ECSP088291A

Methods for genetic control of insect infestations in plants and compositions thereof

Abstract

The present invention relates to the control of pest infestations by inhibiting one or more biological functions. Methods and compositions for said control are provided in the invention, comprising feeding the pest with one or more double-stranded recombinant RNA molecules provided by the invention, whereby a reduction in pest infestation is obtained by suppressing gene expression The invention also relates to methods for preparing transgenic plants that express double-stranded RNA molecules, and with particular combinations of transgenic pesticide agents, for use in protecting plants against pest infestations.

Term

No projected expiry on record.

33 claims: 2 independent, 31 dependent

1
Un polinucleótido aislado seleccionado del grupo que consiste en:(a) un polinucleótido que comprende una secuencia de ácido nucleico de SEQ ID N-1 a SEQ ID N 9 906;(b) un polinucleótido que hibridiza con una secuencia de ácido nucleico de SEQ ID N 9 1 a SEQ ID N 9 906, bajo condiciones de lavado de SSC 5X, 50% de formamida y 42 9 C por 10 minutos;(c) un polinucleótido que comprende al menos 70% de identidad de secuencia con una secuencia de ácido nucleico de SEQ ID N s 1 a SEQ ID N 9 906;(d) un fragmento de al menos 21 nucleótidos contiguos de una secuencia de ácido nucleico de SEQ ID N 9 1 a SEQ ID N 9 906, donde la ingestión por un coleóptero plaga de plantas de una secuencia de ribonucleótidos de cadena doble que comprende al menos una cadena que es complementaria con dicho fragmento inhibe el crecimiento de dicha plaga;y (e) un complemento de la secuencia de (a), (b), (c) o (d).
2
El polinucleótido aislado de la reivindicación 1, seleccionado del grupo que consiste en SEQ ID N 9 697, SEQ ID N 9 813-819, SEQ ID N 9 841 y SEQ ID N 9 874.
3
El polinucleótido aislado de la reivindicación 1, definido como unido operativamente a un promotor heterólogo.
4
El polinucleótido aislado de la reivindicación 1, definido como incluido en un vector para transformar plantas.
5
Una secuencia de ribonucleótidos de cadena doble producida a partir de la expresión de un polinucleótido de acuerdo con la reivindicación 1, donde la ingestión de dicha secuencia de ribonucleótidos por un coleóptero plaga de plantas inhibe el crecimiento de dicha plaga.
6
La secuencia de ribonucleótidos de cadena doble de la reivindicación 5, definida como una secuencia que se produce preparando una secuencia de polinucleótidos recombinante, que comprende una primera, una segunda y una tercera secuencia de polinucleótidos, donde la primera secuencia de polinucleótidos comprende el polinucleótido aislado de la reivindicación 1, donde la tercera secuencia de polinucleótidos está unida a la primera secuencia de polinucleótidos a través de la segunda secuencia de polinucleótidos, y donde la tercera secuencia de polinucleótidos es sustancialmente el complemento inverso de la primera secuencia de polinucleótidos, de modo la primera y la tercera secuencias de polinucleótidos hibridizan cuando se transcriben en un ácido ribonucleico para formar la molécula de ribonucleótido de cadena doble estabilizada por la segunda secuencia de ribonucleótidos unida.
7
La secuencia de ribonucleótidos de cadena doble de la reivindicación 5, donde la ingestión de la secuencia de polinucleótidos por la plaga inhibe la expresión de una secuencia de nucleótidos sustancialmente complementaria con dicha secuencia de polinucleótidos.
8
Una célula transformada con el polinucleótido de la reivindicación 1.
9
La célula de la reivindicación 8, definida como una célula procariota.
10
La célula de la reivindicación 8, definida como una célula eucariota.
11
La célula de la reivindicación 8, definida como una célula vegetal o bacteriana.
12
Una planta transformada con el polinucleótido de la reivindicación 1.
13
Una semilla de la planta de la reivindicación 12, donde la semilla comprende el polinucleótido.
14
Una planta transformada con el polinucleótido de la reivindicación 2.
15
La planta de la reivindicación 12, donde dicho polinucleótido se expresa en una célula de la planta como una secuencia de ribonucleótidos de cadena doble, y la ingestión de una cantidad capaz de inhibir insectos de dicha secuencia de ribonucleótidos de cadena doble en una dieta inhibe la alimentación ulterior de la plaga con dicha dieta.
16
La planta de la reivindicación 15, donde el insecto plaga se selecciona del grupo que consiste en Diabrotica virgifera, Diabrotica virgifera virgifera, Diabrotica virgifera zea, Diabrotica balteata, Diabrotica barberi, Diabrotica viridula, Diabrotica speciosa y Diabrotica undecimpunctata.
17
La planta de la reivindicación 15, donde la ingestión de la cantidad capaz de inhibir insectos de la secuencia de ribonucleótidos de cadena doble detiene el crecimiento de la plaga.
18
Un producto básico producido a partir de una planta de acuerdo con la reivindicación 12, donde dicho producto básico comprende una cantidad detectable del polinucleótido de la reivindicación 1, o un ribonucleótido expresado a partir de él.
19
Un método para controlar una infestación de coleópteros plaga, que comprende proporcionar en la dieta de un coleóptero plaga un agente que comprende una primera secuencia de polinucleótidos, que al ser ingerida por la plaga, funciona inhibiendo una función biológica de dicha plaga, donde dicha secuencia de polinucleótidos tiene entre aproximadamente 95 y aproximadamente 100% de identidad de secuencia de nucleótidos, entre al menos aproximadamente 19 y aproximadamente 25 nucleótidos contiguos, con una secuencia codificante derivada de dicha plaga, e hibridiza con una segunda secuencia de polinucleótidos que es complementaria con dicha primera secuencia de polinucleótidos, y donde dicha secuencia codificante que deriva de dicha plaga se selecciona del grupo que consiste en SEQ ID N s 1 a SEQ ID N s 906, y sus complementos.
20
El método de la reivindicación 19, donde dicho coleóptero plaga es un Diabrotica spp. seleccionado del grupo que consiste en Diabrotica virgifera, Diabrotica virgifera virgifera, Diabrotica virgifera zea, Diabrotica balteata, Diabrotica barberi, Diabrotica viridula, Diabrotica speciosa y Diabrotica undecimpunctata.
21
Un método para controlar una infestación de coleópteros plaga, que comprende proporcionar en la dieta de un coleóptero plaga una célula vegetal que expresa una secuencia de polinucleótidos de acuerdo con la reivindicación 1, donde el polinucleótido se expresa para producir un ácido ribonucleico de cadena doble, que al ser ingerido por la plaga, funciona inhibiendo la expresión de una secuencia blanco en dicha plaga, y resulta en una alimentación reducida con dicha dieta, con relación a una dieta que carece de la célula vegetal.
22
El método de la reivindicación 21, donde la plaga presenta un crecimiento reducido después de la ingestión de la célula.
23
