from a larger set of genes from that genome. Leptinotarsa. A specific modality refers to a set of target genes for RNA mediated silencing in a Leptinotarsa species that is selected from a Leptinotarsa genome by identifying single or low copy number target genes from a larger set of genes from that genome. Leptinotarse, where the set of sequences is the group consisting of SEQ ID NO: 1-725 or its DNA complement.
Related aspects of the present invention are methods and compositions using the target gene set consisting of SEQ ID NO: 1-725, or the DNA complement thereof. These include: (i) a method of controlling an infestation with Leptinotarsa species from a plant comprising contacting the Leptinotarsa species with a polynucleotide comprising at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with a segment of equivalent length of a DNA having a sequence selected from the group consisting of: SEQ ID NO: 1-725, or the DNA complement thereof; (I) a method of controlling a Leptinotarsa species infestation of a plant comprising providing in the diet of a Leptinotarsa species an agent comprising a polynucleotide having at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% identity with a segment of equivalent length of a DNA that has a sequence that is selected from the group consisting of: SEQ ID NO: 1-725, or the DNA complement of these, where the agent works after the Ingestion by the Leptinotarsa species to Inhibit a biological function within the Leptinotarsa species thus controlling the infestation with the Leptinotarsa species ·, (iii) a method of causing mortality or weakening in Leptinotarsa species larvae comprising providing in the diet of Leptinotarsa species larvae at least one recombinant RNA comprising at least one silencing element essentially identical or essentially complementary to a target gene from the Leptinotarsa species larva, where the target gene sequence is selected from the group consisting of SEQ ID NO: 1-725; (V) a method of providing a plant having improved resistance to an infestation with the Leptinotarsa species comprising applying topically to the plant a composition comprising at least one polynucleotide having the
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minus a segment of 18 or more contiguous nudeotides with a sequence of about Jé y5%? r about 100% identity with a segment of DNA equivalent length that has a sequence that is selected from the group consisting of: SEQ ID NOs : 1-725, or the DNA complement thereof; (v) a composition for controlling a Leptinotarsa species comprising at least one recombinant polynucleotide comprising at least one segment of 18 or more contiguous nudeotides that is essentially identical or complementary to a segment of equivalent length of DNA having a sequence that is select from the group consisting of SEQ ID NOs: l 725; (vi) a method of providing a plant having improved resistance to an infestation with the Leptinotarsa species comprising expressing in the plant at least one polynucleotide comprising at least a segment of 18 or more contiguous nudeotides that is essentially identical or complementary to a equivalent length segment of a DNA having a sequence that is selected from the group consisting of SEQ ID NO: 1-725; (vii) a recombinant DNA construct comprising a heterologous promoter operably linked to a DNA comprising at least a segment of 18 or more contiguous nudeotides with a sequence of about 95% to about 100% identity with a length segment DNA equivalent having a sequence selected from the group consisting of: SEQ ID NO: 1-725, or the DNA complement thereof; and (viii) a transgenic solanaceous plant cell that has in its genome an RNA that encodes recombinant DNA that suppresses the expression of a target gene in a Leptinotarsa species that contacts or ingests the RNA, where the RNA comprises at least one element. silencing complementary to the target gene and wherein the sequence of the target gene is a sequence selected from the group consisting of: SEQ ID NO: 1-725, or the complement thereof.
Another modality refers to a set of target genes for RNA mediated silencing that is selected from a genome by estimating the diversity of nudeotides for a given set of genes in a population of individuals of the species that has that genome and selecting genes with the least diversity of nudeotides. One modality refers to a set of target genes for RNA mediated silencing that is selected from an invertebrate genome by estimating the diversity of nudeotides for a given set of genes in a population of invertebrate individuals that has that genome and select genes with the least diversity of nudeotides. Another modality refers to a set of target genes for RNA mediated silencing that is selected from an invertebrate genome by estimating nudeotide diversity for a low / single copy gene in a population of invertebrate individuals that has that genome. and select the low / single couplet genes that also have the least diversity of nudeotides.
Another modality refers to a set of target genes for mediated silencing.
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104 by iRNA that is selected from a genome by comparing the ratio of synonymous nucleotide changes (K<sub>s</sub>) to no synonyms (K<sub>to</sub>) in genes of that genome and the selection of genes where K<sub>s</sub> is at least equal to or greater than K<sub>to</sub>. In one embodiment, the set of target genes for iRNA-mediated silencing are genes where K<sub>s</sub> is at least equal to or greater than K<sub>to</sub>. In one embodiment, the set of target genes for iRNA mediated silencing are genes where<sub>K</sub>s »<sub>K</sub>to. One modality refers to a set of target genes for iRNA mediated silencing of an Invertebrate genome and where K<sub>s</sub> >> K<sub>to</sub> for the selected genes.
In one embodiment, the single or low copy number target genes are a subset of target genes from a first invertebrate species that is selected from a larger set of genes from the first invertebrate species, where selection is by a computerized sequence comparison between the largest gene set of the first invertebrate species and a gene set of a second invertebrate species that were identified as single or low copy number in the second species of invertebrates. In a specific embodiment, the single or low copy number target genes are a subset of the Leptinotarsa decemiineata target genes that is selected from a larger set of Leptinotarsa decemiineata target genes, where selection is by a computer-based sequence comparison between a larger set of Leptinotarsa decemiineata target genes and a set of genes from a second invertebrate species that were identified as single or low copy number in the second species of invertebrates. The single or low copy number target genes of Leptinotarsa decemiineata that are selected by the method are particularly useful for making polynudeotides of the present invention, Including useful recombinant DNA constructs, for example, to provide plants that have increased resistance to infestation. with Leptinotarsa species and useful isolated recombinant RNA molecules, for example, to make compositions for the topical treatment of a Leptinotarsa plant or species to provide prevention or control of Leptinotarsa species infestations. In one embodiment, the Leptinotarsa decemiineata single or low copy number target genes that are selected by the method are genes that have a sequence that is selected from the group consisting of SEQ ID NO: 1-725.
A further aspect of the present invention are polyclonal or monoclonal antibodies that bind to a protein encoded by a sequence or a fragment of a sequence that is selected from the group consisting of the Diana Gene Sequence Group, and polyclonal or monoclonal antibodies that are bind to a protein encoded by a sequence or a fragment of a sequence that is selected from the Trigger Sequence Group, or the complement thereof; These antibodies are made by routine methods known to one skilled in the art, for example using routine protocols as described in Antibody.
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105
Methods and Protocols (Proetzel and Ebersbach, editors, 2012, ITtlllldlld Puass, Nucuu Yerk) or Making and Using Antibodies (Howard and Kaser, editors, 2006, CRC Press, Boca Raton).
Selection of effective polynucleotides by overlap
The polynucleotides for use in the embodiments described herein need not be the full length of a target gene, and in many embodiments are much shorter than the target gene. An example of a technique that is useful for selecting effective polynucleotides is the overlapping or evaluation of polynucleotides that correspond to adjacent or partially overlapping segments of a target gene.
In some embodiments, effective polynucleotide triggers can be identified by overlapping target genes on fragments of selected length, eg, fragments 200-300 nucleotides in length, with partially overlapping regions, eg, around 25 nucleotides, together with the length of the target gene. In some embodiments, the polynucleotide trigger sequences are designed to correspond to (have a nucleotide identity or complementarity with) regions that are unique to the target gene. In some embodiments, the selected region of the target gene may include a coding sequence or non-coding sequence (eg, promoter regions, 3 'untranslated regions, introns, and the like), or a combination of both.
When it is of interest to design an efficient target to suppress multiple target genes, the sequences of multiple target genes are aligned and the polynucleotide triggers are designed to correspond to regions with high sequence homology in common between the multiple targets. In contrast, when it is of interest to design an efficient target to selectively suppress one among multiple target sequences, the sequences of multiple target genes are aligned and polynucleotide triggers are designed to correspond to regions with no sequence homology or with low homology of common sequence between multiple targets.
Thermodynamic considerations in the selection of effective polynucleotides
In some embodiments, polynucleotide triggers can be designed or their sequence can be optimized using thermodynamic considerations. For example, polynucleotide triggers can be selected according to thermodynamics that control hybridization between one nucleic acid chain (eg, a polynucleotide trigger or a single siRNA) and another (eg, a transcript of a target gene).
Methods and algorithms for predicting nucleotide sequences that are likely to be effective in iRNA mediated silencing of a target gene are known in the art. Non-exhaustive examples of such methods and algorithms include i ratings, described
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Allowed pairing errors
Essentially identical or essentially complementary means that a polynucleotide (or at least one strand of a double-stranded polynucleotide) has sufficient identity or complementarity to the target gene or to the transcribed RNA of a target gene (eg, transcription) to suppress the expression of a target gene (eg, to effect a reduction in transcription levels or activity of the target gene and / or encoded protein). Polynucleotides as described herein need not have 100 percent identity or complementarity to a target gene or to the transcribed RNA of a target gene to suppress expression of the target gene (eg, to effect a reduction in levels or transcription activity of the target gene or encoded protein, or to provide control of a Leptinotarsa species}. In some embodiments, the polynucleotide or part thereof is designed to be essentially identical to, or essentially complementary to, a sequence of at least 18 or 19 contiguous nucleotides in the target gene or RNA transcribed from the target gene. In some embodiments, the polynucleotide or a portion thereof is designed to be 100% identical to, or 100% complementary to, one or more contiguous 21 nucleotide sequences in the target gene or the transcribed RNA of the target gene. In certain embodiments, an essentially identical polynucleotide has 100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent sequence identity when compared to the sequence of 18 contiguous nucleotides or more, in either the endogenous target gene or an RNA transcribed from the target gene. In certain embodiments, an essentially complementary polynucleotide has 100 percent sequence complementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent sequence complementarity when compared to the sequence of 18 contiguous nucleotides or more, in either the target gene or an RNA transcribed from the target gene.
Polynucleotides that contain mismatches to the target gene or transcription can be used in certain embodiments of the compositions and methods described herein. In some embodiments, the polynucleotide includes at least 18 or at least 19 or at least 21 contiguous nucleotides that are essentially identical or essentially complementary to a segment of equivalent length in the target gene or transcription of the target gene. In certain embodiments, a polynucleotide of 21 or more contiguous nucleotides that is
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107 essentially identical or essentially complementary to a pnuivalent Innnitnd segment in the target gene or the transcription of the target gene may have 1 or 2 mismatches to the target gene or transcription (i.e. 1 or 2 mismatches between the 21 contiguous polynucleotide nucleotides and the segment of equivalent length in the target gene or the target gene transcript). In certain embodiments, a polynucleotide of about 50, 100, 150, 200, 250, 300, 350 or more nucleotides that contains an extension of 21 contiguous nucleotides of identity or complementarity to a segment of equivalent length in the target gene or transcript the target gene may have 1 or 2 or more mismatches to the target gene or transcript.
When designing polynucleotides with mismatches to an endogenous target gene or to a transcribed RNA of the target gene, mismatches of certain types and at certain positions can be used that are more likely to be tolerated. In certain embodiments, mismatches formed between adenine and cltosine residues or guanosine and uracil are used as described in Du et al. (2005) Nudeic Acíds Res., 33: 1671-1677. In some embodiments, mating errors in overlapping regions of 19 base pairs are located at positions 5, 7, 8, or 11 of low tolerance (from the 5 'end of a 19 nucleotide target), at positions 3, 4 , and 12-17 of medium tolerance (from the 5 'end of a 19 nucleotide target) and / or in positions of high tolerance at either end of the complementary line, that is, at positions 1, 2, 18 and 19 (from the 5 'end of a 19 nucleotide target) as described in Du et al. (2005) Nudeic Adds Res., 33: 1671-1677. Empirically tolerated mating errors can be determined by routine tests, for example, in vitro feeding tests on Leptinotarsa species larvae.
Inlay of mute elements in neutral sequence
In some embodiments, a silencing element comprising a sequence that corresponds to the target gene and that is responsible for an observed deletion of the target gene is embedded in the neutral sequence, that is, it is inserted into additional nucleotides that do not have identity or complementarity of sequence to the target gene. The neutral sequence may be desirable, for example, to increase the total length of a polynucleotide. For example, it might be desirable for a polynucleotide to be of a particular size for reasons of stability, cost-effectiveness of manufacture, or biological activity. In some embodiments, the neutral sequence is also useful for forming the loop in a hairpin trigger or as a spacer between trigger regions.
In another kleoptera species, Diabrotica virgifera, cDNAs greater than or equal to approximately 60 base pairs (bp) have been reported to be required for biological activity in artificial food bioassays; see Bolognesi et al. (2012) PLoS ONE 7 (10): e47534. doi: 10.1371 / journal.pone.0047534. Therefore, in one embodiment, an element of
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108 silencing of 21 base pair dsRNA corresponding to a target gene in Table 1 and pl? was found to provide control of an infestation with Leptinotarse Embedded in a neutral sequence of another 39 base pairs, thereby forming a polynucleotide of about 60 base pairs. In some embodiments, the dsRNA trigger includes a neutral sequence of between about 60 and about 500 or between 100 and about 450 base pairs, where at least one segment of 21 nucleotides contiguous to a sequence of 100% is embedded. Identity or 100% complementarity to a fragment of equivalent length of a target gene that has a sequence that is selected from the group consisting of SEQ ID NO: 1-725 and SEQ ID NO: 726 830 and SEQ ID NO: 1087-1094 . In another embodiment, a single 21 base pair silencing element with a sequence of 100% identity or 100% complementarity to a fragment of equivalent length of a target gene is found to be effective when embedded in larger sections of neutral sequence, for example, where the total polynucleotide length is from about 60 to about 300 base pairs. In another embodiment, at least a segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of: or the complement of SEQ ID NO: 831-1085, 1095-1104, and 1110-1114 Embed in larger sections of neutral sequence to provide an efficient polynucleotide. In another embodiment, the segments of multiple sequences (or multiple copies of a segment of one or more sequences) that are selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the In addition to this, they are embedded in larger sections of neutral sequence to provide an efficient polynucleotide. In modalities where the pollnucleotide includes neutral sequence strings, the pollnucleotide will have a relatively low overall sequence identity compared to the target gene; for example, a cDNA with a total length of 210 base pairs, containing a single trigger of 21 base pairs (100% Identity or complementary to a 21 nucleotide fragment of a target gene) Embedded in another 189 pairs of Neutral sequence bases will have a total sequence identity with the target gene of about 10%.
Insecticidal double-stranded RNA molecules
Another aspect of the present invention provides an Insecticidal double-stranded RNA molecule that causes mortality or growth impairment in a Leptinotarse species when it is ingested or contacted by I specle Leptinotarse, wherein the insecticidal double-stranded RNA molecule comprises at least one segment of 18 or more contiguous nucleotides that is essentially Identical or essentially complementary to a segment of equivalent length of a target gene or DNA (cDNA) having a sequence that is selected from the Gen Diana Sequence Group. In some embodiments, the insecticidal double-stranded RNA molecule
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109 has between 50 and around 500 base pairs rip jnngitiiH rn <sub>3</sub>ig.πα ^ -. ιιγι ^^ the insecticidal double-stranded RNA molecule comprises at least one segment of at least 30 contiguous nucleotides in length. In some embodiments, the Insecticidal double-stranded RNA molecule comprises multiple segments of 18 or more contiguous nucleotides that are essentially identical or essentially complementary to a segment of equivalent length of a target gene or DNA (cDNA) having a sequence that is selected of the Target Gene Sequence Group, where the segments are from different regions of the target gene (for example, the segments may correspond to different exon regions of the target gene and the spacer nucleotides that do not correspond to a target gene may optionally be used between or adjacent to the segments) or are from different target genes. In some embodiments, the Insecticidal double-stranded RNA molecule comprises multiple segments of 18 or more contiguous nucleotides that are essentially identical to or essentially complement one segment of equivalent length of a target gene or DNA (cDNA) that has a sequence that is selected from the Diana Gen Sequence Group, where the segments are from different regions of the target gene and are arranged on the insecticidal double-stranded RNA molecule in a different order than the order in which the segments naturally occur in the target gene. In some embodiments, the insecticidal double-stranded RNA molecule comprises multiple segments each of 21 contiguous nucleotides with a sequence of 100% identity or 100% complementarity to a segment of equivalent length of a target gene or DNA (cDNA) that has a sequence that is selected from the Diana Gene Sequence Group, where the segments are from different regions of the target gene and are arranged on the insecticidal double-stranded RNA molecule in a different order than the order in which the segments naturally occur in the target gene. In some embodiments, the insecticidal double-stranded RNA molecule comprises a strand that comprises a sequence that is selected from the group consisting of: SEQ ID NO: 831-1085, 1095-1104, and 1110-1114, or the complement thereof , or comprises an RNA hairpin encoded by a sequence selected from the group consisting of SEQ ID NO: 1105-1109. In some embodiments, the insecticidal double-stranded RNA comprises a stranded dsRNA having a sequence that is selected from the group consisting of the Trigger Sequence Group. The insecticidal double-stranded RNA molecule can be applied topically to a plant, especially a nightshade plant such as tomato, eggplant, or potato, to control or prevent infestation with a Leptinotarsa species. The insecticidal double-stranded RNA molecule can be provided in a form suitable for ingestion or direct contact by a Leptinotarsa species, eg, in the form of a spray or powder or bait. Other suitable methods and compositions for providing the insecticidal double-stranded RNA molecule are similar to those described in the preceding paragraphs for other aspects of the present invention.
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110
IMPI fNSTITVTO MEXICANO Ofi LA PROHCDAP INDUSTRIAL
Various embodiments relate to a tank mixture comprising one or more insecticidal polynucleotides and water or another solvent, optionally including a cationic lipid or an organosilicone surfactant or both. Modalities include polynucleotide tank mix formulations and optionally at least one pesticidal agent that is selected from the group consisting of a patatin, a plant lectin, a phytoecysteroid, an insecticidal protein Bacillus thuríngiensis, an insecticidal protein Xenorhabdus, an insecticidal protein Photorhabdus , an insecticidal protein Bacillus taterosporous and an insecticidal protein Bacillus sphaerícus. Modalities of such compositions include those where one or more insecticidal polynucleotides are provided in a living or dead microorganism such as a bacterial or fungal or yeast cell, or are provided as a microbial fermentation product or provided in a living plant cell. either killed or provided as a synthetic recombinant polynucleotide. In one embodiment, the composition includes a non-pathogenic strain of a microorganism containing a polynucleotide as described herein; ingestion or absorption of the microorganism results in the weakening or mortality of the Leptinotarsa species; Non-exhaustive examples of suitable microorganisms include E. coii, B. thuríngiensis, Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Serraría entomophiia and species Serratia sp., B. sphaerícus, B. cereus, B. iaterosporus, B. popilliae, related Ciostrídium bifermentans and other Ciostrídium species, or other gram positive spore forming bacteria. In one embodiment, the composition includes a plant virus vector comprising a polynucleotide as described herein; feeding by a Leptinotarsa species with a plant treated with a plant virus vector results in weakening or mortality of the Leptinotarsa species. In one embodiment, the composition includes a baculovirus vector that includes a polynucleotide as described herein; ingestion or absorption of the vector results in the weakening or mortality of the Leptinotarsa species. In one embodiment, a polynucleotide as described herein is encapsulated in a synthetic matrix such as a polymer or bound to particles and applied topically to the surface of a plant; feeding a Leptinotarsa species with a topically treated plant results in weakening or mortality of the Leptinotarsa species. In one embodiment, a polynucleotide as described herein is provided in the form of a plant cell (eg, a transgenic solanaceous plant cell of the present invention) that expresses the polynucleotide; ingestion of the plant cell or plant cell contents by a Leptinotarsa species results in the weakening or mortality of the Leptinotarsa species.