El método de la reivindicación 21, donde la célula vegetal comprende además una secuencia de polinucleótidos que codifica un agente plaguicida seleccionado del grupo que consiste en una patatina, una proteína insecticida de Bacillus thuringiensis, una proteína insecticida de Xenorhabdus, una proteína insecticida de Photorhabdus, una proteína insecticida de Bacillus laterosporous y una proteína insecticida de Bacillus sphearicus.
24
El método de la reivindicación 23, donde dicha proteína insecticida de Bacillus thuringiensis se selecciona del grupo que consiste en Cry1, Cry3, TIC851, CryET70, Cry22, TIC901, TIC201, TIC407, TIC417, la proteína insecticida binaria CryET33 y CryET34, la proteína insecticida binaria CryET80 y CryET76, la proteína insecticida binaria TIC100 y TIC101, la combinación de las proteínas insecticidas ET29 o ET37 con las proteínas insecticidas TIC810 o TIC812, y la proteína insecticida binaria PS149B1.
25
El método de la reivindicación 21, donde la secuencia blanco codifica una proteína cuya función predicha se selecciona del grupo de funciones que consiste en la formación de los músculos, la formación de hormona juvenil, la regulación de hormona juvenil, la regulación y el transporte de iones, la síntesis de enzimas digestivas, el mantenimiento del potencial de la membrana celular, la biosíntesis de aminoácidos, la degradación de aminoácidos, la formación de esperma, la síntesis de feromonas, la percepción de feromonas, la formación de las antenas, la formación de las alas, la formación de las patas, el desarrollo y la diferenciación, la formación de los huevos, la maduración de las larvas, la formación de enzimas digestivas, la síntesis de la hemolinfa, el mantenimiento de la hemolinfa, la neurotransmisión, la división celular, el metabolismo energético, la respiración, y la apoptosis.
26
El método de la reivindicación 21, donde dicho coleóptero plaga es un Diabrotica spp. seleccionado del grupo que consiste en Diabrotica virgifera, Diabrotica virgifera virgifera, Diabrotica virgifera zea, Diabrotica balteata, Diabrotica barberi, Diabrotica viridula, Diabrotica speciosa y Diabrotica undecimpunctata. 2J. El método de la reivindicación 21, donde el polinucleótido, al ser ingerido por la plaga, funciona suprimiendo un gen que cumple una función esencial para la supervivencia del insecto, donde dicha función se selecciona del grupo que consiste en la alimentación de la plaga, la apoptosis celular, la diferenciación celular y el desarrollo de la plaga, la capacidad o el deseo de reproducción sexual, la formación de los músculos, la activación muscular, la contracción muscular, la formación de hormona juvenil, la regulación de hormona juvenil, la regulación y el transporte de iones, el mantenimiento del potencial de la membrana celular, la biosíntesis de aminoácidos, la degradación de aminoácidos, la formación de esperma, la síntesis de feromonas, la percepción de feromonas, la formación de las antenas, la formación de las alas, la formación de las patas, la formación de huevos, la maduración de las larvas, la formación de enzimas digestivas, la síntesis de la hemolinfa, el mantenimiento de la hemolinfa, la neurotransmisión, la transición del estado larval, la formación de pupas, la emergencia de la forma de pupa, la división celular, el metabolismo energético, la respiración, y la formación de la estructura del citoesqueleto.
27
28. Un método para mejorar el rendimiento de un cultivo producido a partir de una planta de cultivo sometida a una infestación de insectos plaga, donde dicho método comprende los pasos de a) Introducir un polinucleótido de acuerdo con la reivindicación 1 en dicha planta de cultivo, b) cultivar la planta de cultivo para permitir la expresión de dicho polinucleótido, donde la expresión del polinucleótido inhibe la alimentación por los insectos plaga y la pérdida de rendimiento debida a la infestación de la plaga.
28
29. El método de la reivindicación 28, donde la expresión del polinucleótido produce una molécula de ARN que suprime al menos un primer gen blanco en un insecto plaga que ha ingerido una porción de dicha planta de cultivo, donde el gen blanco cumple al menos una función esencial seleccionada del grupo que consiste en la alimentación de la plaga, la viabilidad de la plaga, la apoptosis celular de la plaga, la diferenciación y el desarrollo de la plaga o de cualquier célula de la plaga, la reproducción sexual de la plaga, la formación de los músculos, la activación muscular, la contracción muscular, la formación y/o la reducción de hormona juvenil, la regulación de hormona juvenil, la regulación y el transporte de iones, el mantenimiento del potencial de la membrana celular, la biosíntesis de aminoácidos, la degradación de aminoácidos, la formación de esperma, la síntesis de feromonas, la percepción de feromonas, la formación de las antenas, la formación de las alas, la formación de las patas, la formación de huevos, la maduración de las larvas, la formación de enzimas digestivas, la síntesis de la hemolinfa, el mantenimiento de la hemolinfa, la neurotransmisión, la transición del estado larval, la formación de pupas, la emergencia de la forma de pupa, la división celular, el metabolismo energético, la respiración, la síntesis y el mantenimiento de la estructura del citoesqueleto, el metabolismo de los nucleótidos, el metabolismo del nitrógeno, el uso del agua, la retención de agua, y la percepción sensorial.
29
30. El método de la reivindicación 28, donde el insecto plaga es un gusano de la raíz del maíz plaga seleccionada del grupo que consiste en Diabrotica undecimpunctata howardi (gusano de la raíz del maíz del sur (SCR)), Diabrotica virgifera virgifera (gusano de la raíz del maíz occidental (WCR)), Diabrotica barberi (gusano de la raíz del maíz del norte (NCR)), Diabrotica virgifera zea (gusano de la raíz del maíz mexicana (MCR)), Diabrotica balteata (gusano de la raíz del maíz brasilera (BZR)), Diabrotica viridula (gusano de la raíz del maíz brasilera (BZR)) y Diabrotica speciosa (gusano de la raíz del maíz brasilera (BZR)).
30
31. Un método para mejorar la tolerancia a la sequía de un cultivo producido a partir de una planta de cultivo sometida a una infestación de insectos plaga, donde dicho método comprende los pasos de a) introducir un polinucleótido de acuerdo con la reivindicación 1 en dicha planta de cultivo, b) cultivar la planta de cultivo para permitir la expresión de dicho polinucleótido, donde la expresión del polinucleótido inhibe la alimentación por los insectos plaga y la pérdida de tolerancia a la sequía debida a la infestación de la plaga.
31
32. Un método para producir un producto básico, que comprende obtener una planta de acuerdo con la reivindicación 12, o una parte de ésta, y preparar un producto básico a partir de la planta, o la parte de ésta.
32
33. Un método para producir alimento para seres humanos o animales, que comprende obtener una planta de acuerdo con la reivindicación 12, o una parte de ésta, y preparar un alimento para seres humanos o animales a partir de dicha planta, o la parte de ésta.
33
34. El método de la reivindicación 33, donde el alimento para seres humanos o animales se define como aceite, harina, proteína, almidón, harina o forraje ensilado.

Independent claims33

818 paragraphs in 13 sections, as filed

SIGNATURE OF THE APPLICANT

SIGNATURE OF ATTORNEY

CASE 5853

TECHNICAL MEMORY

METHODS AND COMPOSITIONS FOR THE GENETIC CONTROL OF INSECT INFESTATIONS IN PLANTS

INCORPORATION AS A REFERENCE OF THE LIST OF SEQUENCES PRESENTED IN

A COMPACT DISC

The Sequence Listing is presented on a compact disc (Copy 1), along with a duplicate thereof (Copy 2), each created on September 15, 2006, each containing a 669 kb file titled “MNDE002 .APP.TXT.” The material contained on the compact disc is specifically incorporated herein by reference.

INVOKED PRIORITY

This application invokes the priority of US Provisional Application No. 60/718,034, filed September 16, 2005, which is incorporated herein in its entirety by reference.

BACKGROUND OF THE INVENTION

1. field of invention

The present invention relates generally to the genetic control of pest infestations. More specifically, the present invention relates to recombinant DNA technologies for post-transcriptionally repressing or inhibiting the expression of target coding sequences in a pest cell to provide a protective effect against the pest.

2. Description of Related Art

The environment in which humans live is teeming with pest infestations. Pests, including insects, arachnids, crustaceans, fungi, bacteria, viruses, nematodes, flatworms, roundworms, spindleworms, hookworms, tapeworms, trypanosomes, schistosomes, parasitic flies, fleas, ticks, mites and lice, and the like, are ubiquitous in the human environment. A multitude of means have been used in attempts to control infestations by these pests. Compositions for controlling infestations of microscopic pests, such as bacteria, fungi, and viruses, have been provided in the form of antibiotic compositions, antiviral compositions, and antifungal compositions. Compositions for controlling infestations by larger pests such as nematodes, flatworms, roundworms, spindleworms, hookworms, tapeworms, trypanosomes, schistosomes, and the like typically take the form of chemical compositions that can be applied to surfaces on which the pests are present in or can be administered to infested animals in the form of pellets, powders, tablets, pastes, or capsules and the like. There is a great need in the art for an improvement in these methods and in particular for methods that benefit the environment in relation to prior art.

Commercial crops are commonly the targets of insect attack. In the last decades, substantial progress has been achieved, aimed at the development of more efficient methods and compositions to control insect infestations in plants. Chemical pesticides have been very effective in eradicating pest infestations. However, there are several disadvantages associated with the use of chemical pesticidal agents. Chemical pesticidal agents are not selective. Chemical pesticide applications attempt to control invertebrate pests such as coleopteran insects including corn rootworm species that are injurious to different crops and other plants, they also exert their effects on non-target fauna, often effectively sterilizing a field during a period of time during which the pesticidal agents have been applied. Chemical pesticidal agents persist in the environment, and are generally metabolized slowly, if at all. They accumulate in the food chain, and particularly in the top predatory species. The accumulation of these chemical pesticidal agents results in the development of resistance to the agents, and in species higher in the evolutionary chain, they can act as mutagens and/or carcinogens and cause irreversible and deleterious genetic modifications. Therefore, for a long time there has been a need for methods that are not harmful to the environment that allow controlling or eradicating insect infestations on or inside plants, that is, methods that are selective, inert to the environment, non-persistent and biodegradable, and that fit into pest resistance management schemes.

Compositions including Bacillus thuringiensis (Bt) bacteria have been commercially available and used as safe and acceptable insecticides for over thirty years. The insecticidal effect of Bt bacteria does not persist in the environment, is highly selective for the affected pest species, exerts its effects only through ingestion by a target pest, and has been shown to be non-detrimental to plants and other non-target organisms. , including humans. There are also transgenic plants that contain one or more genes encoding Bt insecticidal proteins available in the art, and are remarkably efficient in controlling insect pest infestation. A substantial result of the use of recombinant plants expressing Bt insecticidal proteins is a marked decrease in the amount of chemical pesticidal agents that are applied to the environment to control pest infestation on crop plants, in areas where these transgenic crops are used. The decrease in the application of chemical pesticidal agents has resulted in cleaner soils and waters within canals, rivers, puddles and lakes. In addition to these environmental benefits, a notable increase in the number of beneficial insects has been observed in crop fields where insect-resistant transgenic crops are grown, due to the decrease in the use of chemical insecticidal agents.

Antisense methods and compositions have also been described in the art, and are believed to exert their effects via the synthesis of a single-stranded RNA molecule that, theoretically, hybridizes in vivo to an in-frame RNA molecule. substantially complementary reading. Antisense technology has proven difficult to employ in many systems for three main reasons. First, the antisense sequence expressed in the transformed cells is unstable. Second, the instability of the antisense sequence expressed in the transformed cells creates difficulty in delivery of the sequence to the host, cell type, or biological system remote from the transgenic cell. Third, the difficulties encountered with instability and delivery of the antisense sequence create difficulties in trying to provide a dose within the recombinant cell expressing the antisense sequence that can effectively modulate the level of expression of the nucleotide sequence in the sense of the desired reading frame.

There have been few improvements in the technology to modulate the level of gene expression within a cell, tissue, or organism, and in particular, a lack of development technologies to delay, repress, or reduce the expression of specific genes using recombinant DNA technology. . Furthermore, as a consequence of the lack of predictability of these approaches, there is no commercially viable means of modulating the expression level of a specific gene in a eukaryotic or prokaryotic organism.

Double-stranded RNA-mediated inhibition of specific genes has previously been demonstrated in several pests. Double-stranded RNA-mediated approaches to genetic control in the fruit fly Drosophila melanogaster have been evaluated (Kennerdell and Carthew, 1998; Kennerdell and Carthew, 2000). Kennerdell and Carthew (1998) describe a method for administering dsRNA, which comprises generating transgenic insects expressing dsRNA molecules, or injecting dsRNA solutions into the insect's body or into the egg cavity. , before or during embryonic development.

Researchers have previously shown that double-stranded RNA-mediated gene suppression in nematodes can be effected by feeding or immersing nematodes in solutions containing small interfering or double-stranded RNA molecules, and injecting double-stranded RNA molecules. Rajagopal et. to!. (2002) describe failed attempts to suppress an endogenous gene in larvae of the insect pest Spodoptera litura by feeding on or dipping neonate larvae in solutions with double-stranded RNAs specific for the target gene, but which were successful in suppressing after larvae were injected with dsRNA into the hemolymph of 5th instar larvae using a microapplicator. Recently, Yadav et al. (2006) reported that host-generated double-stranded RNA produced in a plant can protect the plant from nematode infection. Similarly, US Patent Application Publication No. 2003/0150017 predictively describes a preferred locus for inhibition of larvae of the lepidopteran Helicoverpa armigera using double-stranded RNA administered to the larvae by ingestion of a transformed plant. to produce a double-stranded RNA. WO 2005/110068 discloses the provision, in the corn rootworm (CRW) diet, of CRW-specific double-stranded RNA targeting essential CRW genes. Double-stranded RNA is provided in the diet of CRW in-vitro and in-planta, with the result that CRW larvae retard their growth or die after feeding on the diet, and this effect was demonstrated for several different genes. .

Therefore, there has been a need to identify effective nucleotide sequences for use in better methods of modulating gene expression by repressing, retarding, or any other type of reduction of gene expression within a particular pest coleopteran for the purpose of controlling infection. ingestion by pests or to introduce new phenotypic characteristics.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for inhibiting the expression of a target gene in a coleopteran pest. In certain embodiments, the method comprises modulating or inhibiting the expression of one or more target genes in a pest beetle, causing disruption of feeding, growth, development, reproduction, and/or infectivity, and eventually resulting in the death of the insect. The method comprises introducing partially or fully stabilized double-stranded RNA (dsRNA), including its modified forms, such as small interfering RNA (siRNA) sequences, into cells or the extracellular environment, such as in the mesenteron, in the body of a pest beetle, where the dsRNA enters cells and inhibits the expression of at least one or more target genes, and where the inhibition exerts a detrimental effect on the pest beetle. The methods and associated compositions can be used to limit or eliminate pest coleopteran infestation in or on any pest host, pest symbiont, or environment where the pest is present, and comprise providing one or more compositions comprising the pest molecules. dsRNA described in the present documentation in the diet of the pest. The method will be particularly beneficial in protecting plants from insect attack. In one embodiment, the pest is defined as having a digestive tract with a pH in the range of about 4.5 to about 9.5, about 5 to about 9, about 6 to about 8, and about pH 7. ,0.