In some embodiments, one or more polynucleotides as described herein are provided with adhesives and humectants necessary for effective foliar coverage as well as UV protectors to protect polynucleotides such as dsRNA from UV damage.
<img file="MX359191B_D0115.tif" />
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MEXICAN INSTITUTE OF PROPERTY lll industrial
Such additives are commonly used in the bioinsecticide industry and are known to a person skilled in the art. Compositions for soil application may include granular formulations that serve as a bait for Leptinotarsa species larvae. In some embodiments, one or more polynucleotides as described herein are also provided with a carrier agent, a surfactant, a cationic lipid (such as that described in Example 18 of US Patent Application Publication 2011/0296556. , Incorporated herein by this reference), an organosilicone, an organosilicone surfactant, a a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a protector and a regulator of insect growth. In some embodiments, the composition also includes at least one pesticidal agent that is selected from the group consisting of a patatin, a plant lectin, a phytoecysteroid, an insecticidal protein Bacillus thuríngiensis, an insecticidal protein Xenorhabdus, an insecticidal protein Photorhabdus, a protein Bacillus iaterosporous insecticide and a Bacillus sphaericus insecticidal protein.
These compositions are applied in any conventional way, for example, by spraying or dusting the Leptinotarsa species directly, or by spraying or dusting a plant or environment where prevention or control of infestation with that Leptinotarsa species is desired, or by applying a coating to a surface of a plant, or by applying a coating to a seed (or seed potato) in preparation for planting the seed, or by soaking the soil around the roots of a plant for which prevention or control of infestation with that Leptinotarsa species is desired.
An effective amount of a polynucleotide as described herein is an amount sufficient to provide control of the Leptinotarsa species, or to avoid infestation with the Leptinotarsa species], determination of effective amounts of a polynucleotide is performed using routine assays such as those described in Examples 5 and 6. While there is no upper limit to the concentrations and dosages of an insecticidal polynucleotide that may be useful in the methods and compositions provided herein, lower effective concentrations and dosages will generally be sought for purposes of efficacy and economy. Non-exhaustive modalities of effective amounts of a polynucleotide include a range from about 10 nanograms per milliliter to about 100 micrograms per milliliter of a polynucleotide in liquid spray form for a plant, or from about 10 milligrams per acre to about 100 grams per acre of polynucleotide applied to a field of plants, or from about 0.001 to about 0.1 micrograms per milliliter of polynucleotide in an artificial diet to feed the Leptinotarsa species. When polynucleotides as described herein are applied topically to a plant, the concentrations can be adjusted in consideration of the spray or treatment volume applied to plant leaves or surfaces
<img file="MX359191B_D0116.tif" />
112
IMPI ~ from other parts of plants, such as petals, stems, tubers, fnd-ac ant-ora<sub>C |</sub> pni ^ n hniag raírpg n flower seeds. In one embodiment, a useful treatment for herbaceous plants using 25-mer pollnucleotides as described herein is about 1 nanomol (nmol) of pollnucleotides per plant, eg, about 0.05 to 1 nmol of pollnucleotides per plant . Other modalities for herbaceous plants include useful ranges from about 0.05 to about 100 nmol, or from about 0.1 to about 20 nmol, or from about 1 nmol to about 10 nmol of pollnucleotides per plant. In certain embodiments, about 40 to about 50 nmol of a cDNA polynucleotide are applied. In certain modalities, about 0.5 nmol to about 2 nmol of dsRNA is applied. In certain embodiments, a composition containing about 0.5 to about 2.0 milligrams per milliliter, or about 0.14 milligrams per milliliter of a cDNA or cDNA (21-mer) is applied. In certain embodiments, a composition of from about 0.5 to about 1.5 milligrams per milliliter of a dsRNA polynucleotide of the present invention of about 50 to about 200 or more nucleotides is applied. In certain embodiments, from about 1 mole to about 5 nmol of dsRNA of the present invention are applied to a plant. In certain embodiments, the composition of pollnucleotides as topically applied to the plant contains at least one polynucleotide of the present invention at a concentration of from about 0.01 to about 10 milligrams per milliliter, or from about 0.05 to about 2 milligrams per milliliter, or from about 0.1 to about 2 milligrams per milliliter. Very large plants, trees, or vines may require higher amounts of polynucleotides due to their size. When using large dsRNA molecules of the present invention that can be processed as multiple ollgonucleotides (eg, multiple triggers encoded by a single recombinant DNA molecule of the present invention), it is possible to use lower concentrations. Non-exhaustive examples of effective polynucleotide treatment regimens include treatment of between about 0.1 and about 1 nmol of polynucleotide molecule per plant, or between about 1 nmol and about 10 nmol of polynucleotide molecule per plant, or between about 10 nmol and approximately 100 nmol of polynucleotide molecule per plant.
In some embodiments, one or more polynucleotides are provided with a transfer agent which is an agent that allows a topically applied polynucleotide to enter cells of an organism. These transfer agents can be Incorporated as part of a composition comprising a polynucleotide as described herein, or can be applied before, along with, or after application of the polynucleotide. In some embodiments, a transfer agent is an agent that enhances the absorption of a polynucleotide of the present invention by a Leptinotarsa species. In some ways, a transfer agent is a
<img file="MX359191B_D0117.tif" />
113 agent that conditions the surface of plant tissue, by 'rjcmp *<sup>1</sup>. jiiilluj, liujx, talh<sup>1</sup>· Raírps. flowers or fruits, for impregnation by a polynucleotide in plant cells. In some embodiments, the transfer agent allows a pathway for a polynucleotide through wax barriers, stomata, and / or cell wall or membrane barriers in plant cells.
Suitable transfer agents include agents that increase the permeability of the body's exterior or that increase the permeability of the body's cells to polynucleotides. Suitable transfer agents include a chemical agent or a physical agent, or combinations of both. Chemical agents for conditioning or transfer include (a) surfactants, (b) an organic solvent or aqueous solution or aqueous mixtures of organic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils , (g) enzymes or any combination of these. In some embodiments, the application of a polynucleotide and a transfer agent optionally includes an incubation step, a neutralization step (for example, to neutralize an acid, base or oxidation agent, or to activate an enzyme), a step rinse or combinations of these. Suitable transfer agents may be in the form of an emulsion, a reverse emulsion, a liposome, or other micellar-like composition, or may cause the polynucleotide to take the form of an emulsion, a reverse emulsion, a liposome, or other micellar-like composition. Modalities of transfer agents include counterions or other molecules known to associate with nucleic acid molecules, for example, inorganic ammonium ions, alkyl ammonium ions, lithium ions, polyamines such as spermine, spermidine, or putrescine, and other cations. Modes of transfer agents include organic solvents such as DMSO, DMF, pyridine, / V-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, or other solvents that are water soluble or phosphonudeotid dissolving in non-aqueous systems (as used in reactions synthetic). Modes of transfer agents include synthetic or naturally derived oils with or without surfactants or emulsifiers, for example vegetable oils, crop oils (such as those listed in 9<sup>th</sup> Compendium of Herbicide Adjuvants, available to the public online at herbicide.adjuvants.com), paraffinic oils, polyol fatty acid esters, or oils with short chain molecules modified with amides or polyamines, such as polyethyleneimine or / V-pyrrolidine.
Transfer agent modalities include organosilicone preparations. For example, a suitable transfer agent in an organosilicone preparation that is commercially available as SILWET L-77®, a brand name surfactant having CAS number 27306-78-1 and EPA number: CAL.REG. No. 5905-50073-AA, and currently available from Momentive Performance Materials, Albany, New York. One embodiment includes a composition comprising a polynucleotide and a transfer agent that includes
114
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<img file="MX359191B_D0119.tif" />
to an organosllicone preparation such as Silwet L-77 at about 2 weight percent (weight percent) (eg, about 0.01, 0.015, 0.02,
<img file="MX359191B_D0120.tif" />
2.5 percent by weight). One embodiment includes a composition comprising a polynucleotide of the present invention and a transfer agent that includes the SILWET L77® brand surfactant in the range of about 0.3 to about 1 weight percent (weight percent) or about 0.5 to about 1%. by weight (weight percent).
Organosllicone compounds useful as transfer agents for use in the present invention include, but are not limited to, compounds that include: (a) a major group of trisiloxane that is covalently linked to (b) an alkyl linker which includes, but is not limited to, a linker / hpropyl, which is covalently linked to (c) a polyglycol chain, which is covalently linked to (d) a terminal group. The major trisiloxane groups of such organosllicone compounds include, but are not limited to, heptamethyltrisiloxane. Alkyl linkers may include, but are not limited to, a / 7-propyl linker. Polyglycol chains Include, but are not limited to, polyethylene glycol or pollpropylene glycol. The polyglycol chains can comprise a mixture that provides an average chain length n of about 7.5. In certain embodiments, the average chain length n can range from about 5 to about 14. Terminal groups can include, but are not limited to, alkyl groups such as a methyl group. Organosllicone compounds useful as transfer agents include, but are not limited to, trisiloxane ethoxylate surfactants or polyalkylene oxide modified heptamethyltrisiloxane. An example of a transfer agent to be used in the present invention is Compound I:
<img file="MX359191B_D0121.tif" />
OR
<img file="MX359191B_D0122.tif" />
(Compound I: polyalkylene oxide heptamethyltrisiloxane, average n = 7.5).
Organosllicone compounds useful as transfer agents are used, for example, as freshly prepared concentrations in the range of about 0.015 to about
<img file="MX359191B_D0123.tif" />
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115 2 percent by weight (percent by weight) (for example, alrtitltídUf 'tlt! U.Ü1, U.U15, 0.02, 0.025,
<img file="MX359191B_D0124.tif" />
weight percent).
Transfer agent modalities Include one or more salts such as ammonium chloride, tetrabutylphosphonium bromide and ammonium sulfate, which are provided in or used with a composition that includes a polynucleotide. In some embodiments, ammonium chloride, tetrabutylphosphonium bromide, and / or ammonium sulfate are used at a concentration of about 0.5% to about 5% (w / v) or about 1% to about 3% (w / v) or about 2% (w / v). In certain embodiments, the composition that includes a polynucleotide includes an ammonium salt at a concentration greater than or equal to 300 mlllol. In certain embodiments, the composition including a polynucleotide includes an organosilicone transfer agent in a concentration of about 0.015 to about 2 weight percent (weight percent) as well as ammonium sulfate at concentrations of about 80 to about 1200mM or about 150mM to about 600mM.
Modes of transfer agents include a phosphate salt. Phosphate salts useful in a composition that Include a polynucleotide Include, but are not limited to, calcium, magnesium, potassium or sodium phosphate salts. In certain embodiments, a composition includes a polynucleotide. Includes a phosphate salt at a concentration of at least about 5 millilol, at least about 10 millol or at least about 20 millol. In certain embodiments, a composition that includes a phosphate salt a polynucleotide in a range of from about 1mM to about 25mM or in a range of from about 5mM to about 25mM. In certain embodiments, the composition includes a polynucleotide sodium phosphate at a concentration of at least about 5 millilol, at least about 10 millol or at least about 20 millimolar. In certain embodiments, a composition that includes a polynucleotide includes a sodium phosphate at a concentration of about 5 millimolar, about 10 mlmolar, or about 20 mlmolar. In certain embodiments, a composition that includes a polynucleotide includes a sodium phosphate salt in a range of from about 1mM to about 25mM or in a range of from about 5mM to about 25mM. In certain embodiments, a composition that includes a polynucleotide includes a sodium phosphate salt in a range from about 10mM to about 160mM or in a Range from about 20mM to about 40mM. In certain embodiments, a composition that includes a polynucleotide includes a sodium phosphate buffer at a pH of about 6.8.
Transfer agent modalities Include surfactants and / or effective molecules contained therein. The surfactants and / or effective molecules contained therein Include, of
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116
INSTITUTO MtXICANC í> í LA FkOFiEDAL
INDUSTRIAL
<img file="MX359191B_D0126.tif" />
phospholipids) and organosilicone surfactants. In certain embodiments, a composition that includes a pollnucleotide is formulated with contralons or other molecules known to associate with nucleic acid molecules. Non-limiting examples include Tetraalkullamonlo Ions, Trialkylammon Ions, Sulfonon Ions, lithium ions, and pollamines such as spermine, sperm, or putresin. In certain embodiments, a composition that includes a polynucleotide is formulated with a non-polynucleotide herbicide, for example, glyphosate, auxlna-type benzoic acid herbicides, including dlcamba, chloramben, and TBA, gluphoslonate, auxin-type herbicides including phenoxy-carboxylic acid herbicide, herbicide of carboxylic acid, carboxylic quinoline acid herbicide, carboxylic acid herbicide and benazolinethyl herbicide, sulfonureureas, mldazollnones, bromoxlnll, delapon, clclohezanodlone, protoporphrinogen oxidase inhibitors and herbicides that inhibit 4-hldroxlfenll-plruvate-dloxlgenase. In certain embodiments, a composition that includes a polynucleotide is formulated with a non-polynucleotide pesticide, for example, a potato, a plant lectin, a fltoecdlsterol, an Insecticidal protein Bacillus thuringiensis, an Insecticidal protein Xenorhabdus, an Insecticidal protein Photorhabdus, an Insecticidal protein Bacillus laterosporous and a Bacillus sphaericus Insecticide protein. In some embodiments, a composition that includes a pollnucleotide and a non-pollnucleotide pesticide provides a significant improvement in the prevention or control of Leptinotarsa species infestations, compared to the effect obtained with the pollnucleotide alone or the non-pollnucleotide pesticide alone. In some embodiments, a composition comprising a double-stranded RNA with a strand having a sequence that is selected from the group consisting of the Trigger Sequence Group is combined with a non-polynucleotide pesticide (eg, a protein, a vegetable lectlna, a fltoecdlsterolde, a Bacillus thuringiensis Insecticidal protein, a Xenorhabdus Insecticidal protein, a Photorhabdus Insecticidal protein, an insecticidal protein Bacillus laterosporous and an insecticidal protein Bacillus sphaericus}, where the combination was found to effect a highly improved prevention or control of Leptinotarsa species infestations, compared to the effect obtained with double-stranded RNA alone or the non-pesticide polynucleotide alone.
Related techniques
Modalities of Pollnucleotides and Nucleic Acid Molecules as Described herein may Include additional elements, such as promoters, small RNA recognition sites, aptamers or ribozymes, additional expression cassettes to express coding sequences (eg, to express a transgene such as an insecticidal protein or marker to be selected) or non-coding sequences (eg, to express
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117 additional suppression elements). For example, one aspect of the préUdUm * li iveiilIói ι 'μιυμυι ύυι iu a recombinant DNA construct comprising a heterologous promoter operably linked to a DNA comprising at least a segment of 18 or more contiguous nucleotides with a sequence of about 95 % to about 100% identity with a fragment of equivalent length of DNA that has a sequence that is selected from the Diana Gene Sequence Group or its DNA complement. Another aspect of the invention provides a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an RNA hairpin having an antisense region having a sequence, or a fragment of a sequence, that is selected from the group that is selected from the Trigger Sequence Group. In another embodiment, a recombinant DNA construct comprising a promoter operably linked to DNA encodes: (a) an RNA silencing element to suppress a target gene that is selected from the group consisting of the genes identified in Table 1) and (b) an aptamer, is stably integrated into the plant genome from which RNA transcripts are expressed including the RNA aptamer and the RNA silencing element in plant cells; the aptamer serves to guide the RNA silencing element to a desired location in the cell. In another embodiment, the inclusion of one or more recognition sites for binding and cleavage by small RNA (eg, by a miRNA or siRNA that is expressed only in a particular cell or tissue) allows for more accurate expression patterns in a plant, where the expression of the recombinant DNA construct is suppressed where the small RNA is expressed. Such additional elements are described below.
Promoters
Promoters for use in the invention are functional in the cell where the construct is intended to be transcribed. These promoters are generally heterologous promoters, as used in recombinant constructs, that is, they are not found in nature to be operably linked to the other nucleic elements used in the constructs described herein. In various embodiments, the promoter is selected from the group consisting of a constitutive promoter, a spatially specific promoter, a time-specific promoter, a development-specific promoter, and a promoter inducible. In many embodiments the promoter is a functional promoter in a plant, for example, a pol II promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.
Non-constitutive promoters suitable for use with the recombinant DNA constructs of the present invention include spatially specific promoters, promoters
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118 Temporarily specific and inducible promoters. Spatially, the pmmnt-ηι-ρς pcpprífíros can include promoters specific for organelles, cells, tissues or organs (for example, a specific promoter for plastids, roots, pollen or seeds for expression in plastids, roots, pollen or seeds, respectively). In many cases a specific promoter for seeds, embryos, aleurones or endosperm is especially useful. Temporally specific promoters may include promoters that tend to promote expression during certain stages of development in a plant growth cycle or during different times of the day or night or at different seasons in a year. Inducible promoters include promoters induced by chemicals or environmental conditions, such as, but not limited to, biotic or abiotic stress (for example, water deficit or drought, heat, cold, or high or low levels of nutrients or salt, high levels or low light or pathogen or pest infections). MicroRNA promoters are useful, especially those that have specific expression patterns from a temporal, spatial or inducible point of view; Examples of microRNA promoters, as well as methods for identifying microRNA promoters having specific expression patterns, are provided in US Patent Application Publications 2006/0200878, 2007/0199095 and 2007/0300329, which are specifically incorporated. hereby by reference. A specific expression promoter may also include promoters that are generally constitutively expressed but to varying degrees or strengths of expression, including promoters that are commonly considered to be strong promoters or weak promoters.