In another aspect, examples of nucleic acid compositions that are homologous to at least a portion of one or more native nucleic acid sequences in a target pest are provided herein. In certain embodiments, the pest is selected from Diabrotica sp. including western corn rootworm (WCR, Diabrotica virgifera or Diabrotica virgifera virgifera), southern corn rootworm (SCR, Diabrotica undecimpunctata howardi), Mexican corn rootworm (MOR, Diabrotica virgifera zea), Brazilian corn rootworm (BZR, Diabrotica balteata, Diabrotica viridula, Diabrotica speciosa), Northern corn rootworm (NCR, Diabrotica barberi), Diabrotica undecimpunctata; and also the Colorado potato beetle (CPB, Leptinotarsa decemlineata), the red flour beetle (RFB, Tribolium castaneum) and the Mexican bean beetle (Epilachna varivestis). In other embodiments, the pest is selected from among lepidopteran insects, including the European corn borer (ECB, Ostrinia nubilalis), black cutter caterpillar (BCW, Agrotis ipsilon), corn ear caterpillar (CEW, Helicoverpa zea), the autumn walker caterpillar (FAW, Spodoptera frugiperda), the cotton boll weevil (BWV, Anthonomus grandis), silkworms (Bombyx morí) and Manduca sexta, and among dipterous insects, including Drosophila melanogaster, Anopheles gambiae and Aedes aegypti. Specific examples of the nucleic acids provided by the invention are provided in the attached sequence listing, viz., SEQ ID N91 to SEQ ID No.9 906.

In yet another aspect, the invention provides a method for suppressing gene expression in a coleopteran pest, such as the corn rootworm, or related species, comprising the step of providing in the pest's diet an amount gene suppressor of at least one dsRNA molecule transcribed from a nucleotide sequence described herein, at least one segment of which is complementary to an mRNA sequence within the pest cells. In addition, the method may comprise observing the death, inhibition, vanishing or cessation of feeding of the pest. A dsRNA molecule, including its modified form, such as a siRNA molecule, used to feed a pest according to the invention, can be at least about 80, 81, 82, 83, 84, 85, 86, 87, 88 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or approximately 100% identical to an RNA molecule transcribed from a nucleotide sequence selected from the group consisting of SEQ ID Nyes 1 to SEQ ID Nyes 906. In particular embodiments, the nucleotide sequence may be selected from the group consisting of SEQ ID N9 697, SEQ ID No.9 813-819, SEQ ID No.9 841 and SEQ ID No.9 874.

Accordingly, in another aspect of the present invention, a set of isolated and purified nucleotide sequences is provided as set forth in SEQ ID N91 to SEQ ID No.9 906. In the present invention, a stabilized dsRNA molecule is provided, or the expression of one or more miRNA for the inhibition of the expression of a target gene in a pest beetle, where said gene is expressed from these sequences, and fragments of this. A stabilized dsRNA, including a miRNA or siRNA molecule, may comprise at least two coding sequences, which are arranged in a sense and antisense orientation relative to at least one promoter, where the nucleotide sequences, which comprise a sense strand and an antisense strand, are joined or connected by a gap sequence, comprising between at least about five and about one thousand nucleotides, where the in-frame strand and the antisense strand may be of different length, and where each of the two coding sequences share at least 80% sequence identity , at least 90%, at least 95%, at least 98%, or 100% sequence identity, to any of the nucleotide sequences listed in SEQ ID N91 to SEQ ID No.9 906.

Furthermore, in the invention there is provided a fragment or a concatemer of a nucleic acid sequence selected from the group consisting of SEQ ID N91 to SEQ ID No.9 906. In particular embodiments, the nucleotide sequence may comprise a fragment or concatemer of a sequence selected from the group consisting of SEQ ID N9 697, SEQ ID No.9 813-819, SEQ ID No.9 841 and SEQ ID No.9 874.

The fragment can be defined as one that causes death, inhibition, fainting, or cessation of feeding of a pest, when expressed as dsRNA and provided to the pest. The fragment can comprise, for example, at least about 19, 21, 23, 25, 40, 60, 80,100,125 or more contiguous nucleotides of any of the sequences in SEQ ID N91 to SEQ ID

No.yes 906, or a complement of these. A beneficial DNA segment for use in the present invention is at least about 19 to about 23, or about 23 to about 100 nucleotides, and at most about 2000 nucleotides or more in length. dsRNA sequences that include between about 23 and about 300 nucleotides homologous to the pest target sequence will be particularly useful. Also provided by the invention is a ribonucleic acid that is expressed from any of these sequences, including a dsRNA. A selected sequence for use in expression of a gene suppression agent may be constructed from an individual sequence derived from one or more target pests, intended for use in expression of an RNA that functions to suppress a single gene or family. of genes in one or more target pests, or where the DNA sequence can be constructed as a chimera, from a plurality of DNA sequences.

In yet another aspect, the invention provides recombinant DNA constructs comprising a nucleic acid molecule encoding a dsRNA molecule described herein. The dsRNA can be formed from the transcription of a strand of the dsRNA molecule, from a nucleotide sequence that is between at least about 80% and about 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID No.51 to SEQ ID No.yes 906. These recombinant DNA constructs can be defined as sequences that produce dsRNA molecules capable of inhibiting the expression of one or more endogenous target genes in a pest when ingested. The construct may comprise a nucleotide sequence of the invention, operatively linked to a promoter sequence functioning in the host cell. This promoter may have tissue specificity, and for example, may be specific for a type of tissue that is the object of attack by the pest. In the case of rootworms, for example, it may be desirable to use a promoter that provides root-preferential expression.

Nucleic acid constructs according to the invention may comprise at least one non-naturally occurring nucleotide sequence, which can be transcribed into single-stranded RNA, capable of forming a dsRNA molecule in vivo by hybridization. These dsRNA sequences assemble themselves, and can be introduced into the diet of a coleopteran pest to obtain the desired inhibition.

A recombinant DNA construct may comprise two different non-naturally occurring sequences, which when expressed in vivo as dsRNA sequences and introduced into the diet of a coleopteran pest, inhibit the expression of at least two different target genes in the recombinant DNA construct. pest coleopteran cell. In certain embodiments, at least 3, 4, 5, 6, 8, or 10, or more different dsRNAs are produced in a cell or a plant comprising the cell that has a pest-inhibiting effect. dsRNAs can be expressed from several constructs introduced in different transformation events, or they can be introduced into a single nucleic acid molecule. dsRNAs can be expressed using a single promoter or multiple promoters. In one embodiment of the invention, individual dsRNAs comprising nucleic acids homologous to multiple loci in a pest are produced.