Promoters of particular interest include the following examples: an opaline synthase promoter isolated from Agrobacteriurrr T-DNA, a cauliflower mosaic virus 35S promoter; Enhanced promoter elements or chimeric promoter elements such as an improved cauliflower mosaic virus (CaMV) 35S promoter linked to an enhancement element (an intron from Zea mays heat shock protein 70); root specific promoters such as those described in US Patents 5,837,848; 6,437,217 and 6,426,446; a corn L3 oleosin promoter described in US Patent 6,433,252; a promoter for a plant nuclear gene encoding a plastid localized aldolase described in US Patent Publication 2004/0216189; cold inducible promoters described in US Patent 6,084,089; salt inducible promoters described in US Patent No. 6,140,078; light inducible promoters described in US Patent 6,294,714; pathogen inducible promoters described in US Patent 6,252,138; and water-inducible promoters described in US Patent Publication 2004/0123347 Al. All patents and patent publications described above that disclose promoters and their use, especially in DNA constructs
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119 recombinant functional in plants, are incorporated herein by reference.
Specific vascular or plant phloem promoters of interest include a rolC or rolA promoter from Agrobacterium rhizogenes, a promoter from a T-DNA 5 gene of Agrobacterium tumefaciens, the promoter of the RSsl rice sucrose synthase gene, a promoter of Yellow spot Commelina badnavlrus, a promoter of the coconut leaf rot virus, a promoter of the rice tungro bacilliform virus, the promoter of a pea glutamine synthase GS3A gene, invCDlll and invCD141 promoters of the potato invertase gene, a promoter isolated from Arabidopsis that is shown to have phloem-specific expression in tobacco by Kertbundit et al., (1991) Proc. Nati. Acad. Sci. USA, 88: 5212-5216, a VAHOX1 promoter region, a pea cell wall invertase gene promoter, a promoter of the carrot acid invertase gene, a promoter of a Sultrl sulfate transporter gene; 3 , a promoter of a plant sucrose synthase gene and a promoter of a plant sucrose transporter gene.
Promoters suitable for use with a recombinant DNA or polynucleotide construct of the present invention include polymerase II (pol II) promoters and polymerase III (pol III) promoters. RNA polymerase II transcribes structural or catalytic RNAs that are typically shorter than 400 nucleotides in length and recognizes a single pass of T residues as a termination signal; has been used to transcribe siRNA duplexes (see, for example, Lu et al. (2004) Nucieic Acids Res., 32: el71). Pol II promoters are therefore in certain embodiments where a short RNA transcript must be produced from a recombinant DNA construct of the present invention. In one embodiment, the recombinant DNA construct comprises a pol II promoter to express an RNA transcript flanked by self-cleaving ribozyme sequences (eg, self-cleaving hammerhead ribozymes), resulting in processed RNA, such as RNA from A single strand that binds to the transcription of the Leptinotarse target gene, with defined 5 'and 3' ends, without potentially interfering flanking sequences. An alternative approach uses pol III promoters to generate transcripts with relatively defined 5 'and 3' ends, that is, to transcribe an RNA with minimal 5 'and 3' flanking sequences. In some embodiments, Pol III promoters (eg, U6 or Hl promoters) are useful for adding a short AT-rich transcription termination site that results in excess of 2 base pairs (UU) in the transcribed RNA; this is useful, for example, for the expression of siRNA constructs. The use of pol III promoters for driving expression of siRNA constructs has been reported; see van de Wetering et al. (2003) EMBO Rep., 4: 609-615, and Tuschl (2002) Nature Biotechnoi., 20: 446-448. Baculovirus promoters such as baculovirus polyhedrin and plO promoters are known in the art and are commercially available; see,
<img file="MX359191B_D0130.tif" />
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120 M PROPERTY
INDUSTRIAL eg Gulde to Baculovirus Expresslon Vector <sup><</sup>5γςΐρηις. ^ ΒΕ \ / ς) and incprt ΓρΙΙ ΓνΙΗιγρ Techniques from Invltrogen, 2002 (Life Technologies, Carlsbad, CA) and FJ Haines et al. Baculovirus Expression Vectors, updated (Oxford Expression Technologies, Oxford, UK).
The promoter element may include nucleic acid sequences that are not naturally occurring promoters or promoter elements or homologs thereof but that can regulate the expression of a gene. Examples of such gene-independent regulatory sequences include artificially designed or naturally occurring RNA sequences that include an aptamer or ligand-binding region (see aptamers, below) and a regulatory region (which can act on ds). See, for example, Isaacs et al. (2004) Nat. BiotechnoL, 22: 841-847, Bayer and Smolke (2005) Nature BiotechnoL, 23: 337-343, Mandal and Breaker (2004) Nature Rev. Mo !. Ceii BioL, 5: 451-463, Davidson and Ellington (2005) Trends BiotechnoL, 23: 109-112, Winkler et al. (2002) Nature, 419: 952-956, Sudarsan et al. (2003) RNA, 9: 644-647, and Mandal and Breaker (2004) Nature Struct. Mol. BioL, 11: 29-35. These riboregulators could be selected or designed for specific temporal or spatial specificity, for example, to regulate the translation of DNA encoding a silencing element to suppress a Leptinotarse target gene only in the presence (or absence) of a given concentration of the appropriate ligand . An example is a riboregulator that responds to an endogenous ligand (eg, jasmonic acid or salicylic acid) produced by the plant when under stress (eg, abiotic stress such as water, temperature or nutrient stress, or biotic stress such as that related to pests or pathogens); under stress, the level of endogenous ligand increases to a level sufficient for the riboregulator to begin transcription of the DNA encoding a silencing element to suppress a Leptinotarse target gene.
Recombinase sites
In some embodiments, the recombinant DNA or polynucleotide construct of the present invention comprises DNA encoding one or more site-specific recombinase recognition sites. In one embodiment, the recombinant DNA construct comprises at least one pair of loxP sites, where the DNA site specific recombination between the loxP sites is mediated by a Cre recombinase. The position and relative orientation of the loxP sites is selected to achieve the desired recombination; for example, when the loxP sites are in the same orientation, the DNA between the loxP sites is drawn in a circular fashion. In another embodiment, the recombinant DNA construct comprises DNA encoding a loxP site; in the presence of Cre recombinase and another DNA with a loxP site, the two DNAs recombine.
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121
Aptamers
In some embodiments, the recombinant DNA or polynucleotide construct of the present invention comprises DNA that is processed to an RNA aptamer, i.e., an RNA that binds to a ligand through a non-primarily binding mechanism. in Watson-Crick base pairing (in contrast, for example, to base pairing that occurs between antiparallel and complementary nucleic acid chains to form a double chain nucleic acid structure). See, for example, Ellington and Szostak (1990) Nature, 346: 818822. Examples of aptamers can be found, for example, in the Public Aptamer Database, available online at aptamer.icmb.utexas.edu (Lee et al. (2004) Nudeic Acids Res., 32 (l): D95- 100). The aptamers useful in the invention, however, can be monovalent (that binds to a single ligand) or multivalent (that binds to mpas of a single ligand, for example, that binds to a unit of two or more different ligand ).
Ligands useful in the invention include any molecule (or part of a molecule) that can be recognized and linked by a secondary nucleic acid structure by a mechanism that is not primarily based on Watson-Crick base pairing. Thus, the recognition and binding of the ligand and the aptamer are analogous to that of the antigen and the antibody, or of the biological effector and the receptor. Ligands can include individual molecules (or part of a molecule) or a combination of two or more molecules (or parts of a molecule) and can include one or more macromolecular complexes (for example, polymers, lipid bilayers, liposomes, cell membranes or other cellular structures or cellular surfaces). Examples of specific ligands include vitamins, such as coenzyme B<sub>12</sub> and thiamine pyrophosphate, flavin mononucleotide, guanine, adenosine, S-adenosylmethionine, S-adenosylhomocysteine, coenzyme A, Usin, tyrosine, dopamine, glucosamine-6-phosphate, caffeine, theophylline, antibiotics such as chloramphenicol and neomycin, glyphosate and dicamba, proteins including viral or phage coating proteins and surface proteins of the digestive or epidermal tract of invertebrates and RNA including viral RNA, transfer RNA (tRNA), Ribosomal RNA (rRNA) and RNA polymerases such as RNA-dependent RNA polymerase (RdRP). One class of RNA aptamers useful in the invention are thermo switches that do not bind to a ligand but are thermally responsive, that is, the conformation of the aptamer is determined by temperature; see, for example, Box 3 in Mandal and Breaker (2004) Nature Rev. Mol. Ce // Bio /., 5: 451-463.
Transqén transcription units
In some embodiments, the recombinant DNA or polynucleotide construct of the present invention comprises a transgene transcription unit. A transcription unit
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122
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Transgene IMPI comprises a DNA sequence encoding a gene of interest, eg filfl, natural protein linar or a heterologous protein. A gene of interest can be any coding or non-coding sequence of any species (including, but not limited to, non-eukaryotes such as bacteria and viruses; fungi, protists, plants, invertebrates, and vertebrates). Particular genes of interest are genes encoding at least one pesticidal agent that is selected from the group consisting of a patatin, a plant lectin, a phytoecysteroid, an insecticidal protein Bacillus thurlnglensis, an insecticidal protein Xenorhabdus, an insecticidal protein Photorhabdus, a Bacillus laterosporous insecticidal protein and a Bacillus sphaericus insecticidal protein. The transgene transcription unit may also include 3 'or 5' sequence or both as necessary for transcription of the transgene.
Introns
In some embodiments, the recombinant DNA or polynucleotide construct of the present invention comprises DNA that encodes a cleavable intron. Intron generally means a segment of DNA (or the RNA transcribed from such a segment) that is located between exons (segments that encode DNA proteins or the corresponding transcribed RNA), where, during maturation of the messenger RNA, the intron present is enzymatically cleaved or it is removed from the RNA strand by a cleavage / ligaclone process that occurs in the nucleus in eukaryotes. The term intron also applies to non-coding DNA sequences that are transcribed to RNA segments that can be cut from a mature RNA transcript, but are not introns that lie between exons that encode proteins. An example of these are cuttable sequences that have the ability to enhance expression in plants (in some cases, especially monocotyledons) of a subsequent coding sequence; these cuttable sequences are naturally located in the 5 'untranslated region of some plant genes as well as some viral genes (for example the 5' tobacco mosaic virus leader sequence or omega leader described as improving plant gene expression by Gallie and Walbot (1992) Nudeic Acids Res., 20: 4631-4638). These cleavable sequences or expression-enhancing introns can be artificially inserted into the 5 'untranslated region of a plant gene between the promoter but before any protein-encoding exons. Examples of these expression-enhancing introns include, but are not limited to, corn alcohol dehydrogenase (Zm-Adhl), an intron that enhances the expression of corn Bronze-1, a rice actin intron 1 (Os-Actl ), a Shrunken-1 intron (Sh-1), a corn sucrose synthase intron, a heat shock protein 18 intron (hspl8), and an 82 kilodalton heat shock protein intron (hsp82) . US Patents 5,593,874 and 5,859,347, which are specifically incorporated herein by this reference, describe methods for improving DNA constructs
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123 recombinant for use in plants by inclusion of a tntron that mcjorüi I? pyprpqíón derived from the heat shock protein of maize of 70 kllodaltons (hsp70) in the untranslated leader located 3 'with respect to the gene promoter and 5' with respect to the first exon that encodes proteins.
Rlbozlmas
In some embodiments, the recombinant DNA or polynucleotide construct of the present invention comprises DNA encoding one or more rbobozymes. Particles of Special Interest Include a self-indexing pitch, a hammerhead talon, or a fork pitch. In one embodiment, the recombinant DNA construct comprises DNA encoding one or more ribozymes that serve to cleave the transcribed RNA to provide defined segments of RNA, such as silencing elements to suppress a Leptinotarse target gene.
Gene deletion elements
In some embodiments, the recombinant DNA or polynucleotide construct of the present invention comprises DNA encoding an additional gene deletion element to suppress a target gene other than a Leptinotarse target gene. The target gene to be deleted may include a coding sequence or a non-coding sequence or both.
Suitable gene deletion elements are described in detail in US Patent Application Publication 2006/0200878, the disclosure of which is specifically incorporated herein by this reference and includes one or more of:
(a) DNA comprising at least one antisense DNA segment that is antisense to at least one segment of the gene to be deleted;
(b) DNA comprising multiple couplets of at least one antisense DNA segment that is antisense to at least one segment of the gene to be deleted;
(c) DNA comprising at least one sense DNA segment that is at least one segment of the gene to be deleted;
(d) DNA comprising multiple couplets of at least one sense DNA segment that is at least one segment of the gene to be deleted;
(e) DNA that is transcribed into RNA to suppress the gene to be deleted forming double-stranded RNA and comprising at least one segment of antisense DNA that is antisense to at least one segment of the gene to be deleted and at least one segment DNA sense that is at least one segment of the gene to be deleted;
(f) DNA that is transcribed into RNA to suppress the gene to be deleted
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forming a single double stranded RNA and comprising multiple segments of serial sense DNA that are antisense to at least one segment of the gene to be deleted and multiple sense DNA segments to be deleted that are at least one segment of the gene to be deleted ;
(g) DNA that is transcribed into RNA to suppress the gene to be deleted forming multiple double strands of RNA and comprising multiple antisense DNA segments that are antisense to at least one segment of the gene to be deleted and multiple sense DNA segments that they are at least one segment of the gene to be deleted and where the multiple antisense DNA segments and the multiple sense DNA segments are arranged in a series of inverted repeats;
(h) DNA comprising nucleotides derived from a plant miRNA;
(I) DNA comprising nucleotides of a siRNA;
0) DNA that is transcribed to an RNA aptamer capable of binding to a ligand;
and (k) DNA that is transcribed to an RNA aptamer capable of binding to a ligand and DNA that is transcribed to regulatory RNA capable of regulating the expression of the gene to be deleted, where regulation depends on the conformation of regulatory RNA. and the conformation of regulatory RNA is affected from the allosteric point of view by the binding state of the RNA aptamer.
In some embodiments, an intron is used to deliver a gene deletion element in the absence of any protein-encoding exons (coding sequence). In one example, an intron, such as an expression-enhancing intron, is interrupted by embedding in the Intron of a gene deletion element, where, after transcription, the gene deletion element is excised from the intron. Therefore, the exons encoding proteins do not need to provide the gene deletion function of the recombinant DNA constructs described herein.
Transcriptional regulatory elements
In some embodiments, the recombinant DNA or polynucleotide construct of the present invention comprises DNA encoding a transcriptional regulatory element. Transcriptional regulatory elements include elements that regulate the level of expression of the recombinant DNA construct of the present invention (relative to its expression in the absence of such regulatory elements). Examples of suitable transcriptional regulatory elements include rlbointerruptors (cis- or trans-acting), transcription stabilizing sequences, and miRNA recognition sites, as described in detail in US Patent Application Publication 2006/0200878, which is It specifically incorporates hereby
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this reference.
Embodiment and use of cells of transgenic plants and transgenic plants
The transformation of a plant can include any of several known methods and compositions. Suitable methods for plant transformation Include virtually any method by which DNA can be introduced into a cell. A plant transformation method is bombardment of microprojectiles, for example, as illustrated in US Patents 5,015,580 (soy), 5,538,880 (corn), 5,550,318 (corn), 5,914,451 (soy),
6,153,812 (wheat), 6,160,208 (corn), 6,288,312 (rice), 6,365,807 (rice), and 6,399,861 (corn), and
6,403,865 (corn), all of which are incorporated by this reference to allow the production of transgenic plants.
Another useful method of plant transformation is transformation mediated by
Agrobacterlum via Agrobacterium containing a binary Ti plasmid system, where the Agrobacterium carries a first Ti plasmid and a second chimeric plasmid containing at least one T-DNA border of a wild-type Ti plasmid, a functional promoter in the plant cell transformed and operably linked to a polynucleotide or recombinant DNA construct of the present invention. See, for example, the binary system described in US Patent 5,159,135, which is incorporated by this reference. Also see De Framond (1983) Biotechnology, 1: 262-269; and Hoekema et al<sub>v</sub> (1983) Nature, 303: 179. In such a binary system, the smaller plasmid, which contains the T-DNA border or borders, can be conveniently constructed and manipulated in a suitable alternative host, such as £ coli, and then transferred to Agrobacterium.