In still another aspect, the invention provides a recombinant host cell that has in its genome at least one recombinant DNA sequence that is transcribed in the host cell to produce at least one dsRNA molecule, which once ingested by a beetle pest , works by inhibiting the expression of a target gene in the pest. The dsRNA molecule may be encoded by any of the nucleic acids described herein and indicated in the sequence listing. Also provided in the present invention is a transformed plant cell having in its genome at least one recombinant DNA sequence described in the present documentation. Also provided are transgenic plants comprising this transformed plant cell, including progeny plants of any generation, seeds, and plant products, each comprising the recombinant DNA.

The methods and compositions of the present invention can be applied to any monocot or dicot plant, depending on the desired control of coleopteran pests. Specifically, plants include, without limitation, alfalfa, anet, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, kidney bean, jute, blackberry, cranberry, broccoli, Brussels sprout, cabbage, canola, cantaloupe, carrot , cassava, cauliflower, celery, cherry, coriander, grapefruit, clementine, coffee, peanut, cotton, cucumber, Douglas fir, eggplant, endive, endive, eucalyptus, fennel, fig, pumpkin, grape, pineapple, honeydew, jicama, kiwi, lettuce, leek, lemon, lime, Loblolly pine, mango, melon, mushroom, walnut, oats, okra, onion, orange, an ornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, banana, plum, pomegranate, steel , potato, pumpkin, quince, radish pine, radish, radish, raspberry, rice, rye, sorghum, southern pine, soybean, spinach, pumpkin, strawberry, beet, sugar cane, sunflower, sweet potato, sweet gum, tangerine, tea , tobacco, tomato, grass, a vineyard, watermelon, wheat, yams and long zucchini. Therefore, a plant transformed with a recombinant DNA sequence is also provided by the invention as one of among

SEQ ID NO:1 through SEQ ID NO:906, or a concatemer, fragment, or complement thereof, that is transcribed to produce at least one double-stranded RNA molecule that functions when ingested by a beetle pest to inhibit the expression of a target gene in the pest. In particular embodiments, the recombinant DNA sequence may be selected from the group consisting of SEQ ID NO:697, SEQ ID NOs:813-819, SEQ ID NO:841, and SEQ ID NO:874, or a fragment, complement or concatemer thereof.

The invention also provides combinations of methods and compositions for controlling infestations of coleopteran pests. By an alternative, a double-stranded RNA method is provided as described herein for protecting plants against insect infestation together with one or more insecticidal agents that exhibit different characteristics than those exhibited by double-stranded RNA methods and compositions. chain. For example, one or more Bt protein can be provided in the diet of insect pests in combination with one or more double-stranded RNAs as described herein. A composition formulated for topical application or derived from the use of a transgenic method that combines methods and compositions with Bt double-stranded RNA can be used to provide synergisms not previously known in the art to control insect infestation. A synergism is the reduction in the expression level required for the double-stranded RNAs or Bt proteins. When combined together, a lower effective dose of each pest control agent can be used. Bt insecticidal proteins are thought to create entry pores through which double-stranded RNA molecules can more effectively penetrate into spaces remote from the insect pest's mesenteron, or can more efficiently penetrate cells close to the insects. lesions created by Bt proteins, thus less Bt protein or dsRNA is needed to obtain the desired insecticidal result or the desired inhibition or suppression of a desired biological function in the target pest.

The present invention therefore provides a composition containing two or more different pesticidal agents where each is toxic to the same pest or insect species, where at least one comprises a double-stranded RNA described herein. In certain embodiments, the second agent may be an agent selected from the group consisting of a patatin, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, an insecticidal protein from Bacillus sphaericus, and a lignin. A Bacillus thuringiensis insecticidal protein can be any of a number of insecticidal proteins including, but not limited to, a Cry1, a Cry3, a TIC851, a CryET70, a Cry22, a TIC901, a TIC1201, a TIC407, a TIC417, a CryET33 and CryET34 binary insecticidal protein, a CryET80 and CryET76 binary insecticidal protein, a TIC100 and TIC101 binary insecticidal protein, a PS149B1 binary insecticidal protein, a VIP insecticidal protein, a TIC900 or related protein, or combinations of the insecticidal proteins ET29 or ET37 with insecticidal proteins TIC810 or TIC812, and insecticidal chimeras of any of the above insecticidal proteins.

A ribonucleic acid that is provided in a diet can be provided in an artificial diet that is formulated to meet particular nutritional requirements for maintaining a pest on said diet. The diet may be supplemented with a pest control amount of an RNA that has been purified from a separate expression system to determine a pest control amount of RNA composition or to determine the extent of suppression activity against ingestion of the supplemented diet by the pest. The diet can also be a recombinant cell transformed with a DNA sequence constructed to express the gene suppression agent, RNA or agent. Once one or more of these transformed cells is ingested by the pest, a desired phenotypic result is observed, indicating that the agent has served to inhibit the expression of a target nucleotide sequence within the pest cells.

A target gene to be deleted may encode an essential protein, the predicted function of which is selected from the group consisting of muscle building, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, protein synthesis and transport, enzyme synthesis digestive, maintenance of cell membrane potential, amino acid biosynthesis, amino acid degradation, sperm formation, pheromone synthesis, pheromone sensing, antennae formation, wing formation, leg formation, development and differentiation, egg formation, larval maturation, digestive enzyme formation, hemolymph synthesis, hemolymph maintenance, neurotransmission, cell division, energy metabolism, respiration, an unknown function , and apoptosis.

Another aspect of the present invention also provides methods for improving the yield of a crop produced from a cultivated plant subjected to infestation by an insect pest, wherein said method comprises the steps of a) introducing a polynucleotide comprising a sequence that is chosen from SEQ ID NO:1 to SEQ ID NO:906 or a complement or concatemer or fragment thereof in said crop plant; and b) cultivating the crop plant to allow expression of said polynucleotide, wherein expression of the polynucleotide inhibits its ingestion by insect pests and loss of yield due to infestation by pests.

In certain embodiments, expression of the polynucleotide produces an RNA molecule that suppresses at least one first target gene in a pest insect that has ingested a portion of said crop plant, wherein the target gene performs at least one function. essential which is chosen from the group consisting of pest feeding, pest viability, pest cell apoptosis, differentiation and development of the pest or any pest cell, sexual reproduction of the plague, muscle formation, muscle spasm, muscle contraction, formation and/or reduction of juvenile hormone, regulation of juvenile hormone, regulation and transport of ions, cell membrane maintenance, biosynthesis of amino acids, degradation of amino acids, formation sperm formation, pheromone synthesis, pheromone sensing, antennae formation, wing formation, leg formation, egg formation, larval maturation, digestive enzyme formation, hemolymph synthesis, hemolymph maintenance, neurotransmission, transition between larval stages, pupal formation, emergence from pupae, cell division, energy metabolism, respiration, synthesis and maintenance of cytoskeletal structure, nucleotide metabolism, nitrogen metabolism, usa of water, water retention and sensory perception.

In other embodiments, the pest insect is a corn rootworm pest selected from the group consisting of Diabrotica undecimpunctata howardi (Southern Corn Rootworm (SCR)), Diabrotica virgifera virgifera (Western Corn Rootworm (WCR)), Diabrotica barberi (Northern Corn Rootworm (NCR)), Diabrotica virgifera zea (Mexican Corn Rootworm (MCR)), Diabrotica balteata (Brazilian Corn Rootworm (BZR)), Diabrotica viridula (Brazilian Corn Rootworm (BZR)), and Diabrotica speciosa (Brazilian Corn Rootworm (BZR)).