Detailed procedures for Agrobacterium-mediated transformation of plants, especially crop plants, include procedures described in US Patents 5,004,863, 5,159,135, and 5,518,908 (cotton); 5,416,011, 5,569,834, 5,824,877 and 6,384,301 (soybean); 5,591,616 and 5,981,840 (corn); 5,463,174 (brasslcas including canela), 7,026,528 (wheat), and 6,329,571 (rice), and in US patent application publications 2004/0244075 (corn) and 2001/0042257 Al (sugar beet), all of which are specifically incorporated by this reference to allow the production of transgenic plants. US Patent Application Publication 2011/0296555 describes transformation vectors (including vector sequences) and detailed protocols for transforming corn, soybean, cane, cotton, and sugar cane in Example 5 and is specifically incorporated by this reference to allow the production of transgenic plants. Similar methods have been reported for many plant species, both dicotyledonous and monocotyledonous, including, but not limited to, peanuts (Cheng et al. (1996) Piant Ceii Rep., 15: 653); asparagus (Bytebier et
OR
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to the. (1987) Proc. Nati. Acad. Sci. USA, 84: 5345); barley (Wan and Lemaux (I9! W) Pldrit PtiyM :; '104: 37); rice (Toriyama et al. (1988) Bio / Technology, 6:10; Zhang et al. (1988) Plant Cell Rep., 7: 379; wheat (Vasll et al. (1992) Bio / Technology, Ity.ttT, Becker et al. (1994) Plant J., 5: 299), alfalfa (Masoud et al. (1996) Transgen. Res., 5: 313); and tomato (Sun et al. (2006) Plant Cell Physioi., 47: 426-431). See also a description of vectors, transformation methods, and production of transformed Arabidopsis tha / iana plants where transcription factors are constitutively expressed by a CaMV35S promoter, in US Patent Application Publication 2003/0167537 Al, which is incorporated herein by reference. Transformation methods specifically useful for nightshade plants are known in the art. See, for example, the publicly described transformation methods for tomato (Sharma et al. (2009), J. Biosci., 34: 423-433), aubergine (Arpaia et al. (1997) Theor. AppL Genet., 95: 329-334), potato (Bannerjee et al. (2006) Plant Sci., 170: 732-738; Chakravarty et al. (2007) Amer. J. Potato Res., 84: 301-311; S. MiHam 'Agrobacterlum-medlated transformatlon of potato. Chapter 19 (pp. 257 - 270), Transgenlc Crops of the World: Essentlal Protocols, Ian S. Curtís (editor), Sprlnger, 2004), and peppers (L¡ et al. (2003) Plant Cell Reports, 21: 785-788). Stably transgenic potatoes, tomatoes and aubergines have been commercially introduced in several areas; see, for example, K. Redenbaugh et al. Safety Assessment of Genetlcally Engineered Frults and Vegetables: A Case Study of the FLAVR SAVR ™ Tomato, CRC Press, Boca Raton, 1992, and in the extensive publicly available documentation of commercially genetically modified crops in the GM Crop Database; see: WAX. (2012). GM Crop Database. Center for Envlronmental Rlsk Assessment (CERA), ILSI Research Foundation, Washington DC, available electronically at www.ceragmc.org/?actlon=gm_crop_database. Various methods of transforming other plant species are known in the art, see, for example, the encyclopedic reference, Compendlum of Transgenic Crop Plants, edited by Chittaranjan Kole and Tlmothy C. Hall, Blackwell Publlshing Ltd., 2008; ISBN 978-1-405-16924-0 (available electronically at mrw.lntersclence.wlley.com/emrw/9781405181099/hpt/toc), which describes procedures for the transformation of cereals and forage grasses (rice, maize, wheat, barley, oat bran, sorghum, pearl millet, finger millet, cold season forage grasses, and bay grass), oilseed crops (soybeans, oilseed brassicas, sunflower, peanut, flax, sesame, and safflower), grains legume and fodder (common peas, cow beans, pea, beans, lentil, teparl bean, Asian beans, pigeon pea, pea, chickpea, lupine, alfalfa, and clovers), seasonal fruits and nuts (apple, pear, peach, plum, berry, cherry, grape, olive, almond and persian walnut), tropical and subtropical fruits and nuts (cltrus, grapefruit, banana and plantain, pineapple, papaya, mango, avocado, kiwi, granadilla, and plantain), vegetable crops (tomato, eggplant, peppers, vegetable brassicas, radish, carrot, cucurbit, alllum, asparagus and leafy vegetables),
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127 sugar, tubers and fiber crops (sugar cane, beets, stevia, potatoes, sweet potatoes, cassava and cotton), plantation crops, ornamentals, and grass (tobacco, coffee, cocoa, tea, rubber tree, medicinal, ornamental plants, and lawns), and forest tree species.
Transformation methods to provide transgenic plant cells and transgenic plants containing stably integrated recombinant DNA are preferably practiced in tissue culture in a medium and in a controlled environment. Medium refers to the various nutrient mixtures that are used to grow cells in vitro, that is, outside the intact living organism. Receptor cell targets include, but are not limited to, meristematic cells, calluses, immature embryos, or parts of embryos and gametes such as microspores, pollen, sperm, and ova. Any cell from which a plant can be regenerated is contemplated as a useful recipient cell for practicing the present invention. Calluses can be initiated from various tissue sources, including, but not limited to, immature embryos or parts of embryos, apical seedling meristems, microspores, and the like. Those cells that can proliferate as calluses can serve as receptor cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of the present invention (eg, various media and receptor target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are described, for example, in US Patents 6,194,636. and 6,232,526 and US Patent Application Publication 2004/0216189, which are specifically incorporated by this reference.
In general transformation practice, DNA is introduced into only a small percentage of target cells in any transformation experiment. Marker genes are generally used to provide an efficient system for the identification of those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers that confer resistance to a selective agent, such as an antibiotic or herbicide. Any of the antibiotics or herbicides to which a plant cell is resistant can be a useful agent for selection. Possibly transformed cells are exposed to the selective agent. In the surviving cell population will be those cells in which, generally, the gene that provides resistance is Integrated and expressed at sufficient levels to allow cell survival. The cells can be evaluated additionally to confirm the stable integration of the recombinant DNA. Commonly used selective marker genes include those that provide resistance to antibiotics such as kanamycin or paromomycin fnptll), hlgromlcin B (aph IV), and gentamicin faac3 and aacC4J or resistance to herbicides such as giufosinate / bar or pat) and glyphosate (EPSPS). Examples of useful selective marker genes and selection agents are illustrated in
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US Patents 5,550,318, 5,633,435, 5,780,708, and 6,118,047, all of which are specifically incorporated by this reference. Markers or indicators that can be evaluated can also be used, such as markers that provide an ability to visually identify transformants. Examples of markers that can be evaluated that are useful include, for example, a gene that expresses a protein that produces a detectable color by acting on a chromogenic substrate (for example, beta glucuronidase (GUS) (uldA) or luciferase (luc)) or which is itself detectable, such as green fluorescent protein (GFP) (gfp) or an immunogenic molecule. Those of skill in the art will recognize that there are many other useful markers or Indicators available for use.
Detection or transcription measurement of a recombinant DNA construct in a transgenic plant cell can be accomplished by any suitable method, including protein detection methods (eg, western blots, ELISA, and other immunochemical methods), activity measurements enzymatic or nucleic acid detection methods (eg, Southern blots, northern blots, PCR, RT-PCR, fluorescence in situ hybridization).
Other suitable methods for detecting or measuring transcription in a plant cell of a recombinant polynucleotide of the present invention that targets a target gene of the species Leptinotarsa Includes measurements of any other trait that is a direct Indicator or of substitute level variables of expression of the target gene in the Leptinotarsa species, with respect to the level of expression observed in the absence of the recombinant polynucleotide, for example, growth rates, mortality or reproductive or recruitment rates of the Leptinotarsa species or measurements of injury (eg root injury) or loss of yield in a plant or plant field Infected with the Leptinotarsa species. In general, suitable methods for detecting or measuring transcription in a plant cell of a recombinant pollnucleotide of interest include, for example, gross or microscopic morphological traits, growth rates, yield rates, reproductive or recruitment, resistance to pests or pathogens or resistance to blot or ablotic stress (for example, water deficit stress, salt stress, nutrient stress, heat or cold stress). These methods may use direct measurements of a phenotypic trait or surrogate variable tests (eg, in plants, these tests include plant part tests such as leaf or root tests to determine tolerance to abiotic stress). These methods include direct measurements of resistance to an invertebrate pest or pathogen (for example, damage to plant tissues) or tests of surrogate variables (for example, plant performance tests or bloensays such as the bioassay of worm larvae of the root of western corn (Diabrotica virgifera virgifera LeConte) described in International Patent Application Publication W02005 / 110068 A2 and Application Publication of
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129 US patent US 2006/0021087 Al, which incorporates * épec'hcaíiiéhlé IIItíllluiiLl ljIference, or the soybean cyst nematode bioassay, described by Steeves et al. (2006) Funct. Piant Bioi., 33: 991-999, where cysts are measured per plant, cysts per gram of root, eggs per plant, eggs per gram of root, and eggs per cyst, or the Colorado potato beetle bioassay {Leptinotarsa decemiineata ') described herein in the Working Examples.
The recombinant DNA constructs of the present invention can be stacked with other recombinant DNAs to provide additional traits (for example, in the case of transformed plants, traits include herbicide resistance, pest resistance, tolerance to cold germination, tolerance to water deficit and the like) eg, expressing or deleting other genes. Constructs for the coordinated decrease or increase in gene expression are described in US Patent Application Publication 2004/0126845 Al, which is specifically incorporated by this reference.
The seeds of fertile transgenic plants can be harvested and used to cultivate generations of progeny, including hybrid generations of transgenic plants of the present invention that include the construction of recombinant DNA in their genome. Therefore, in addition to direct transformation of a plant with a recombinant DNA construct of the present invention, transgenic plants of the present invention can be prepared by crossing a first plant having recombinant DNA with a second plant that does not have the construct. . For example, the recombinant DNA can be introduced into a plant line that is susceptible to transformation to produce a transgenic plant, which can be crossed with a second plant line to introduce the recombinant DNA into the resulting progeny. A transgenic plant of the present invention can be crossed with a plant line that has another recombinant DNA that provides one or more additional traits (such as, non-exhaustively, resistance to herbicides, resistance to pests or diseases, resistance to tension environmental, modified nutrient content, and yield enhancement) to produce progeny plants that have recombinant DNA that provides both the desired target sequence expression behavior and one or more additional traits.
In this reproduction to combine traits the transgenic plant that donates the additional trait can be a male line (pollinator) and the transgenic plant that carries the base traits can be the female line. The progeny of this cross is segregated in such a way that part of the plant will carry the DNA for the traits of both parents and part will carry the DNA of the traits of one parent; these plants can be identified with markers associated with recombinant progenitor DNA. Progeny plants that have DNA from the traits of both parents can be crossed back into the female parent line multiple times, for example, usually 6 to 8 generations, to produce a homozygous progeny plant with virtually the same
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130 genotype than an original transgenic progenitor line as well as the ADlTT ^ omBíñáñfe'dé la 011 * 3 transgenic progenitor line.
Still another aspect of the present invention is a transgenic plant grown from the transgenic seed (or in the case of potatoes, a transgenic seed potato) of the present invention. The present invention contemplates transgenic plants grown directly from transgenic seeds containing the recombinant DNA as well as generations of plant progeny, including inbred or hybrid plant lines, made by crossing a transgenic plant grown directly from the transgenic seed to a second plant. uncultivated from the same transgenic seed. Crossing may include, for example, the following steps:
(a) plant seeds from the first parent plant (eg, non-transgenic or transgenic) and a second parent plant that is transgenic according to the invention;
(b) cultivating the seeds of the first and second parent plants to be flowering plants;
(c) pollinate a flower of the first parent with pollen from the second parent; and (d) harvest seeds produced in the parent plant that has the fertilized flower.
It is often desired to introduce recombinant DNA into exclusive varieties, for example, by backcrossing, to transfer a specific desirable trait from one source to an inbred plant or other plant that does not have that trait. This can be accomplished, for example, by first crossing a superior inbred plant (A) (recurrent parent) to a donor inbred plant (B) (non-recurrent parent) carrying one or more genes appropriate for the trait in question, for example, a construction prepared in accordance with the present invention. The progeny of this cross is first selected from the resulting progeny so that the desired trait is transferred from the non-recurring progenitor B and then the selected progeny is paired with the superior recurrent progeny A. After five or more generations of backcrossing with selection of the desired trait, the progeny may be essentially hemzygous with respect to the loci that control the trait being transferred, but they are like the parent superior to most or almost all other genes. The latest generation of backcrossing would self-cross to give offspring that are a pure reproduction of the gene (s) being transferred, for example, one or more transformation events.
Through a series of breeding manipulations, a selected DNA construct can be moved from one line to an entirely different line without the need for additional recombinant manipulation. Therefore inbred plants can be produced that are actual replicas of one or more DNA constructs. By crossing different inbred plants, a large number of different hybrids can be produced with different combinations of DNA constructs. In this way plants can be produced that have the
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131 desirable agronomic properties frequently associated with hybrid C'vígor hibrioo) as well as the desired characteristics provided by one or more DNA constructs.
In certain cells of transgenic plants and transgenic plants of the present invention, it is sometimes desired to simultaneously express a gene of Interest while modulating the expression of a Leptinotarse target gene. Therefore, in some embodiments, the transgenic plant contains recombinant DNA further comprising a gene expression element for expression of at least one gene of interest and transcription of the recombinant DNA construct of the present invention is performed with the simultaneous transcription of the gene expression element.
In some embodiments, the recombinant DNA constructs of the present invention can be transcribed into any plant cell or tissue or to an entire plant at any stage of development. Transgenic plants can be derived from any monocotlledon or dicotyledon plant such as, but not limited to, plants of commercial or agricultural interest, such as crop plants (especially crop plants used for human or animal food), wood-producing trees, or pulp, vegetable plants, fruit plants and ornamental plants. Examples of plants of interest include grain crop plants (such as wheat, bran, barley, corn, rye, triticale, rice, millet, sorghum, qulnoa, amaranth, and buckwheat); forage crop plants (such as forage grasses and forage dicotyledons including alfalfa, pea, clover and the like); oilseed crop plants (such as cotton, safflower, sunflower, soybean, cane, rapeseed, flax, peanuts, and palm oil); tree nuts (such as walnut, cashew, hazelnut, pecan, almond, and the like); sugar cane, coconut, date palm, olive, beet, tea and coffee; trees that produce wood or pulp; Vegetable plants such as legumes (for example, beans, peas, lentils, alfalfa, peanuts), lettuce, asparagus, artichoke, celery, carrots, radish, brassicas (for example, cabbage, kale, mustards, and other broadleaved brassicas , broccoli, cauliflower, Brussels sprouts, turnip, kohlrabi), edible curcubltáceas (for example, cucumbers, melons, summer squash, winter squash), edible allium (for example, onions, garlic, leeks, shallots, chives), edible members of the Solanaceae (for example, tomatoes, aubergines, potatoes, peppers, alquequenje), and edible members of Chenopodiaceae (for example, fodder beet, chard, spinach, quinoa, amaranth); fruit growing plants such as apple, pear, citrus fruit (eg, orange, lime, lemon, grapefruit, and others), stone fruit (eg, apricot, peach, plum, nectarine), banana, pineapple, grape , kiwi, papaya, avocado, and berries; plants cultivated for blomass or biofuel (for example, Miscanthus grasses, rod grass, jatropha, oil palm, eukaryotic microalgae such as Botryococcus braunii, Chioreiia spp., and Dunaliella spp., and eukaryotic macroalgae such as Graciiaria spp., and Sargas ); and ornamental plants
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132 INDUSTRIAL including ornamental flower plants, ornamental trees and shrubs, ornamental ground covers, and ornamental grasses.
The present invention also provides basic products produced from a transgenic plant cell, plant, or seed of the present invention that include, but are not limited to, leaves, roots, shoots, tubers, stems, fruits, seeds, or other parts harvested from a plant, food, oils, extracts, fermentation or digestion products, ground or whole grains or seeds of a plant, or any food or non-food product Including the basic products produced from a transgenic plant cell, plant or seed of the present invention. Detection of one or more nucleic acid sequences of the recombinant DNA constructs of the present invention in one or more commodities contemplated herein is de facto evidence that the commodities contain or are derived from a transgenic plant cell, plant or seed of the present invention.
Generally a transgenic plant that has in its genome a recombinant DNA construct of the present invention exhibits a higher resistance to an Infestation with the Leptinotarsa species. In various embodiments, for example, where the transgenic plant expresses a recombinant DNA construct of the present invention that is stacked with another recombinant DNA to provide additional traits, the transgenic plant has at least one additional altered trait, relative to a plant that is not it has the recombinant DNA construct, which is selected from the group of traits consisting of:
(a) better tolerance to abiotic stress;
(b) better tolerance to blural stress;
(c) modified primary metabolite composition;
(d) modified secondary metabolite composition;
(e) trace element, carotenolde or modified vitamin composition;
(f) better performance;
(g) better ability to use nitrogen, phosphate or other nutrients;
(h) modified agronomic characteristics;
(i) modified growth or reproductive characteristics; and (j) better quality of harvest, storage or processing.
In some modalities, the transgenic plant is characterized by: better tolerated to abiotic stress (for example, tolerance to water deficit or levels of drought, heat, cold, non-optimal nutrient or salt, non-optimal light levels) or tension blotlca (eg, masses, allelopathy, or injury); by a modified primary metabolite composition (eg, fatty acid, oil, amino acid, protein, sugar or carbohydrate); a modified secondary metabolite composition (for example, alkaloids, terpenoldes, polyptylates, non-rhobosomal peptides, and
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133 secondary metabolites of mixed biosynthetic origin); a modified trace element (eg, iron, zinc), carotenoid composition (eg, beta-carotene, lycopene, lutein, zeaxanthin, or other carotenoids and xanthophylls) or vitamin (eg, tocopherols); better performing compositions (eg better performance under non-stress conditions or better performance under biotic or abiotic stress); better able to use nitrogen, phosphate or other nutrients; modified agronomic characteristics (eg, delayed maturation; delayed senescence; early or late maturity; better shade tolerance; improved resistance to root or stem curl; better resistance to green breakage of roots; modified response to photoperiod) ; modified growth or reproductive characteristics (eg, intentional dwarfism; intentional male sterility, useful, eg, in improved hybridization procedures; better vegetative growth rate; better germination; better fertility of the male or female); better harvest, storage or processing quality (eg, improved resistance to pests during storage, improved resistance to breakdown, better attractiveness to consumers) or any combination of these traits.
In another embodiment, the transgenic seed, or seed produced by the transgenic plant, has a modified primary metabolite composition (eg, fatty acid, oil, amino acid, protein, sugar, or carbohydrate), a modified secondary metabolite composition, a trace element modified, carotenoid or vitamin composition, better harvest, storage or processing quality or a combination of these. In another embodiment, it may be desired to change the levels of the natural components of the transgenic plant or transgenic plant seed, for example, to reduce the levels of an allergenic protein or glycoprotein or of a toxic metabolite.
Generally, the classification of a regenerated transgenic plant population each from a transgenic plant cell is performed to identify transgenic plant cells that develop and become transgenic plants with desired trait. Transgenic plants are tested for an improved trait, eg, better water use efficiency, better cold tolerance, higher yield, better nitrogen use efficiency, better seed protein, and better seed oil. Classification methods include direct classification to determine the trait in a greenhouse or field trial or classification to determine a surrogate trait. These analyzes aim to detect changes in the chemical composition, biomass, physiological or morphological properties of the plant. Changes in chemical compositions such as nutritional composition of the grain are detected by analysis of the composition of the seed and the content of proteins, free amino acids, oil, free fatty acids, starch, tocopherols or other nutrients. Changes in growth characteristics or
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134 Biomass are detected by measuring the height of the plant, diameter of t5T1ü, lUlltjllud ¡i ilu i mdoo, pococ. root and stem dried and (for plants producing grains such as corn, rice or wheat) length and diameter of seed head or ear. Changes in physical properties are identified by evaluating responses to stress conditions, for example, tests under imposed stress conditions such as water deficit, nitrogen or phosphate deficiency, growth conditions with hot or cold, pathogen attack, or insect, light deficiency or higher plant density. Other selection properties include days to flower, days for pollen spread, days for fruit ripening, quality or quantity of fruit or tuber produced, days for stigma in maize, leaf spread rate, chlorophyll content, leaf temperature, posture, seedling vigor, length of the plant, height of the plant, number of leaves, area of the leaves, tillering, supporting roots, if they remain green, root curl, plant health, fertility, green break, and pest resistance. Furthermore, the phenotypic characteristics of the harvested fruit, seeds or tubers can be evaluated; for example, in tomato and eggplant this may include the total amount or weight of the harvested fruit or the color, acidity, sugar content or flavor of such fruit and in the potato this may include the amount or total weight of the tubers harvested and the quality of such tubers.