Methods for improving the drought tolerance of a crop produced from a crop plant subjected to infestation by insect pests are also provided, wherein said method comprises the steps of a) introducing a polynucleotide sequence that is chosen from SEQ ID NO:1 through SEQ ID NO:906, or a fragment thereof, in said crop plant; and b) cultivating the crop plant to allow expression of said polynucleotide, wherein expression of the polynucleotide inhibits ingestion by insect pests and loss of drought tolerance due to ingestion by pests.

Yet another aspect of the invention further provides agronomically and commercially important products and/or compositions of importance including, but not limited to, animal feeds, commercial commodities, products and by-products that are for use as human food or for use in compositions. and commercial commodities intended for human consumption including, but not limited to, cornmeal, cornmeal, corn syrup, corn oil, corn starch, puffed corn, corn cakes, cereals, and the like. Such compositions can be defined as containing detectable amounts of a nucleotide sequence set forth herein, and therefore also containing any transgenic events contained in such nucleotide sequences. These products are useful at least because they are likely derived from crops propagated with less pesticides and organophosphates as a result of the incorporation of the nucleotides of the present invention to control ingestion by coleopteran plant pests. Said commercial primary products and commercial derivative products may be produced from seeds produced from a transgenic plant, wherein the transgenic plant expresses RNA from one or more contiguous nucleotides of the present invention or nucleotides from one or more coleopteran pests and the their complements. These commercial primary products and commercial by-products may also be useful in controlling beetle pests of these commercial primary products and commercial by-products, such as, for example, the control of flour weevils, due to the presence in the commercial primary products. and commercially derived products of RNA capable of suppressing pest genes expressed from a genetic sequence detailed in the present invention.

Also provided is a method of producing said commercial derivative products which comprises obtaining a plant transformed with a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:906, or a concatemer or fragment or complement thereof, and preparing a commercial derivative product from the plant or a part thereof. In addition, still another aspect of the invention is a method for producing food or feed, which comprises obtaining a transformed plant with a polynucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:906 or a fragment or complement thereof, and preparing food or feed from said plant or part thereof.

The invention also provides a computer readable medium having recorded one or more of the nucleotide sequences indicated in SEQ ID N9 1 to SEQ ID N9 906, or complements thereof, for use in a number of computer-based applications, including, without limitation, DNA identity and similarity searches, protein identity and similarity searches, transcriptional profiling characterizations, between genomes and artificial hybridization analyses.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Bioassay of F1 events from maize plants transformed with pMON98503 (SEQ ID N5 820) and attacked by the western corn rootworm (WCR).

FIG. 2: Bioassay of F1 events from maize plants transformed with pMON98504, comprising the C1 concatemer (SEQ ID Ng 821), attacked by the western corn rootworm (WCR).

FIG. 3: Selection of Dv49 and Dv248 fragments, and schematic design of the Dv49Dv248 C38 concatemer.

FIG. 4: dsRNA F1-F13, synthesized on the basis of the C38 concatemer.

FIG. 5:Dose response of the DV49-DV248 concatemer 38 (Fragments F1-F6).

FIG. 6:Dose response of the DV49-DV248 concatemer 38 (Fragments F7-F10).

FIG. 7:DV49 Dose response of the DV49-DV248 concatemer 38 (Fragments F11-F13). DETAILED DESCRIPTION OF THE INVENTION

A detailed description of the invention is provided below, in order to assist those skilled in the art with the practice of the present invention. Those skilled in the art may make modifications and variations to the embodiments described herein, without departing from the spirit and scope of the present invention.

The present invention provides methods and compositions for the genetic control of pest infestations. For example, the present invention provides recombinant DNA technologies for repressing or inhibiting by post-transcriptional means the expression of a target coding sequence in a pest cell, to provide a protective effect against the pest, where the pest you are given one or more molecules of a double-stranded ribonucleic acid (RNA) or small interfering ribonucleic acid, transcribed from all or a portion of a target coding sequence, in order to control the infestation. Thus, the present invention relates to the sequence-specific inhibition of coding sequences using double-stranded RNA (dsRNA), including small interfering RNA (siRNA), to achieve desired pest control levels.

Isolated and substantially purified nucleic acid molecules, including, without limitation, non-naturally occurring nucleotide sequences and recombinant DNA constructs, are provided for transcribing the dsRNA molecules of the present invention, which suppress or inhibit the expression of a endogenous coding sequence or a target coding sequence in the pest when introduced into it. Also provided are transgenic plants that (a) contain nucleotide sequences encoding isolated and substantially purified nucleic acid molecules, and non-naturally occurring recombinant DNA constructs, for transcribing dsRNA molecules to control pest infestations in plants, and (b) exhibit improved resistance and/or tolerance to insect infestations. Also disclosed are compositions containing the dsRNA nucleotide sequences of the present invention, for use in topical applications on plants or animals, or in the animal's environment, to achieve elimination or reduction of pest infestation.

In the present documentation, the inventors have discovered that, contrary to the teachings of the technical background, feeding a coleopteran species with a composition containing double-stranded RNA molecules, consisting of sequences found in one or more sequences of nucleotides expressed by the beetle species from which said sequences were obtained, results in the inhibition of one or more biological functions in the beetle species. In particular, the inventors have discovered that feeding a crop pest species, such as corn rootworms, the RNA molecules described herein, results in death or inhibition of development and the differentiation of pest insects that ingest these compositions.

The inventors have identified the nucleotide sequences described herein as sequences capable of providing protective effects against pest Coleopteran species. The amino acid sequences encoded by the cDNA sequences have been deduced, and compared with known amino acid sequences. It is predicted that many of the sequences will encode proteins that will have some associated annotation information. The annotation information that is associated with a particular nucleotide sequence, and the protein sequence encoded by it, is based on the homology or similarity between the amino acid sequences deduced from translation of the coding sequences described in the submit documentation, as listed, and amino acid sequences known in the art, from publicly available databases.

DNA sequences encoding proteins or portions of proteins known in the art to be essential for survival were selected, such as amino acid sequences involved in various metabolic or catabolic biochemical pathways, cell division, reproduction, energy metabolism, digestion, neurological function and the like, for use in the preparation of double-stranded RNA molecules that were introduced into the diet of pest Coleoptera. As described herein, ingestion by a target pest of compositions containing one or more dsRNAs, at least one segment of which corresponds to at least one substantially identical RNA segment produced in cells of the target pest, resulted in death, fainting, or other inhibition of the pest insect. These results indicate that it is possible to use a nucleotide sequence, either DNA or RNA, derived from a coleopteran pest, to construct plant cells resistant to pest infestation. The pest host can be transformed, for example, to contain one or more of the nucleotide sequences derived from the pest beetle. The transformed nucleotide sequence in the host or pest symbiont encodes one or more RNAs that form a dsRNA sequence in cells or biological fluids within the transformed host or symbiont, making the dsRNA available in the host. pest diet if/when the pest feeds on the transgenic host or symbiont, which will result in suppression of expression of one or more genes in the pest cells, and ultimately in death, the vanishing or other type of inhibition of the pest.

In general, the present invention relates to the genetic control of coleopteran pest infestations in host organisms. More particularly, the present invention includes methods of administering pest control agents to a beetle pest. These pest control agents cause, directly or indirectly, an alteration in the ability of the pest to maintain or grow, or otherwise infest the host or symbiont of the pest. The present invention provides methods for employing stabilized dsRNA molecules in the pest's diet as a means of suppressing desired genes in the pest, thereby allowing control of pest infestations in or around the host or symbiont attacked by the pest. .

By allowing the above to be achieved, the present invention provides a method for inhibiting the expression of a target gene in a coleopteran pest, including, for example, corn rootworms or other coleopteran insect species, resulting in Interruption of feeding, growth, development, reproduction, affectivity, and may eventually result in the death of the pest. The method comprises introducing partially or completely stabilized double-stranded RNA nucleotides (dsRNA) molecules into a nutritional composition used by the pest as a food source, and making the nutritional composition available for the pest to feed on. Ingestion of the nutritional composition containing double-stranded RNA or siRNA molecules results in the uptake of the molecules by the pest cells, resulting in the inhibition of the expression of at least one target gene in the pest cells. the plague. Inhibition of the target gene has a detrimental effect on the pest.

In certain embodiments, the dsRNA molecules provided in the invention comprise nucleotide sequences complementary to a sequence as set forth in any of SEQ ID N9 1 to SEQ ID N9 906, the inhibition of which in a pest organism results in the reduction or removal of an agent based on a protein or a nucleotide sequence that is essential for the growth and development of the pest, or for other biological functions. The selected nucleotide sequence has between about 80% and at least about 100% sequence identity to one of the nucleotide sequences listed in SEQ ID N91 to SEQ ID No.9 906, as detailed in the sequence listing, or their complement. This inhibition is specific, since a nucleotide sequence of a portion of the target gene is selected from which the inhibitory dsRNA or siRNA is transcribed. The method is effective in inhibiting the expression of at least one target gene, and can be used to inhibit many different types of target genes in the pest. In particular embodiments, the nucleotide sequence may be selected from the group consisting of SEQ ID N9 697, SEQ ID No.9 813-819, SEQ ID No.9 841 and SEQ ID No.9 874.

Sequences identified as having a protective effect against the pest can be easily expressed as dsRNA molecules through the creation of appropriate expression constructs. For example, these sequences can be expressed as hairpin and stem-and-loop structures, taking a first segment that corresponds to a sequence selected from SEQ ID N91 to SEQ ID No.9 906, or a fragment thereof, joining this sequence to a second segment, which consists of a gap region that is not homologous or complementary to the first segment, and joining it to a third segment that transcribes an RNA, where at least one portion of the third segment is substantially complementary to the first segment. This construct forms a stem and curl structure by hybridizing the first segment with the third segment, where the curl structure comprises the second segment (W094/01550, W098/05770, US 2002/0048814A1 and US 2003/0018993A1).

A. Constructions and Compositions of Nucleic Acids

The invention provides recombinant DNA constructs that can be used to effect stable transformation of a particular desired pest host or symbiont. Desired hosts or pest symbionts can express pesticidally effective levels of preferred siRNA or double-stranded RNA molecules from the recombinant DNA constructs, and introduce the molecules into the pest's diet. Pairs of nucleotide sequences isolated and purified from information from cDNA libraries and/or genomic libraries can be provided. The pairs of nucleotide sequences may be derived from any preferred pest beetle for use as thermal amplification primers to generate DNA templates for the preparation of double-stranded RNA and siRNA molecules of the present invention.

As used herein, the term "nucleic acid" refers to a polymer of single- or double-stranded ribonucleotide or deoxyribonucleotide bases, which reads from the 5' end to the 3' end. The nucleic acid can also optionally contain nucleotide bases that are not naturally occurring or are altered, which allow their correct reading by a polymerase and do not reduce the expression of a polypeptide encoded by said nucleic acid. The term nucleotide sequence or "nucleic acid sequence" refers to the in-frame and antisense strand of a nucleic acid, as single strands or as double strands. The term "ribonucleic acid" (RNA) includes RNAi (inhibitory RNA), double-stranded RNA (double-stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro-RNA), tRNA ( blot RNA, loaded or unloaded with a corresponding acetylated amino acid), and cRNA (complementary RNA), and the term "deoxyribonucleic acid" (DNA) includes cDNA and genomic DNA, and DNA-RNA hybrids. The terms "nucleic acid segment", "nucleotide sequence segment" or more generally "segment" will be interpreted by those trained in the art as a functional term including genomic sequences, ribosomal RNA sequences, blot RNA sequences, sequences of messenger RNA, operon sequences, and modified small nucleotide sequences that express or can be adapted to express proteins, polypeptides, or peptides.

According to the invention there are provided nucleotide sequences, the expression of which results in an RNA sequence that is substantially homologous to an RNA molecule of a target gene in an insect comprising an RNA sequence encoded by a nucleotide sequence within the genome. of the insect Therefore, after ingestion of the stabilized RNA sequence, an inactivation of the nucleotide sequence of the target gene can be obtained in the insect cells, resulting in a deleterious effect for maintenance, viability, proliferation, reproduction. and insect ingestion.

As used herein, the term "substantially homologous" or with substantial homology", applied to a nucleic acid sequence, includes a nucleotide sequence that hybridizes under stringent conditions to the coding sequence indicated in any of SEQ

ID No.51 up to SEQ ID Nyes 906, as detailed in the list of sequences, or with their complements. Sequences that hybridize under stringent conditions with any of SEQ ID NO:1 through SEQ ID NO:906 as set forth in the sequence listing, or with complements thereof, are those that allow antiparallel alignment to take place between the two sequences, and then the two sequences are capable, under severe conditions, to form hydrogen bonds with corresponding bases on the opposite strand to form a duplex molecule that is stable enough, under stringent conditions, to be detected using methods well known in the art. Substantially homologous sequences preferably have between about 70% and about 80% sequence identity, or more preferably between about 80% and about 85% sequence identity, or more preferably between about 90% and about 95% sequence identity. , and approximately 99% sequence identity, with the reference nucleotide sequences as set forth in any of SEQ ID NO:1 through SEQ ID NO:906 as set forth in the sequence listing, or the complements thereof.

As used herein, the term "sequence identity", "sequence similarity" or homology describes sequence relationships between two or more nucleotide sequences. Percent "sequence identity" between two sequences is determined by comparing two optimally aligned sequences in a comparison window, where the portion of the sequence in the comparison window may comprise additions or deletions (i.e., mismatches). compared to the reference sequence (which does not comprise additions or deletions), in order to effect optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which an identical nucleic acid base exists in both sequences, to obtain the number of matching positions, by dividing the number of matching positions by the total number of positions in the window comparison, and multiplying the total number of positions by 100 to obtain percent sequence identity. A sequence that is identical at any position, as compared to a reference sequence, is said to be identical to the reference sequence, and vice versa. A first nucleotide sequence, when viewed in the 5' to 3' direction, is said to be "complementary" or complementary to a second nucleotide sequence or reference sequence, viewed in the 3' to 5' direction , if the first nucleotide sequence exhibits complete complementarity with the second nucleotide sequence or the reference sequence. As used herein, the sequence of nucleic acid molecules is said to exhibit "complete complementarity" when each nucleotide of one of the sequences, read in the 5' to 3' direction, is complementary to each nucleotide of the other sequence, read in the 3' to 5' direction. A nucleotide sequence that is complementary to a reference nucleotide sequence will have a sequence identical to the sequence of the reverse complement of the reference nucleotide sequence. These terms and descriptions are well defined in the art and are readily understood by those skilled in the art.