Specific tests with the compositions and methods of the present invention can be performed on nightshade plants including potato, tomato, eggplant and peppers, either as hybrids or inbred plants; These assays are useful, for example, to identify or select plants with improved resistance to the Colorado potato beetle (larvae or adults), to determine insecticide-effective amounts of a given composition, or to determine effective treatment regimens. Non-exhaustive examples of these tests include the following.
An in planta Colorado potato beetle test (larva or adult) is performed on tomato plants with 6 copies per treatment. Large Cherry tomato plants are sown in Readl-Earth soil containing 6 llbras / cubic yard of fertilizer 14-14-14 and kept in a growth chamber at 27 degrees Celsius, 50% relative humidity for three weeks. On the day of the assay, double-stranded RNA is diluted in 25 milliliters of spray solution (20 ml of sodium phosphate buffer (pH 6.8), optionally containing a surfactant, eg 0.2% Sllwet L77) at the concentration desired and applied to plants using a monitored sprayer at a rate of 15 gallons per acre. A higher concentration (for example, 100 ml / gram / ml) can be used to test primarily a polynucleotide for activity, and lower concentrations (for example, between about 0.1 and about 1 ml / gram per milliliter) in subsequent tests such as those that determine the relative efficacy of various pollnucleotides. Plants are caged individually with
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135 mesh sleeves and are infected with 12 Leptinotarsa ¢ larvae. <sub>r</sub><sub>Ίι</sub> φ i<sub>to</sub> p<sub>3</sub>p<sub>to</sub> h<sub>p</sub>
Colorado) newborn. Infected plants are incubated in the growth chamber (27 degrees Celsius, 50% relative humidity) for 12-14 days. At the end of this period, plants are evaluated to determine the level of defoliation, classified as a control percentage, and insects are collected from the plants and soil to evaluate the percentage of viable insects recovered and the average weight of viable insects recovered.
An in planta Colorado potato beetle test (larva or adult) is performed on potato plants with 9 copies per treatment. Cuttings of mature Atlantic potato plants are prepared by cutting the stem at an angle less than the second node of the youngest growth. The clipping is soaked in rooting hormone (Rhizopon # 1, 0.1% IBA) and inserted immediately into previously moistened Readi-Earth soil containing 6 lbs / cubic yard of fertilizer 14-14-14. The cutout strips are covered to decrease light exposure and placed in a sealed plastic bag to increase humidity. Over the next week, the cover is removed and the strips are removed from the plastic bags. Plants that are 6 - 9 inches tall (generally 3 weeks from the cut date) are used in the trial. On the day of the assay, double-stranded RNA is diluted in 25 milliliters of spray solution (20 millimolar sodium phosphate buffer (pH 6.8), optionally containing a surfactant, eg 0.2% Silwet L77) at the concentration desired and applied to plants using a monitored sprayer at a rate of 15 gallons per acre. A higher concentration (eg, 100 micrograms / milliliter) can be used to assay a polynucleotide online for activity, and lower concentrations (eg, between about 0.1 and about 1 microgram per milliliter) in subsequent tests such as those that determine the relative efficacy of various polynucleotides. The plants are individually caged with mesh sleeves and infected with 6 newborn Leptinotarsa decemiineata (Colorado potato beetle) larvae. Infected plants are Incubated in the growth chamber (27 degrees Celsius, 50% relative humidity) for 12-14 days. At the end of this period, plants are evaluated to determine the level of defoliation, classified as a control percentage, and insects are collected from the plants and soil to evaluate the percentage of viable insects recovered and the average weight of viable insects recovered.
The following Examples are presented for illustration purposes and should not be considered as limitations.
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EXAMPLES
EXAMPLE 1
Leptinotarse cDNA library generation
A cDNA library was generated from newborn Leptinotarsa decemiineata larvae (Colorado potato beetle, CPB), as follows. Total RNA was isolated from 800 third instar Leptinotarsa decemiineata larvae (whole body) using an Ambion Totally RNA Isolation Kit (catalog number AM1910, Life Technologies, Carlsbad, CA) with the optional LiCL precipitation procedure. PoIyA RNA was isolated using Ambion MicroPoly (A) Purist (catalog number AM1919, Life Technologies, Carlsbad, CA). Random primed cDNA synthesis was performed using a Superscript double-stranded cDNA synthesis kit (catalog number 11917-010, Life Technologies, Carlsbad, CA) with a random hexamer kit (catalog number 12328-032, Life Technologies, Carlsbad, CA). The cDNA library was obtained by high-throughput sequencing using commercially available 454 technology (454 Life Sciences, 15 Commercial St., Branford, CT 06405, USA), as described in Margulies et al. (2005) Nature, 437: 376-380. This provided 1,446,014 readings (an average of ~ 350 base pairs in length), supplemented by publicly available Leptinotarsa decemiineata sequence data from NCBI (including 8,835 expressed tag sequence sequences, 150 full-length cDNAs, 839,061 sequence reads high-throughput DNA and RNA) to provide a total of 2294087 combined readings. The combined sequence data was assembled into de novo contuses using the Newbler software package (version 2.3) (454 Life Sciences, 15 Commercial St., Branford, CT 06405, USA). Approximately 38,164 armed contigs were identified from the sequence data.
EXAMPLE 2
Low copy target gene selection
The predicted Leptinotarse gene sequences that were predicted to be effective targets for RNA mediated silencing were identified as follows. Low-copy genes, and in particular single-coupled genes, were selected as targets for RNA-mediated silencing since a paralogue is unlikely to recapitulate the function of this gene. A public OrthoDB6 orthologous gene database was filtered (available at cegg.unige.ch/orthodb6 and described in Waterhouse et al. (2012) Nucieic Acids Res., PMID: 23180791; doi: 10.1093 / nar / gkslll6) to select a subset of 766 genes that had a single copy or
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INDUSTRIAL low copy of Triboiium castaneum ^ red flour beetle, a S-güpédb of LuleúptciOs) or as a single couplet or low copy in all the arthropod genomes available in the database (at the time this application was submitted, there were other 33 arthropod genomes available). Triboiium castaneum is a species of beetles and is therefore closely related to Leptinotarsa, making it likely that a single copy or low copy gene present in the Triboiium castaneum genome database is also a single copy gene. or low copy in the Leptinotarsa decemiineata genome, at least for genes that have high sequence similarity between the two organisms. Of the 38,164 unigenes obtained from the sequencing and assembly of Leptinotarsa decemiineata (Colorado potato beetle, CPB) described in Example 1, a subset of 725 genes was identified using a BLAST search for translated nucleotides (tblastx) as genes having high sequence similarity (importance or value e less than or equal to 1 X 10 '<sup>15</sup>) to the 766 single copy or low copy Triboiium castaneum genes in the OrthoDB database.
For sequence annotation, SmartBIast annotation was done using NCBI Blastall 2.2.21 software to search for Leptinotarsa decemiineata contigs against the publicly available database unlref90.fasta (ftp.unlprot.org/pub/databases/unlprot/current_release/ uniref / uniref90 /). The blast search was performed in blastx mode (translated Leptinotarsa decemiineata nucleotide queries searched against the uniref90 protein database). Only blast hits with an e value less than or equal to 9e-9 were retained. For each Leptinotarsa decemiineata con, the description line of the best hit uniref90 was used as an annotation. When no SmartBIast hits were found, the sequence underwent a supplemental Pfam search. To achieve this, the longest open reading frame (ORF) for each Leftinotarsa decemiineata was identified and used to query the publicly available Pfam-A database (ftp.sanger.ac.uk/pub/databases/Pfam / current_release) using the publicly available HMMER 3.0 software package (hmmer.janelia.org/). Leptinotarsa decemiineata contigs with a Pfam hit with an e value less than or equal to le-5 were noted with the protein family name and Pfam identifier. Leptinotarsa decemiineata contigs without SmartBIast or Pfam hits were scored as novel protein.
The 725 Leptinotarsa decemiineata genes identified as having high sequence similarity to the single copy or low copy Triboiium castaneum genes as described above are provided as SEQ ID NO: 1-725, where each gene is annotated based on the Sequence similarity with the sequences of Triboiium castaneum and / or OrthoDB or by conserved Pfam domains. For each Leptinotarsa decemiineata gene, the homologous Triboiium castaneum gene is also identified in the annotation, along with the similarity e value for
<img file="MX359191B_D0150.tif" />
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EXAMPLE 3
Selection of Leptinotarsa target genes
The cDNA sequences corresponding to target genes useful to control the Leptinotarsa species by RNA mediated silencing were selected from the sequences obtained from the sequencing and assembly of Leptinotarsa decemlineata (Colorado potato beetle, CPB) described in Example 1 This subset of the target gene cDNA sequences is provided in SEQ ID NO: 726-830. It is recognized that it is possible to obtain analogous sequences from any other Leptinotarsa species referred to herein.
EXAMPLE 4
Selection of overlapping polynucleotide triggers
A non-exhaustive example of a method of selecting a polynucleotide trigger to be expressed in a transgenic plant or used in a composition for topical application to the surface of a transgenic or non-transgenic plant involves mapping efficient polynucleotide sequences (or segments of sequences ) using a full gene overlap assay approach (or full length reference sequence). The sequences that are selected from SEQ ID NO: 1-725 and SEQ ID NO: 726-830 and SEQ ID NO: 1087-1094 are divided into overlapping sequences or 200-300 contiguous nucleotide segments along the entire length. of the selected target sequence. Overlapping sequences can be designed to be contiguous segments of the selected sequence without overlapping or to overlap about 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides on adjacent segments of the selected sequence. The polynucleotide triggers that correspond to each overlapping sequence of 200-300 nucleotides (in sense, antisense, or both sense and antisense orientation) are synthesized to classify their efficacy.
Polynucleotide triggers are evaluated by any convenient means to determine the silencing efficiency of the target gene for the Leptinotarsa species. A suitable test example is a food bioassay such as that described in Examples 5 and 6. Another suitable test involves the topical application of polynucleotide triggers either directly to Leptinotarsa individuals or to the surface of a plant to be protected from an infestation with the Leptinotarsa species. A desired result of treatment with a polynucleotide trigger is the prevention or control of an infestation with the Leptinotarsa species, for example by
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INDUSTRIAL induction in an insect Leptinotarsa a physiological or behavioral change such as, of iViótlU iiütaxative, growth weakening, increased mortality, decreased reproductive capacity, decreased or ceased movement or eating behavior or decreased or ceased development in the stage of metamorphosis. Another desired result of treatment with a polynucleotide trigger is to provide a nightshade plant that exhibits better resistance to an infestation with the Leptinotarsa species such as a potato, tomato, eggplant or pepper plant that exhibits better resistance to an infestation with Leptinotarsa decemiineata ( Colorado potato beetle, CPB) or other Leptinotarsa species. Polynucleotide triggers can be classified into sets. For example, the sets of five individual polynucleotide triggers are grouped into a single polynucleotide composition and applied topically to plants. Those pools that show better efficacy are reclassified by evaluating the individual component polynucleotide triggers to determine their efficacy.
The overlap procedure can be repeated, if desired. A polynucleotide trigger that is found to provide the desired activity can overlap itself. The original polynucleotide trigger is divided into smaller overlapping or non-overlapping segments over the entire length of the parent polynucleotide trigger. For example, the parent polynucleotide trigger is divided into segments 50-60 nucleotides in length over the entire length of the parent polynucleotide trigger. The polynucleotide triggers that correspond to each overlap sequence of 50-60 nucleotides (in sense, antisense, or both sense and antisense orientation) are synthesized to classify their efficacy. Additional rounds of overlap analysis can be performed, where triggers as short as 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides are evaluated.
Effective polynucleotide triggers of any size are used to make a composition for topical application or a recombinant DNA construct useful for making a transgenic plant.
EXAMPLE 5
This example illustrates a non-exhaustive assay useful for evaluating the efficacy in the control of Leptinotarsa of polynucleotide triggers. More specifically, this example illustrates double-stranded RNA triggers that comprise a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a Leptinotarse target gene (eg, a target gene that is selected from the Target Gene Sequence Group. or having a DNA sequence that is selected from the group consisting of: SEQ ID NO: 1-725 and SEQ ID NO: 726-830 and SEQ ID NO: 1087 - 1094, or its DNA complement), and a useful bioassay to assess efficacy
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<img file="MX359191B_D0152.tif" />
in Leptinotarsa control of these cdRNA triggers. —--—____
Triggers between about 50 and about 500 base pairs (more specifically, between about 100 and about 450 base pairs) in length were designed for Leptinotarse target genes (see Examples 2 and 3). Blunt-ended double-stranded RNAs (dsRNAs) with the antisense strand sequences provided in SEQ ID NO: 8311085 were made for the target genes listed in Table 1.
The cDNA triggers (Table 1) to suppress the Leptinotarse target genes were evaluated using the following methodology to test for mortality or weakness of Leptinotarsa decem / ineata due to contact with or ingestion of polynucleotide triggers. Bioassays with the Colorado potato beetle (CPB), Leptinotarsa decemiineata, were performed using an artificial diet consisting of 13.2 grams / liter of agar (Serva 11393), 140.3 grams / liter of Bio-Serve pre-mix (F9380B ), 5 mih'ülitres / liter of KOH (18.3% w / w), and 1.25 milliliters / liter of formalin (37%). The diet was provided in 200 microliter aliquots in 96-well plates and dried briefly prior to sample application. Twenty microliters of test sample per well was applied with sterile water as the untreated control (UTC). The plates were allowed to dry before adding insect larvae. A newborn CPB larva was added per well with a fine brush. The plates were sealed with Mylar and vented using an insect pin. Thirty-two larvae were evaluated per treatment. The bioassay plates were incubated at 27 degrees Celsius, 60% relative humidity, in total darkness for 10-12 days. The plates were scored according to the weakening and mortality of the larvae. Data were analyzed using JMP®4 statistical software (SAS Institute, 1995) and a full factorial ANOVA with a Dunnet test was performed to search for treatment effects compared to the untreated control (P <0.05). A Tukey-Kramer post hoc test was performed to compare all pairs of treatments (P <0.05). The results are provided in Table 1.
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TABLE 1
<td>I KNOW THAT ID NOT.*</td><td>Target gene</td><td>SEQ ID NOT. of the gen Diana</td><td>Results of CPB diet bioassay **</td><td>DsRNA concentration (Ppm)</td><td>No. of exon</td>
<td> 831</td><td>26S non-ATPase proteasome regulatory subunit 1</td><td> 825</td><td> (+)</td><td> 0.1</td><td> 1</td>
<td> 832</td><td>26S non-ATPase proteasome regulatory subunit 1</td><td> 825</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 833</td><td>26S non-ATPase proteasome regulatory subunit 1</td><td> 825</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 834</td><td>26S non-ATPase proteasome regulatory subunit 1</td><td> 825</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 835</td><td>26S non-ATPase proteasome regulatory subunit 1</td><td> 825</td><td> (-)</td><td> 0.1</td><td> 2</td>
<td> 836</td><td>Actin</td><td> 821</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 837</td><td>Actin</td><td> 821</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 838</td><td>Actin</td><td> 821</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 839</td><td>Actin</td><td> 821</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 840</td><td>Actin</td><td> 821</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 841</td><td>Coatomer beta subunit</td><td> 822</td><td> ()</td><td> 0.1</td><td> 1</td>
<td> 842</td><td>Coatomer beta subunit</td><td> 822</td><td> (+)</td><td> 0.1</td><td> 1</td>
<td> 843</td><td>Coatomer beta subunit</td><td> 822</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 844</td><td>Coatomer beta subunit</td><td> 822</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 845</td><td>Coatomer beta subunit</td><td> 822</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 846</td><td>26S non-ATPase proteasome regulatory subunit 2</td><td> 805</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 847</td><td>26S non-ATPase proteasome regulatory subunit 2</td><td> 805</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 848</td><td>26S non-ATPase proteasome regulatory subunit 2</td><td> 805</td><td> (-)</td><td> 0.1</td><td> 1</td>
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<td> 849</td><td>26S non-ATPase proteasome regulatory subunit 2</td><td> 805</td><td> (+)</td><td> 0.1</td><td> 1</td>
<td> 850</td><td>26S non-ATPase proteasome regulatory subunit 2</td><td> 805</td><td> (-)</td><td> 0.1</td><td> 2?</td>
<td> 851</td><td>26S non-ATPase proteasome regulatory subunit 12</td><td> 806</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 852</td><td>26S non-ATPase proteasome regulatory subunit 12</td><td> 806</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 853</td><td>26S non-ATPase proteasome regulatory subunit 12</td><td> 806</td><td>NT</td><td> 0.1</td><td> 2?</td>
<td> 854</td><td>26S non-ATPase proteasome regulatory subunit 12</td><td> 806</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 855</td><td>26S non-ATPase proteasome regulatory subunit 12</td><td> 806</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 856</td><td>Probable non-ATPase proteasome of 26S regulatory subunit 3</td><td> 807</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 857</td><td>Probable non-ATPase proteasome of 26S regulatory subunit 3</td><td> 807</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 858</td><td>Probable non-ATPase proteasome of 26S regulatory subunit 3</td><td> 807</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 859</td><td>Probable non-ATPase proteasome of 26S regulatory subunit 3</td><td> 807</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 860</td><td>Probable non-ATPase proteasome of 26S regulatory subunit 3</td><td> 807</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 861</td><td>26S non-ATPase proteasome regulatory subunit 7</td><td> 808</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 862</td><td>26S non-ATPase proteasome regulatory subunit 7</td><td> 808</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 863</td><td>26S non-ATPase proteasome regulatory subunit 7</td><td> 808</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 864</td><td>26S non-ATPase proteasome regulatory subunit 7</td><td> 808</td><td>NT</td><td> 0.1</td><td> 1</td>
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<td> 865</td><td>26S non-ATPase proteasome regulatory subunit 7</td><td> 808</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 866</td><td>26S non-ATPase proteasome regulatory subunit 2</td><td> 809</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 867</td><td>26S non-ATPase proteasome regulatory subunit 2</td><td> 809</td><td> (-)</td><td> 0.1</td><td> 2?</td>
<td> 868</td><td>26S non-ATPase proteasome regulatory subunit 2</td><td> 809</td><td> (-)</td><td> 0.1</td><td> 2</td>
<td> 869</td><td>26S non-ATPase proteasome regulatory subunit 2</td><td> 809</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 870</td><td>26S non-ATPase proteasome regulatory subunit 2</td><td> 809</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 871</td><td>265 Non-ATPase Proteasome regulatory subunit 4</td><td> 810</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 872</td><td>26S non-ATPase proteasome regulatory subunit 4</td><td> 810</td><td>NT</td><td> 0.1</td><td> 2</td>
<td> 873</td><td>26S non-ATPase proteasome regulatory subunit 4</td><td> 810</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 874</td><td>26S non-ATPase proteasome regulatory subunit 4</td><td> 810</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 875</td><td>26S non-ATPase proteasome regulatory subunit 4</td><td> 810</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 876</td><td>Protease regulatory subunit 8 from 26S</td><td> 811</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 877</td><td>Protease regulatory subunit 8 from 26S</td><td> 811</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 878</td><td>Protease regulatory subunit 8 from 26S</td><td> 811</td><td>NT</td><td> 0.1</td><td> 2?</td>
<td> 879</td><td>Protease regulatory subunit 8 from 26S</td><td> 811</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 880</td><td>Protease regulatory subunit 8 from 26S</td><td> 811</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 881</td><td>26S non-ATPase proteasome 1 regulatory subunit 13</td><td> 812</td><td>NT</td><td> 0.1</td><td> 2</td>
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<td rowspan="2"> 882</td><td>26S non-ATPase proteasome</td><td rowspan="2"> 812</td><td rowspan="2">NT</td><td rowspan="2"> 0.1</td><td rowspan="2"> 1</td>
<td>regulatory subunit 13</td>
<td> 883</td><td>26S non-ATPase proteasome regulatory subunit 13</td><td> 812</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 884</td><td>26S non-ATPase proteasome regulatory subunit 13</td><td> 812</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 885</td><td>26S non-ATPase proteasome regulatory subunit 13</td><td> 812</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 886</td><td>Putative uncharacterized protein</td><td> 813</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 887</td><td>Putative uncharacterized protein</td><td> 813</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 888</td><td>Putative uncharacterized protein</td><td> 813</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 889</td><td>GTPase factor activating protein ADP ribosllation, putative</td><td> 814</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 890</td><td>GTPase factor activating protein ADP ribosllation, putative</td><td> 814</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 891</td><td>GTPase factor activating protein ADP ribosllation, putative</td><td> 814</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 892</td><td>GTPase factor activating protein ADP ribosllation, putative</td><td> 814</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 893</td><td>Guanine exchange factor with resistance to brefeldin A Golgi specific, putative</td><td> 815</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 894</td><td>Guanine exchange factor with resistance to brefeldin A Golgi specific, putative</td><td> 815</td><td>NT</td><td> 0.1</td><td> 1</td>
useful I Mil II i IWf Ijll 1U.C9WW
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MEXICAN INSTITUTE .. r-eír <sup>1</sup> ’
OF THE C'm PROPERTY »,
INDUSTRIAL ** »
<td> 895</td><td>Guanine exchange factor with resistance to brefeldin A Golgi specific, putative</td><td> 815</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 896</td><td>Guanine exchange factor with resistance to brefeldin A Golgi specific, putative</td><td> 815</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 897</td><td>Guanine exchange factor with resistance to brefeldin A Golgi specific, putative</td><td> 815</td><td>NT</td><td> 0.1</td><td> 2?</td>
<td> 898</td><td>Sec24 protein, putative</td><td> 816</td><td> (+)</td><td> 0.1</td><td> 2</td>
<td> 899</td><td>Sec24 protein, putative</td><td> 816</td><td> (-)</td><td> 0.1</td><td> 2?</td>
<td> 900</td><td>Sec24 protein, putative</td><td> 816</td><td> (-)</td><td> 0.1</td><td> 2?</td>
<td> 901</td><td>Sec24 protein, putative</td><td> 816</td><td> (-)</td><td> 0.1</td><td> 2?</td>
<td> 902</td><td>Sec24 protein, putative</td><td> 816</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 903</td><td>Protein Sec24B transport protein</td><td> 817</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 904</td><td>Protein Sec24B transport protein</td><td> 817</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 905</td><td>Protein Sec24B transport protein</td><td> 817</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 906</td><td>Protein Sec24B transport protein</td><td> 817</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 907</td><td>Protein Sec24B transport protein</td><td> 817</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 908</td><td>Protein sec31A transport protein</td><td> 818</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 909</td><td>Protein sec31A transport protein</td><td> 818</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 910</td><td>Protein sec31A transport protein</td><td> 818</td><td> (+)</td><td> 0.1</td><td> 1</td>
HllIBi l'.H «a»
<img file="MX359191B_D0156.tif" />
<td rowspan="2"> 911</td><td>Transport protein sec31A</td><td rowspan="2">of</td><td rowspan="2"> 818</td><td rowspan="2"> (-)</td><td rowspan="2"> 0.1</td><td rowspan="2"> 2?</td>
<td>protein</td>
<td> 912</td><td>Transport protein sec31A protein</td><td>of</td><td> 818</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 913</td><td>GTP SAR1B binding protein</td><td></td><td> 819</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 914</td><td>GTP SAR1B binding protein</td><td></td><td> 819</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 915</td><td>GTP SAR1B binding protein</td><td></td><td> 819</td><td>NT</td><td> 0.1</td><td> 2</td>
<td> 916</td><td>GTP SAR1B binding protein</td><td></td><td> 819</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 917</td><td>GTP SAR1B binding protein</td><td></td><td> 819</td><td>NT</td><td> 0.1</td><td> 1</td>
<td> 918</td><td>Transport secl3 protein protein</td><td>of</td><td> 820</td><td> (-)</td><td> 0.1</td><td> 2</td>
<td> 919</td><td>Transport secl3 protein protein</td><td>of</td><td> 820</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 920</td><td>Transport secl3 protein protein</td><td>of</td><td> 820</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 921</td><td>Transport secl3 protein protein</td><td>of</td><td> 820</td><td> (-)</td><td> 0.1</td><td> 1</td>
<td> 922</td><td>L13A ribosomal protein</td><td></td><td> 741</td><td>NT</td><td> 1.0</td><td> 2</td>
<img file="MX359191B_D0157.tif" />
IMPI
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY
<td> 923</td><td>L13A ribosomal protein</td><td> 741</td><td>NT</td><td>το—</td><td>z</td>
<td> 924</td><td>60S L5 ribosomal protein</td><td> 728</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 925</td><td>60S r5bosomal L5 protein</td><td> 728</td><td> (+)</td><td> 1.0</td><td> 2?</td>
<td> 926</td><td>Rlbosomal protein S7</td><td> 776</td><td>NT</td><td> 1.0</td><td> 1</td>
<td> 927</td><td>S7 ribosomal protein</td><td> 776</td><td> (-)</td><td> 1.0</td><td> 1</td>
<td> 928</td><td>Rlbosomal protein L9</td><td> 735</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 929</td><td>Rlbosomal protein L9</td><td> 735</td><td>NT</td><td> 1.0</td><td> 1</td>
<td> 930</td><td>L3 ribosomal protein</td><td> 726</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 931</td><td>Rlbosomal protein L3</td><td> 726</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 932</td><td>60S rlbosomal protein L32</td><td> 755</td><td> (+)</td><td> 1.0</td><td> 3</td>
<td> 933</td><td>L8 ribosomal protein</td><td> 734</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 934</td><td>L8 ribosomal protein</td><td> 734</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 935</td><td>S15 ribosomal protein</td><td> 785</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 936</td><td>S15 ribosomal protein</td><td> 785</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 937</td><td>L7A ribosomal protein</td><td> 732</td><td> (+)</td><td> 1.0</td><td> 3</td>
<td> 938</td><td>Rlbosomal protein L7A</td><td> 732</td><td> (+)</td><td> 1.0</td><td> 3</td>
<td> 939</td><td>40S rlbosomal protein S14</td><td> 784</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 940</td><td>40S ribosomal protein S14</td><td> 784</td><td> (+)</td><td> 1.0</td><td> 2</td>
<img file="MX359191B_D0158.tif" />
148
IMPI
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY
<td rowspan="2"> 941</td><td rowspan="2">40S ribosomal protein S24</td><td rowspan="2"> 796</td><td>(_χ_ \</td><td></td><td></td>
<td>(+ J '</td><td>lU</td><td>ΖΓ</td>
<td> 942</td><td>60S L10A ribosomal protein</td><td> 737</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 943</td><td>L13 ribosomal protein</td><td> 740</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 944</td><td>L13 ribosomal protein</td><td> 740</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 945</td><td>S13 ribosomal protein</td><td> 783</td><td> (+)</td><td> 1.0</td><td> 3</td>
<td> 946</td><td>S13 ribosomal protein</td><td> 783</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 947</td><td>L4e ribosomal protein</td><td> 727</td><td> (+)</td><td> 1.0</td><td> 3</td>
<td> 948</td><td>L4e ribosomal protein</td><td> 727</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 949</td><td>S30 ribosomal protein</td><td> 803</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 950</td><td>S30 ribosomal protein</td><td> 803</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 951</td><td>L26 ribosomal protein</td><td> 749</td><td> (+)</td><td> 1.0</td><td> 2?</td>
<td> 952</td><td>L26 ribosomal protein</td><td> 749</td><td> (+)</td><td> 1.0</td><td> 2?</td>
<td> 953</td><td>L31 ribosomal protein</td><td> 754</td><td>NT</td><td> 1.0</td><td> 3</td>
<td> 954</td><td>60S ribosomal IOL protein</td><td> 736</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 955</td><td>60S ribosomal IOL protein</td><td> 736</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 956</td><td>S4 ribosomal protein</td><td>7T2.</td><td> (+)</td><td> 1.0</td><td> 3</td>
<td> 957</td><td>S4 ribosomal protein</td><td>neither</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 958</td><td>Lile ribosomal protein</td><td> 738</td><td> (+)</td><td> 1.0</td><td> 2</td>
IMPI
<img file="MX359191B_D0159.tif" />
149
INSTITUTE M EX ΙΟ N <. or,
OF PROPERTY C> »¿J INDUSTRIAL
<td rowspan="2"> 959</td><td rowspan="2">S6 ribosomal protein</td><td rowspan="2"> 774</td><td></td><td> 1.0</td><td> 1</td>
<td></td><td></td><td></td>
<td> 960</td><td>Sil ribosomal protein</td><td> 782</td><td> (+)</td><td> 1.0</td><td> 3</td>
<td> 961</td><td>Sil ribosomal protein</td><td> 782</td><td> (+)</td><td>Ϊ.0</td><td> 3</td>
<td> 962</td><td>Sil ribosomal protein</td><td> 781</td><td>NT</td><td> 1.0</td><td> 3</td>
<td> 963</td><td>Sil ribosomal protein</td><td> 781</td><td>NT</td><td> 1.0</td><td> 3</td>
<td> 964</td><td>L12e ribosomal protein</td><td> 739</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 965</td><td>L12e ribosomal protein</td><td> 739</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 966</td><td>S5 ribosomal protein</td><td> 773</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 967</td><td>S5 ribosomal protein</td><td> 773</td><td> (+)</td><td> 1.0</td><td> 3</td>
<td> 968</td><td>S18 ribosomal protein</td><td> 790</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 969</td><td>S18 ribosomal protein</td><td> 790</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 970</td><td>L23A ribosomal protein</td><td> 747</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 971</td><td>L23A ribosomal protein</td><td> 747</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 972</td><td>L35A ribosomal protein</td><td> 759</td><td>NT</td><td> 1.0</td><td> 1</td>
<td> 973</td><td>L35A ribosomal protein</td><td> 759</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 974</td><td>L21 ribosomal protein</td><td> 746</td><td>NT</td><td> 1.0</td><td> 2?</td>
<td> 975</td><td>L21 ribosomal protein</td><td> 746</td><td>NT</td><td> 1.0</td><td> 2?</td>
<td> 976</td><td>L21 ribosomal protein</td><td> 745</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 977</td><td>L21 ribosomal protein</td><td> 745</td><td> (-)</td><td> 1.0</td><td> 2?</td>
<td> 978</td><td>S8 ribosomal protein</td><td> 777</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 979</td><td>S8 ribosomal protein</td><td> 777</td><td> (+)</td><td> 1.0</td><td> 3</td>
<td> 980</td><td>S16 ribosomal protein</td><td> 788</td><td>NT</td><td> 1.0</td><td> 1</td>
<td> 981</td><td>S16 ribosomal protein</td><td> 799</td><td>NT</td><td> 1.0</td><td> 2</td>
IMPI
150
MEXICAN INSTITUTE f> í LA AROHEDAD
INDUSTRIAL
<td rowspan="2"> 982</td><td rowspan="2">L18Ae ribosomal protein</td><td rowspan="2"> 744</td><td> (+)</td><td>1-Q</td><td> 9</td>
<td></td><td></td><td></td>
<td> 983</td><td>S6 ribosomal protein</td><td> 775</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 984</td><td>S3 ribosomal protein</td><td> 768</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 985</td><td>S3 ribosomal protein</td><td> 768</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 986</td><td>S17 ribosomal protein</td><td> 789</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 987</td><td>S15A ribosomal protein</td><td> 786</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 988</td><td>L7 ribosomal protein</td><td> 730</td><td> (+)</td><td> 1.0</td><td> 2?</td>
<td> 989</td><td>L7 ribosomal protein</td><td> 730</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 990</td><td>S4 ribosomal protein</td><td> 771</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 991</td><td>S4 ribosomal protein</td><td> 771</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 992</td><td>40S S3A ribosomal protein</td><td> 769</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 993</td><td>40S S3A ribosomal protein</td><td> 769</td><td>NT</td><td> 1.0</td><td> 1</td>
<td> 994</td><td>L36 ribosomal protein</td><td> 760</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 995</td><td>L37 ribosomal protein</td><td> 762</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 996</td><td>L37 ribosomal protein</td><td> 763</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 997</td><td>S19 ribosomal protein</td><td> 792</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 998</td><td>S19 ribosomal protein</td><td> 792</td><td>NT</td><td> 1.0</td><td> 1</td>
<td> 999</td><td>S19 ribosomal protein</td><td> 792</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1000</td><td>S20 ribosomal protein</td><td> 794</td><td>NT</td><td> 1.0</td><td> 1</td>
<td> 1001</td><td>L15 ribosomal protein</td><td> 743</td><td>NT</td><td> 1.0</td><td> 2</td>
151
<img file="MX359191B_D0160.tif" />
<td> 1002</td><td>L35A ribosomal protein</td><td> 758</td><td>NT</td><td>JL.O</td><td> 1</td>
<td> 1003</td><td>L35A ribosomal protein</td><td> 758</td><td>NT</td><td> 1.0</td><td> 1</td>
<td> 1004</td><td>40S S21 ribosomal protein</td><td> 795</td><td>NT</td><td> 1.0</td><td> 3</td>
<td> 1005</td><td>S29 ribosomal protein</td><td> 802</td><td>NT</td><td> 1.0</td><td> 1</td>
<td> 1006</td><td>S8 ribosomal protein</td><td> 778</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1007</td><td>40S S3A ribosomal protein</td><td> 770</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1008</td><td>L24 ribosomal protein</td><td> 748</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1009</td><td>S16 ribosomal protein</td><td> 787</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1010</td><td>L7A ribosomal protein</td><td> 733</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1011</td><td>40S S9 ribosomal protein</td><td> 780</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 1012</td><td>40S ribosomal protein</td><td> 804</td><td>NT</td><td> 1.0</td><td> 1</td>
<td> 1013</td><td>40S ribosomal protein</td><td> 804</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1014</td><td>L37Ae ribosomal protein</td><td> 764</td><td> (-)</td><td> 1.0</td><td> 2?</td>
<td> 1015</td><td>60S L23 ribosomal protein</td><td> 797</td><td>NT</td><td> 1.0</td><td> 1</td>
<td> 1016</td><td>L7 ribosomal protein</td><td> 731</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 1017</td><td>L36 ribosomal protein</td><td> 761</td><td>NT</td><td> 1.0</td><td> 1</td>
<td> 1018</td><td>40S S9 ribosomal protein</td><td> 779</td><td> (+)</td><td> 1.0</td><td> 2?</td>
<td> 1019</td><td>S26 ribosomal protein</td><td> 798</td><td> (+)</td><td> 1.0</td><td> 3</td>
<td> 1020</td><td>L34A ribosomal protein</td><td> 756</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1021</td><td>L27Ae ribosomal protein</td><td> 751</td><td>NT</td><td> 1.0</td><td> 1</td>
<td> 1022</td><td>L27Ae ribosomal protein</td><td> 751</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1023</td><td>40S ribosomal protein S28</td><td> 801</td><td> (-)</td><td> 1.0</td><td> 2?</td>
<td> 1024</td><td>L29 ribosomal protein</td><td> 753</td><td> (-)</td><td> 1.0</td><td> 3</td>
<td> 1025</td><td>L28 ribosomal protein</td><td> 752</td><td> (+)</td><td> 1.0</td><td> 4</td>
<td> 1026</td><td>L28 ribosomal protein</td><td> 752</td><td>NT</td><td> 1.0</td><td> 4</td>
<td> 1027</td><td>Biogenesis ribosomal protein RLP24</td><td> 765</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 1028</td><td>Biogenesis ribosomal protein RLP24</td><td> 765</td><td> (-)</td><td> 1.0</td><td> 1</td>
<td> 1029</td><td>L27 ribosomal protein</td><td> 750</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1030</td><td>L27 ribosomal protein</td><td> 750</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1031</td><td>39S L13 ribosomal protein</td><td> 766</td><td> (-)</td><td> 1.0</td><td> 3</td>
<td> 1032</td><td>39S L13 ribosomal protein</td><td> 766</td><td> (-)</td><td> 1.0</td><td> 3</td>
<img file="MX359191B_D0161.tif" />
152
IMPI
MEXICAN INSTITUTE The INTUSlftiAt PROPERTY
<td> 1033</td><td>S2 ribosomal protein</td><td> 767</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1034</td><td>40S ribosomal protein S28</td><td> 800</td><td> (-)</td><td> 1.0</td><td> 2?</td>
<td> 1035</td><td>L14 ribosomal protein</td><td> 742</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1036</td><td>L6 ribosomal protein</td><td> 729</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1038</td><td>Coatomer gamma subunit</td><td> 828</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1039</td><td>Myosin Vlla</td><td> 824</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1040</td><td>Myosin Vlla</td><td> 823</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1041</td><td>Actin</td><td> 821</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1042</td><td>26S non-ATPase proteasome regulatory subunit 1</td><td> 826</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1043</td><td>26S non-ATPase proteasome regulatory subunit 1</td><td> 825</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1044</td><td>Crooked neck</td><td> 830</td><td>NT</td><td> 1.0</td><td> 1</td>
<td> 1045</td><td>Crooked neck</td><td> 829</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1046</td><td>Putative predlcha protein</td><td> 827</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1047</td><td>26S non-ATPase proteasome regulatory subunit 2</td><td> 805</td><td> (+)</td><td> 1.0</td><td> 2</td>
<img file="MX359191B_D0162.tif" />
153
IMPI
M EX INSTITUTE (CA no
M LA MONEDAD O »industrial
<td rowspan="2"> 1048</td><td>Regulatory subunit 2 of</td><td rowspan="2"> 806</td><td rowspan="2"> (-)</td><td rowspan="2"> 1.0</td><td rowspan="2"> 2</td>
<td>26S non-ATPase proteasome</td>
<td> 1049</td><td>Probable non-ATPase proteasome of 26S regulatory subunit 3</td><td> 807</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1050</td><td>26S non-ATPase proteasome regulatory subunit 7</td><td> 808</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1051</td><td>26S non-ATPase proteasome regulatory subunit 2</td><td> 809</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 1052</td><td>26S non-ATPase proteasome regulatory subunit 4</td><td> 810</td><td> (-)</td><td> 1.0</td><td> 3</td>
<td> 1053</td><td>Protease regulatory subunit 8 from 26S</td><td> 811</td><td> (+)</td><td> 1.0</td><td> 3</td>
<td> 1054</td><td>26S non-ATPase proteasome regulatory subunit 13</td><td> 812</td><td> (+)</td><td> 1.0</td><td> 3</td>
<td> 1055</td><td>Putative uncharacterized protein</td><td> 813</td><td> (-)</td><td> 1.0</td><td> 2</td>
<td> 1056</td><td>GTPase activating protein ADP ribosiladon factor, putative</td><td> 814</td><td> (-)</td><td> 1.0</td><td> 2</td>
<td> 1057</td><td>Guanine exchange factor with resistance to brefeldin A Golgi specific, putative</td><td> 815</td><td> (-)</td><td> 1.0</td><td> 2?</td>
<td> 1058</td><td>Sec24 protein, putative</td><td> 816</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1059</td><td>Protein Transport Protein Sec24B</td><td> 817</td><td> (-)</td><td> 1.0</td><td> 1</td>
<td> 1060</td><td>Protein sec31A transport protein</td><td> 818</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1061</td><td>GTP SAR1B binding protein</td><td> 819</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1062</td><td>Protein secl3 protein transport</td><td> 820</td><td> (-)</td><td> 1.0</td><td> 2?</td>
<td> 1063</td><td>Protein Sec24B</td><td> 817</td><td> (-)</td><td> 1.0</td><td> 1</td>
<td> 1064</td><td>Coatomer beta subunit</td><td> 822</td><td> (+)</td><td> 1.0</td><td> 2</td>
<img file="MX359191B_D0163.tif" />
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<td> 1065</td><td>Coatomer gamma subunit</td><td> 828</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1066</td><td>Myosin Vlla</td><td> 824</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1067</td><td>Myosin Vlla</td><td> 823</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1068</td><td>Actin</td><td> 821</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1069</td><td>26S non-ATPase proteasome regulatory subunit 1</td><td> 825</td><td>NT</td><td> 1.0</td><td> 2</td>
<td> 1070</td><td>Crooked neck</td><td> 829</td><td> (+)</td><td> 1.0</td><td> 2</td>
<td> 1071</td><td>26S non-ATPase proteasome regulatory subunit 2</td><td> 805</td><td> ()</td><td> 1.0</td><td> 2</td>
<td> 1078</td><td>26S non-ATPase proteasome regulatory subunit 13</td><td> 812</td><td> (+)</td><td> 1.0</td><td> 3</td>
<td> 1079</td><td>GTPase factor activating protein ADP ribosylation, putative</td><td> 814</td><td> (-)</td><td> 1.0</td><td> 2</td>
<td> 1080</td><td>Guanine exchange factor with specific brefeldin A resistance Golgi, putative</td><td> 815</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1081</td><td>Sec24 protein, putative</td><td> 816</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1082</td><td>Protein Sec24B transport protein</td><td> 817</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1083</td><td>Protein sec31A transport protein</td><td> 818</td><td> (-)</td><td> 1.0</td><td> 1</td>
<td> 1084</td><td>GTP SAR1B binding protein</td><td> 819</td><td> (+)</td><td> 1.0</td><td> 1</td>
<td> 1085</td><td>Protein secl3 transport protein</td><td> 820</td><td> (+)</td><td> 1.0</td><td> 1</td>
* dsRNA trigger antisense chain sequence ** (+) Significant weakening or mortality compared to the water-treated control; (-) there was no significant weakening or mortality compared to the water-treated control; NT = either (1) the trigger was not evaluated or (2) the following two things happened: the sample did not provide significant weakening / mortality and the positive control did not provide significant weakening / mortality in that test. The positive control used in this assay was the dsRNA trigger that targets the beta coatomer and has the sense chain sequence of SEQ ID NO: 1086, previously described as SEQ ID NO: 880 in US Patent No. 7,943,819.
nseeóesaesasaBeraesfflBSBRS *
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<sub>155</sub> ΙΜΡΪ MEXICAN institute OF THE INIMimiAL PHOREPAD
When available genomic sequence data allowed, the number of exons spanning a given trigger sequence was determined and is provided in Table 1: 1 Indicates that the trigger sequence appears to be contained in a single contiguous genomic locus; 2? indicates that the full length of the trigger was not aligned with the genome, where at least 40 base pairs are missing, which may indicate that the available genomic sequence data is not complete.
Additional cDNA sequences encoding subunits of an exocyst complex Leptinotarsa decemiineata (Colorado potato beetle, CPB) were identified from a separate sequencing and assembly project as Leptinotarse target genes. These exocyst Leptinotarse target genes, SEQ ID NO: 1087-1094, are useful for designing polynudeotide triggers comprising at least 21 contiguous nucleotides complementary to an exocyst target gene and useful for controlling infestations of the Leptinotarse species and for making transgenic plants that express these polynudeotide triggers for resistance to infestations of the Leptinotarsa species.
Triggers between about 50 and about 500 base pairs (more specifically, between about 100 and about 450 base pairs) in length were designed for each of the Leptinotarsa exocyst target genes (SEQ ID NO: 1087 1094) as described in Example 4. These triggers are evaluated using the same methodology as that described above for the polynudeotides in Table 1.
In a non-exhaustive example, a pollnucleotide trigger, designed to target the Exo70 gene ae Leptinotarsa decemiineata (SEQ ID NO: 1093), was produced as a blunt-ended double-stranded RNA having the antisense strand sequence of SEQ ID NO: 1095. This trigger gave significant weakening and significant mortality at both evaluated concentrations, using the methodology described above. The results are provided in
Table 2.
<img file="MX359191B_D0165.tif" />
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156
TABLE 2
<td>I KNOW THAT ID NOT:*</td><td>Length of the trigger (bp)</td><td>Target gene</td><td>SEQ ID NO: DEL GEN DIANA</td><td>Results of CPB diet bioassay **</td><td>DsRNA concentration (ppm)</td>
<td> 1095</td><td> 277</td><td>Exo70</td><td> 1093</td><td> (+)</td><td> 0.1</td>
<td> 1095</td><td> 277</td><td>Exo70</td><td> 1093</td><td>í ±)</td><td> 0.033</td>
♦ dsRNA trigger antisense chain sequence ** (+) Significant weakening or mortality compared to the water-treated control; (-) there was no significant weakening or mortality compared to the water-treated control; NT = either (1) the trigger was not evaluated or (2) the following two things happened: the sample did not provide significant weakening / mortality and the positive control did not provide significant weakening / mortality in that test. The positive control used in this assay was the dsRNA trigger that targets the beta coatomer and has the sense chain sequence of SEQ ID NO: 1086, previously described as SEQ ID NO: 880 in US Patent No. 7,943,819.
EXAMPLE 6
This example illustrates non-exhaustive modalities of polynucleotides of the present invention, insecticidal compositions for controlling a Leptinotarse species, and a representative assay useful for evaluating Leptinotarse control efficacy of these polynucleotides.
Five dsRNA triggers (with antisense strand sequences from SEQ ID NOs: 989, 1049, 1050, 1078, and 1084; see Table 1) were evaluated to suppress Leptinotarse target genes using the following leaf disk methodologies to assay for mortality or weakening of Leptinotarsa decemiineata larvae due to contact with or ingestion of polynucleotide triggers.
For the leaf disc bioassay with adult insects, newly emerged Colorado potato beetle (CPB, Leptinotarsa decemiineata) adults were collected and kept in potato foliage for up to 7 days, and then fasted for 6 -8 hours before starting the bioassay. Fifteen adults were used per treatment (trigger / dose). Ten microliters containing 250, 83.3, 27.8, or 9.3 nanograms of dsRNA trigger in a 0.1% Silwet L77 solution in UltraPure water (Invitrogen) were applied to potato leaf discs (Atlantic variety) 15 millimeters in diameter; control sheet discs were treated with either the 0.1% Silwet L77 solution formulation or a negative control trigger designed to silence the green fluorescent protein (GFP). The treated leaf disks were individually placed in 6-well group plate wells containing 2 milliliters / well of a solidified agar agar / 2% distilled water matrix. A single adult CPB was placed in each well and incubated overnight to allow the leaf disk to consume; in cases where the disc
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157 leaf was not fully consumed, the insect probably pstaha. m.iori-n n ipc¡onado by manipulation and was excluded from the trial. The next day, the CPB adults from a given trigger / dose treatment were collectively transferred to a feeding track made of a ventilated, covered, 16-ounce translucent plastic container, with the base lined with filter paper and containing foliage. potato (Atlantic variety) with the stems inserted in a tube filled with water to stay cool. The insects were incubated in the feeding track in an environmental chamber (27 degrees Celsius; 60% relative humidity; 16 hours light / 8 hours dark) with potato foliage filled in as needed. Insect viability was monitored daily. The insects were recorded as active (viable), dying (does not stand on the legs after 10 seconds of being placed on its back) or dead. Feasibility results are provided in Table 3.
TABLE 3
<td rowspan="2">Treatment</td><td rowspan="2">SEQ ID NO of target gene CPB</td><td colspan="9">Days from treatment</td>
<td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td><td> 12</td><td> 14</td><td> 16</td>
<td>Formulation 1</td><td>n / a</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td>
<td>Formulation 2</td><td>n / a</td><td> 93</td><td> 93</td><td> 93</td><td> 93</td><td> 93</td><td> 93</td><td> 86</td><td> 86</td><td> 86</td>
<td>SEQ ID NO: 1115, GFP-1</td><td>n / a</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td><td> 80</td><td> 80</td><td> 60</td>
<td>SEQ ID NO: 1115, GFP-2</td><td>n / a</td><td> 93</td><td> 93</td><td> 87</td><td> 87</td><td> 80</td><td> 80</td><td> 80</td><td> 80</td><td> 80</td>
<td>SEQ ID NO: 989 *, 250 ng</td><td> 730</td><td> 87</td><td> 87</td><td> 80</td><td> 33</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO: 989 *, 83 Ώ3</td><td> 730</td><td> 100</td><td> 100</td><td> 79</td><td> 43</td><td> 29</td><td> 7</td><td> 0</td><td> 0</td><td> 0</td>
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<td rowspan="2">SEQ ID NO: 989 *, 9 ng</td><td rowspan="2"> 730</td><td rowspan="2"> 93</td><td rowspan="2"> 93</td><td rowspan="2"> 73</td><td rowspan="2"> 60</td><td rowspan="2"></td><td rowspan="2"></td><td></td><td></td><td rowspan="2">Θ—</td>
<td>OR</td><td>"Θ '</td>
<td>SEQ ID NO: 1049 *, 250 ng</td><td> 807</td><td> 40</td><td> 13</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO: 1049 *, 83 J2S</td><td> 807</td><td> 80</td><td> 7</td><td> 7</td><td> 7</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO: 1049 *, 28 J22</td><td> 807</td><td> 80</td><td> 13</td><td> 13</td><td> 13</td><td> 13</td><td> 7</td><td> 13</td><td> 7</td><td> 7</td>
<td>SEQ ID NO: 1049 *, 9 QS</td><td> 807</td><td> 87</td><td> 73</td><td> 60</td><td> 60</td><td> 60</td><td> 60</td><td> 53</td><td> 53</td><td> 53</td>
<td>SEQ ID NO: 1050 *, 250 ng</td><td> 808</td><td> 60</td><td> 13</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO: 1050 *, 83 ng</td><td> 808</td><td> 60</td><td> 20</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO: 1050 *, 28 ng</td><td> 808</td><td> 86</td><td> 29</td><td> 29</td><td> 14</td><td> 14</td><td> 14</td><td> 14</td><td> 14</td><td> 14</td>
<td>SEQ ID NO: 1050 *, 9 ng</td><td> 808</td><td> 80</td><td> 60</td><td> 60</td><td> 53</td><td> 53</td><td> 53</td><td> 47</td><td> 40</td><td> 40</td>
<td>SEQ ID NO: 1078 *, 250 ng</td><td> 812</td><td> 67</td><td> 27</td><td> 20</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO: 1078 *, 83 ng</td><td> 812</td><td> 60</td><td> 13</td><td> 7</td><td> 7</td><td> 7</td><td> 7</td><td> 7</td><td> 7</td><td> 7</td>
<td>SEQ ID NO: 1078 *, 28 ng</td><td> 812</td><td> 73</td><td> 33</td><td> 20</td><td> 13</td><td> 13</td><td> 13</td><td> 13</td><td> 13</td><td> 13</td>
<td>SEQ ID NO: 1078 *, 9 ng</td><td> 812</td><td> 100</td><td> 80</td><td> 80</td><td> 67</td><td> 60</td><td> 60</td><td> 53</td><td> 47</td><td> 47</td>
<td>SEQ ID NO: 1084 *, 250 ng</td><td> 819</td><td> 33</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO: 1084 *, 83 ng</td><td> 819</td><td> 73</td><td> 33</td><td> 7</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO: 1084 *, 28 ng</td><td> 819</td><td> 73</td><td> 40</td><td> 33</td><td> 33</td><td> 33</td><td> 33</td><td> 20</td><td> 20</td><td> 20</td>
<td>SEQ ID NO: 1084 *, 9 ng</td><td> 819</td><td> 80</td><td> 60</td><td> 53</td><td> 53</td><td> 53</td><td> 53</td><td> 47</td><td> 47</td><td> 40</td>
* ARNcd trigger antisense chain sequence, unless otherwise specified.
Formulation 1 and Formulation 2 are couplets of a null control (0.1% Sllwet in water). GFP-1 and GFP-2 are couplets of a negative control using a 377 bp dsRNA trigger that targets the green fluorescent protein (GFP) and has the sense chain sequence of SEQ ID NO: 1115. n / a = not applicable.
For larvae of leaf disc with larvae, newborn Colorado potato beetle larvae (CPB, Leptinotarsa decemiineata} incubated within 24 hours after bloensay were used. Sixteen larvae were used per treatment (dlsparador / dosls). Two mlcrollters containing 250, 83.3, 27.8, or 9.3 nanograms of dsRNA trigger in a 0.1% Sllwet L77 solution in UltraPure water (Invitrogen) were applied to potato leaf discs (variety
<img file="MX359191B_D0168.tif" />
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INDUSTRIAL
Atlantic) 7 millimeters in diameter; the control sheet disks are treated with ιυιι lu was Wow of »0.1% Silwet L77 solution or with a negative control trigger designed to silence the green fluorescent protein (GFP). The treated leaf disks were individually placed in wells of 128-well group plates containing 0.5 milliliter / well of a solidified agar agar / 2% distilled water matrix. A single newborn CPB was placed in each well and incubated overnight to allow the leaf disk to consume; In cases where the leaf disk was not fully consumed, the Insect was likely dead or injured by manipulation and was excluded from the trial. The next day, the CPB larvae from a given trigger / dose treatment were collectively transferred to a feeder track made of a ventilated, covered 16-ounce translucent plastic container, with the base lined with filter paper and containing foliage potato (Atlantic variety) with stems Inserted in a tube filled with water to keep it cool. The insects were incubated on the feeder track in an environmental chamber (27 degrees Celsius; 60% relative humidity; 16 hours light / 8 hours dark) with potato foliage filled in as needed. Larvae viability was monitored daily. Larvae were recorded as alive or dead. Feasibility results are provided in Table 4.
TABLE 4
<td rowspan="2">Treatment</td><td rowspan="2">I KNOW THAT NOT: gen CPB</td><td rowspan="2">ID of Diana</td><td colspan="9">Days from treatment</td>
<td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td><td> 12</td><td> 14</td><td> 16</td>
<td>Formulation 1</td><td colspan="2">n / a</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td><td> 92</td><td> 54</td><td> 15</td>
<td>Formulation 2</td><td colspan="2">n / a</td><td> 87</td><td> 87</td><td> 87</td><td> 87</td><td> 73</td><td> 73</td><td> 73</td><td> 27</td><td> 20</td>
<td>SEQ ID NO: 1115, GFP-1</td><td colspan="2">n / a</td><td> 69</td><td> 69</td><td> 69</td><td> 69</td><td> 69</td><td> 69</td><td> 69</td><td> 50</td><td> 38</td>
<td>SEQ ID NO: 1115, GFP-2</td><td colspan="2">n / a</td><td> 100</td><td> 100</td><td> 94</td><td> 94</td><td> 75</td><td> 75</td><td> 56</td><td> 19</td><td> 19</td>
<td>SEQ ID NO: 989 *, 250 ng</td><td colspan="2"> 730</td><td> 44</td><td> 38</td><td> 31</td><td> 13</td><td> 13</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO: 989 *, 83 ng</td><td colspan="2"> 730</td><td> 19</td><td> 19</td><td> 13</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO: 989 * 28 ng</td><td colspan="2"> 730</td><td> 69</td><td> 50</td><td> 38</td><td> 13</td><td> 13</td><td> 6</td><td> 6</td><td> 6</td><td> 6</td>
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<td>SEQ ID NO</td><td>989 *, 9 ng</td><td> 730</td><td> 38</td><td> 13</td><td> 13</td><td> 13</td><td> 6</td><td> 6</td><td> 6</td><td> 6</td><td> 6</td>
<td>SEQ ID NO</td><td>1049 *, 83 ng</td><td> 807</td><td> 38</td><td> 13</td><td> 13</td><td> 13</td><td> 13</td><td> 13</td><td> 13</td><td> 13</td><td> 13</td>
<td>SEQ ID NO</td><td>1049 *, 28 ng</td><td> 807</td><td> 38</td><td> 13</td><td> 13</td><td> 6</td><td> 6</td><td> 6</td><td> 6</td><td> 6</td><td> 6</td>
<td>SEQ ID NO</td><td>1049 *, 9 ng</td><td> 807</td><td> 57</td><td> 21</td><td> 21</td><td> 21</td><td> 21</td><td> 21</td><td> 21</td><td> 21</td><td> 14</td>
<td>SEQ ID NO</td><td>1050 *, 250 ng</td><td> 808</td><td> 44</td><td> 31</td><td> 31</td><td> 25</td><td> 19</td><td> 19</td><td> 19</td><td> 0</td><td> 0</td>
<td>SEQ ID NO</td><td>1050 *, 83 ng</td><td> 808</td><td> 38</td><td> 19</td><td> 19</td><td> 6</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO</td><td>1050 *, 28 ng</td><td> 808</td><td> 13</td><td> 13</td><td> 13</td><td> 13</td><td> 13</td><td> 13</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO</td><td>1050 *, 9 ng</td><td> 808</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO</td><td>1078 *, 250 ng</td><td> 812</td><td> 19</td><td> 13</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO</td><td>1078 *, 83 ng</td><td> 812</td><td> 29</td><td> 14</td><td> 14</td><td> 7</td><td> 7</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td>SEQ ID NO</td><td>1078 *, 28 ng</td><td> 812</td><td> 50</td><td> 31</td><td> 19</td><td> 13</td><td> 13</td><td> 6</td><td> 6</td><td> 0</td><td> 0</td>
<td>SEQ ID NO</td><td>1078 *, 9 ng</td><td> 812</td><td> 60</td><td> 47</td><td> 40</td><td> 27</td><td> 27</td><td> 27</td><td> 27</td><td> 27</td><td> 13</td>
<td>SEQ ID NO</td><td>1084 *, 250 ng</td><td> 819</td><td> 79</td><td> 43</td><td> 43</td><td> 43</td><td> 29</td><td> 21</td><td> 21</td><td> 14</td><td> 14</td>
<td>SEQ ID NO</td><td>1084 *, 83 ng</td><td> 819</td><td> 56</td><td> 38</td><td> 19</td><td> 19</td><td> 19</td><td> 13</td><td> 13</td><td> 13</td><td> 13</td>
<td>SEQ ID NO</td><td>1084 *, 28 ng</td><td> 819</td><td> 50</td><td> 38</td><td> 25</td><td> 19</td><td> 19</td><td> 19</td><td> 19</td><td> 19</td><td> 19</td>
<td>SEQ ID NO</td><td>1084 *, 9 ng</td><td> 819</td><td> 75</td><td> 50</td><td> 44</td><td> 44</td><td> 38</td><td> 38</td><td> 38</td><td> 31</td><td> 31</td>
♦ dsRNA trigger antisense chain sequence, unless otherwise specified.
Formulation 1 and Formulation 2 are copies of a null control (0.1% Silwet in water). GFP-1 and GFP2 are copies of a negative control using a 377 bp dsRNA trigger that targets the green fluorescent protein (GFP) and that has the sense chain sequence of SEQ ID NO: 1115. n / a = not applicable.
EXAMPLE 7
This example illustrates non-exhaustive modalities of polynucleotide triggers to suppress Leptinotarse target genes. More specifically, this example illustrates blunt-ended dsRNA trigger modalities consisting of a sense strand and a separate antisense strand, as well as dsRNA trigger modalities in the form of a hairpin (a single RNA transcript containing both a sense region as an antisense region).
Table 5 provides blunt-ended dsRNA triggers with sequences related to a parent trigger (see Table 1), where the parent trigger had been determined to have insecticidal activity against Leptinotarsa decemiineata (see Tables 1, 3 and 4) and the derived triggers they are blunt-ended dsRNA corresponding to subregions of the parent trigger.
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161
TABLE 5
<td>SEQ ID NO: del trigger*</td><td>Target gene name</td><td>SEQ ID NO: of the gene Diana</td><td>SEQ ID NO: from trigger for rent 1</td><td>Diet activity vs. CPB (0.1 ppm)</td><td>Diet activity vs. CPB (0.025 ppm)</td>
<td> 1096</td><td>GTP SAR1B binding protein</td><td> 819</td><td> 1084</td><td> (-)</td><td> (-)</td>
<td> 1097</td><td>GTP SAR1B binding protein</td><td> 819</td><td> 1084</td><td> (-)</td><td> ±)</td>
<td> 1098</td><td>GTP SAR1B binding protein</td><td> 819</td><td> 1084</td><td> (+)</td><td>or</td>
<td> 1099</td><td>GTP SAR1B binding protein</td><td> 819</td><td> 1084</td><td> (+)</td><td> ±1</td>
<td> 1100</td><td>Probable non-proteasome 26S ATPase Requester Subunit 3</td><td> 807</td><td> 1049</td><td> ±1</td><td> (-)</td>
<td> 1101</td><td>Proteasome noATPase of 26S requisitioning subunit 7</td><td> 808</td><td> 1050</td><td> _</td><td> _</td>
<td> 1102</td><td>Proteasome noATPase of 26S requisitioning subunit 13</td><td> 812</td><td> 1078</td><td> (-)</td><td> _</td>
<td> 1103</td><td>L7 ribosomal protein</td><td> 730</td><td> 989</td><td> _</td><td> (-)</td>
<td> 1104</td><td>L7 ribosomal protein</td><td> 730</td><td> 989</td><td> (+)</td><td> ±]</td>
<td colspan="2">* string sequence antlsentldo c</td><td>the trigger d</td><td>e cRNA</td><td></td><td></td>
Table 6 provides hairpin-shaped dsRNA triggers (a single RNA transcript containing both a sense strand and an antisense region that hybridize to form dsRNA), where the sequences are derived from or related to a parent trigger (see Table 1), where the parent trigger had been determined to have
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insecticidal activity against Leptinotarsa decemiineata (see Tables 1, 3 and 4). Hairpin triggers are suitable for in vitro expression or in vivo expression when provided in an expression construct with appropriate promoters or other elements to allow expression, eg, in a bacterial cell or in a plant cell. The non-exhaustive modalities described in Table 6 each contain a T7 promoter (located at nucleotide positions 1-17 in each hairpin sequence) and a loop or spacer located between the sense and antisense strands; the loop contains non-specific nucleotides (not complementary to or identical to any part of the target gene). One skilled in the art would immediately understand that the hairpin sense and antisense regions are useful in combination with different promoters suitable for expression in a given cell type and with different spacer or loop sequences (or none, where the nucleotides at the junction of the regions sense and antisense form the necessary turn or minimum loop in the fork). One of skill would also recognize that similar recombinant DNA constructs are readily designed to encode hairpin dsRNA triggers that correspond to blunt-ended dsRNA triggers provided in Tables 1-5 or that target the genes provided in the Sequence Group from Gen Diana.
TABLE 6
<td>SEQ ID fork NO: from trigger *</td><td>Position of hairpin trigger antisense region nucleotide</td><td>Fork trigger antisense region SEQ ID NO ::</td><td>Loop nucleotide position or hairpin spacer</td><td>Position of hairpin trigger region nucleotide</td><td>SEQ ID NO: de ends dsRNA trigger blunt</td><td>I KNOW THAT ID NO: of target gene CPB</td>
<td> 1105</td><td> 21-417</td><td> 1110</td><td> 418-566</td><td> 567 - 963</td><td> 989**</td><td> 730</td>
<td> 1106</td><td> 21 - 300</td><td>lili</td><td> 301 - 450</td><td> 451 - 730</td><td> 1086</td><td></td>
<td> 1107</td><td> 21-453</td><td> 1112</td><td> 454 - 603</td><td> 604 - 1036</td><td> 1084**</td><td> 819</td>
<td> 1108</td><td> 21 - 458</td><td> 1113</td><td> 459 - 608</td><td> 609 - 1046</td><td> 1050**</td><td> 808</td>
<td> 1109</td><td> 21 - 448</td><td> 1114</td><td> 449 - 598</td><td> 599 - 1026</td><td> 1038**</td><td> 828</td>
* sequence of DNA constructs encoding the hairpin dsRNA trigger.
** dsRNA trigger antisense chain sequence
SEQ ID NO: 1086 corresponds to the sense chain sequence of a blunt-ended dsRNA targeting a beta coatomer, previously described as SEQ ID NO: 880 in US Patent No. 7,943,819.
The combination of certain recombinant RNAs as described herein (for example, the dsRNA triggers described in Tables 1-6 or their hairpin equivalents or active fragments of these triggers) with one or more non-polynudeotide pesticidal agents is anticipated. will result in a synergistic improvement in the prevention or control of infestations of the Leptinotarsa species, when compared with the effect obtained with the RNA
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recombinant alone or the non-polynucleotide pesticide agent alone. Routine Insect Bioassays such as the Bloensay Using an Artificial Diet Described herein are useful in defining dose responses for larval mortality or Growth Inhibition using combinations of the Pollucleotide triggers and one or more agents. non-polynucleotide pesticides (for example, a patatin, a plant lectin, a phytoecdisterolde, a Bacillus thuringiensis Insecticidal protein, a Xenorhabdus Insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous Insecticidal protein, and a Bacillus sphaericus insecticidal protein). Combinations of polynucleotides and non-polynucleotide pesticidal agents can be evaluated by one skilled in the art in routine blockades to identify combinations of bioactives that are selective and desirable for use in protecting plants from Leptinotarse species infestations.
EXAMPLE 8
Field efficacy of Leptinotarsa decem / ineata RNA mediated control
A field test was performed to evaluate the efficacy of topically applied dsRNA triggers on the control of Leptinotarsa decemllneata (Colorado potato beetle, CPB) infestations of potato plants under field conditions. These cdRNA triggers were evaluated using topical application (foliar spray): a blunt-ended cDNA having an antisense sequence of SEQ ID NO: 989, which targets the L7 ribosomal protein (encoded by SEQ ID NO: 730 ); a blunt-ended dsRNA having an antisense strand sequence of SEQ ID NO: 1049, which targets the probable 26S non-ATPase proteasome regulatory subunit 3 (encoded by SEQ ID NO: 807); and a hairpin dsRNA encoded by the DNA construct of SEQ ID NO: 1105, which targets the L7 ribosomal protein (encoded by SEQ ID NO: 730). SEQ ID NO: 1105 encodes a hairpin dsRNA having an antisense strand that corresponds to SEQ ID NO: 989 (see Example 7). The experiment was designed with 11 treatments arranged in a randomized complete block design with four couplets. The test plots consisted of potato plants (Superior variety) planted in the spring in two 20-foot rows with a 6-foot center row space; plots were maintained according to standard commercial cultivation practices. Two foliar spray treatments were performed: a first treatment 36 days after planting and a second treatment 43 days after planting. All foliar treatments were applied with a 4-nozzle jib fitted with 20-inch 110003VS sprayer tips, spraying 2 rows at a time and Powered by a backpack sprayer Powered by carbon dioxide at 40 pounds per square inch, delivering 38 gallons per acre. All stages of beetle life were recorded
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INSTITUTO MEXICANO r »t LA PROMECW» of the Colorado potato for ten randomly selected stems. pnrUntp .pn 3 ριιηίης rip time: 3 days after the first foliar spray treatment (39 days after planting), 7 days after the first foliar spray treatment (43 days after planting) and 3 days after the second treatment foliar spray (46 days after planting). Defoliation, which is mainly caused by small larvae, was measured 9 days after the first foliar spray treatment (45 days after planting). Two commercial synthetic insecticides (small molecule) were used as positive controls: Coragen® (chlorantraniliprol, DuPont) and Radiant® (spinetoram, Dow AgroSclences). The results are presented in Table 7; Statistically different values are indicated with different letters (a, b, c, d, e). Treatments that share a letter, for example the Untreated Control and 5 grams per acre SEQ ID NO: 989 Treatment 3 days after the first spray shares the letter a, are not statistically different; while the treatments that do not share a letter, for example the Untreated Control and the Coragen® treatment 3 days after the first spray, are statistically different. The effects of the cDNA triggers increased over time and showed a dose dependent response; at 3 days after the second foliar spray, all of the dsRNA trigger treatments except the lowest dose of the dsRNA trigger that has an antisense sequence of SEQ ID NO: 1049 resulted in a decrease in large larvae that were not it was significantly different from the positive controls of synthetic insecticides (Coragen® and Radiant Treatments) and that it was significantly different from the untreated Control. Defoliation also showed a dose-dependent response to dsRNA treatments; several of the dsRNA treatments were significantly different from the Untreated Control, and all the dsRNA triggers at the highest evaluated dose provided defoliation protection that was not statistically different from that provided by the positive controls for synthetic insecticides (Coragen® and Radiant). The lower number of larvae and the less defoliation or damage to the plants indicated a better resistance of the potato plants treated with dsRNA to Leptinotarsa decemiineata-, it is expected that these plants with better resistance to Leptinotarsa decemlineata show a better yield (higher tubers than can be harvested).
<img file="MX359191B_D0174.tif" />
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TABLE 7
<td colspan="2" rowspan="2"></td><td colspan="6">Average number of Colorado potato beetles / 10 stems</td><td rowspan="3">or / or defolia tion</td>
<td colspan="3">Small larvae</td><td colspan="3">Large larvae</td>
<td>Treatment</td><td>Rate (9 by 0.40 hect area s (acre ))</td><td>3 days then of the first spray action</td><td>7 days then of the first pulverize tion</td><td>3 days after the second pulveri zaclón</td><td>3 days then of the first spray ion</td><td>7 days then of the first pulverize tion</td><td>3 days after the second spray</td>
<td>Control without treatment</td><td>nc</td><td>115.8 a</td><td>201.3 a</td><td>72.0 ab</td><td> 0</td><td>45.3 ab</td><td>108.0 a</td><td>72.5 a</td>
<td>SEQ ID NO: 989 *</td><td> 5</td><td>63.5 ab</td><td>146.5 ab</td><td>98.0 ab</td><td> 3</td><td>8.5 bed</td><td>8.3 b</td><td>9.8 of</td>
<td>SEQ ID NO: 989 *</td><td> 1</td><td>93.3 ab</td><td>159.5 a</td><td>144.5 a</td><td> 1.3</td><td>33.0 abed</td><td>21.8 b</td><td>28.8 cd</td>
<td>SEQ ID NO: 989 *</td><td> 0.2</td><td>87.8 ab</td><td>116.0 abe</td><td>118.0 a</td><td> 0</td><td>25.3 abed</td><td>33.8 b</td><td>45.0 abe</td>
<td>SEQ ID NO: 1049 *</td><td> 5</td><td>66.5 ab</td><td>135.5 abe</td><td>126.0 a</td><td> 0</td><td>2.0 cd</td><td>12.8 b</td><td>15.0 ede</td>
<td>SEQ ID NO: 1049 *</td><td> 1</td><td>91.0 ab</td><td>175.0 a</td><td>102.5 ab</td><td> 0</td><td>41.3 abe</td><td>33.8 b</td><td>32.5 bed</td>
<td>SEQ ID NO: 1049 *</td><td> 0.2</td><td>93.5 ab</td><td>113.8 abe</td><td>99.3 ab</td><td> 0.8</td><td>59.0 a</td><td>80.0 a</td><td>68.8 ab</td>
<td>SEQ ID NO: 1105 *</td><td> 5</td><td>61.0 ab</td><td>91.3 abe</td><td>117.8 a</td><td> 0</td><td>9.0 bed</td><td>14.0 b</td><td>12.5 ede</td>
<td>SEQ ID NO: 1105 *</td><td> 1</td><td>72.3 ab</td><td>104.8 abe</td><td>87.3 ab</td><td> 0</td><td>17.8 bed</td><td>8.8 b</td><td>18.8 cd</td>
<td>Coraqen®</td><td> 0.14 (5**)</td><td>9.8 b</td><td>6.0 c</td><td>0.3 b</td><td> 0</td><td>0.0 d</td><td>0.0 b</td><td>0.0 e</td>
<td>Radiant</td><td> 0.23 (8**)</td><td>1.3 b</td><td>16.8 be</td><td>0.0 b</td><td> 0</td><td>0.5 d</td><td>0.0 b</td><td>0.0 e</td>
<td colspan="2">P value according to Anova</td><td> 0.0053</td><td> 0.0004</td><td> 0.0009</td><td>ns</td><td> 0.0001</td><td> <0.0001</td><td> <0.0001</td>
nc, not applicable ns, not significant * dsRNA triggers applied in a formulation containing 3 milliliters of a conventional spray adjuvant, TACTIC ™ (Loveland Products, Loveland, CO 80538) per 1600 milliliters of water ** 0.02 L per 0.40 hectares (fluid ounces per acre)
All of the materials and methods described and claimed herein can be manufactured and used without undue experimentation as indicated in the preceding description. Although the materials and methods of the present invention were described in terms of illustrative embodiments and examples, it will be apparent to those skilled in the art that variations to the materials and methods described herein can be applied without departing from the concept, spirit and scope of the present invention. All similar substitutes and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.
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Contents170
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| Document | Office | Kind | Date |
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| 201361856137 | United States of America | P | |
| 201361856137 | United States of America | P | |
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| 201461980800 | United States of America | P | |
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| UA122662C2 | Ukraine | C2 | |
| CN105980567B | China | B | |
| CA2918387C | Canada | C | |
| MX2018011342A | Mexico | A | |
| US11377667B2 | United States of America | B2 | |
| BR112016000555B1 | Brazil | B1 | |
| US2023035621A1 | United States of America | A1 |
2 legal events, as the office reported them to INPADOC
Over the term
Point at a mark for the eventEvents
| Event | Code | |
|---|---|---|
| Grant or registrationFG | FG | |
| Correction or change in generalHH | HH |
Numbers
- Publication
- 359191
- Publication, DOCDB
- 359191
- Publication, EPODOC
- MX359191
- Application
- 2016000741
- Application, DOCDB
- 2016000741
- Application, EPODOC
- MX20160000741
Titles2
- Spanish
- COMPOSICIONES Y MÉTODOS PARA CONTROLAR LEPTINOTARSA.
- English
- COMPOSITIONS AND METHODS FOR CONTROLLING LEPTINOTARSA.
Classification
- CPC, 7
- C12N15/8286
- A01N57/16
- C07K14/43563
- C12N15/8218
- Y02A40/146
- C12N15/113
- C12N2310/14
- IPC, 7
- C12N15 82
- A01H5 10
- A01N63 00
- A01N63 14
- A01N63 60
- C07K14 435
- C12N15 113