As used herein, a comparison window refers to a conceptual segment of at least 6 contiguous positions, usually between about 50 and about 100, more usually between about 100 and about 150, where a sequence is compared to a sequence. reference sequence with the same number of contiguous positions, once both sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., mismatches) of approximately 20% or less, compared to the reference sequence (which does not comprise additions or deletions), in order to effect optimal alignment of the sequences. two sequences. Those skilled in the art will be able to refer to the detailed methods used to align sequences in the Wisconsin Genetics software package, version 7.0 (Genetics Computer Group, 575 Science Drive Madison, Wis.), or to Ausubel et al. (1998) for a detailed description of sequence analysis.

The present invention provides DNA sequences capable of being expressed as an RNA in a cell or microorganism to inhibit the expression of a target gene in an insect cell, tissue or organ. The sequences comprise a DNA molecule coding for one or more different nucleotide sequences, wherein each of the different nucleotide sequences comprises a sense nucleotide sequence and an antisense nucleotide sequence connected by a spacer sequence coding for a molecule double-stranded RNA of the present invention. The spacer sequence constitutes part of the in-frame nucleotide sequence or the antisense nucleotide sequence, and forms the double-stranded RNA molecule between the in-frame and antisense sequences. The in-frame nucleotide sequence or antisense nucleotide sequence is substantially identical to the nucleotide sequence of the target gene, or a derivative thereof, or a sequence complementary thereto. The dsDNA molecule can be placed under the operative control of a promoter sequence functioning in the host cell, tissue or organ, which expresses the dsDNA to produce double-stranded RNA molecules. In one embodiment, the DNA sequence may be derived from a nucleotide sequence as set forth in SEQ ID NO:1 through SEQ ID NO:906 in the sequence listing.

The invention also provides a DNA sequence that can be expressed in plant cells that, when the DNA is expressed in RNA and the plant is ingested by a target pest, suppression of the target gene is achieved in a cell, tissue, or an organ of the pest insect. The double-stranded RNA comprises at least one or multiple structural gene sequences, where each of the structural gene sequences comprises a sense-of-frame nucleotide sequence and an antisense nucleotide sequence, connected by a spacer sequence that it forms a loop within the complementary and antisense sequences. The in-frame nucleotide sequence or antisense nucleotide sequence is substantially identical to the nucleotide sequence of the target gene, a derivative thereof, or a sequence complementary thereto. The one or more structural gene sequences are placed under the operative control of one or more promoter sequences, at least one of which is operable in the cell, tissue or organ of a prokaryotic or eukaryotic organism, particularly a plant.

A pest control gene sequence or fragment according to the invention can be cloned between two tissue-specific promoters, such as the two root-specific promoters that can operate in a transgenic plant cell and be expressed there to produce mRNA in the cell. transgenic plant, which forms double-stranded RNA molecules in it. Double-stranded RNA molecules contained in plant tissues are ingested by an insect to effect the desired suppression of target gene expression.

A nucleotide sequence provided by the present invention may comprise an inverted repeat separated by a "spacer sequence." The spacer sequence can be a region comprising any nucleotide sequence that facilitates the formation of secondary structures between each repeat, where necessary. In one embodiment of the present invention, the spacer sequence is part of the in-frame or antisense mRNA coding sequence. Alternatively, the spacer sequence can comprise any combination of nucleotides or homologues thereof that are capable of covalently binding to a nucleic acid molecule. The spacer sequence may comprise a nucleotide sequence at least about 10 to 100 nucleotides in length, or alternatively at least about 100 to 200 nucleotides in length, at least about 200 to 400 nucleotides in length, or at least about between 400 and 500 nucleotides in length.

Nucleic acid molecules, or fragments of nucleic acid molecules, or other nucleic acid molecules in the sequence listing are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules are said to be capable of specifically hybridizing to one another if the two molecules are capable of forming an antiparallel double-stranded nucleic acid structure. A nucleic acid molecule is said to be the complement of another nucleic acid molecule if they exhibit complete complementarity. Two molecules are said to be "minimally complementary" if they can hybridize to one another with sufficient stability to remain aligned under at least conventional "low stringency" conditions. Similarly, molecules are said to be complementary if they can hybridize to one another with sufficient stability to remain aligned under standard "high stringency" conditions. Conventional stringency conditions are described in Sambrook, et al. (1989) and Haymes et al. (1985).

Therefore, departures from complete complementarity are permitted, as long as such departures do not completely abrogate the molecules' ability to form a double-stranded structure. Therefore, in order for a nucleic acid molecule or a fragment of the nucleic acid molecule to serve as a probe or primer, it only needs to have sufficiently complementary sequence that it can form a double-stranded structure under the concentrations particulars of solvent and salt used.

Appropriate stringency conditions that promote DNA hybridization, for example, 6.0X sodium chloride/sodium citrate (SSC) at approximately 455C, followed by a 2.0X SSC wash at 50yesC, are known to those of skill in the art and can be found in Current Protocols in Molecular Biology (1989). For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0X SSC to 505C and a high severity of approximately 2.0X SSC at 509C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, approximately 22-C, to high stringency conditions at approximately 655C. The temperature and salt concentration can be varied, or the temperature or salt concentration can be held constant while the other variable is altered. A nucleic acid for use in the present invention can specifically hybridize to one or more WCR nucleic acid molecules or complements thereof under such conditions. Preferably, a nucleic acid for use in the present invention will exhibit at least about 80%, or at least about 90%, or at least about 95%, or at least about 98%, or even about 100% sequence identity with one or plus nucleic acid molecules as set forth from SEQ ID NO:1 through SEQ ID NO:906 as set forth in the sequence listing.

The nucleic acids of the present invention may also be synthesized, in whole or in part, especially when it is desirable to provide plant-preferred sequences, using methods known in the art. Accordingly, all or a portion of the nucleic acids of the present invention can be synthesized using codons preferred by a selected host. Host-preferred codons can be determined, for example, from the most frequently used codons in proteins expressed in a particular host species. Other modifications to the nucleotide sequences may result in mutants with slightly altered activity.

Double-stranded RNA or siRNA nucleotide sequences comprise double strands of polymerized ribonucleotides, and may include sugar phosphate or nucleoside backbone modifications. Modifications in the RNA structure can be designed to allow for specific genetic inhibition. In one embodiment, double-stranded RNA molecules can be modified using an enzymatic process, in order to generate siRNA molecules. siRNA can effectively mediate the downregulation effect of some target genes in some insects. This enzymatic process can be accomplished using an RNase III enzyme or a DICER enzyme, present in the cells of an insect, vertebrate animal, fungus, or plant in the eukaryotic RNAi pathway (Elbashir et al., 2002, Hamilton and Baulcombe, 1999). This process can also use a recombinant DICER or RNase III, introduced into the cells of a target insect using recombinant DNA procedures well known to those of skill in the art. The DICER enzyme and the RNase III enzyme can be naturally occurring in an insect or can be made using recombinant DNA procedures, but in either case they cut large strands of double-stranded RNA into smaller oligonucleotides. DICER enzymes specifically cut double-stranded RNA molecules into pieces of siRNA, each of which is approximately 19-25 nucleotides in length, whereas RNase III enzymes normally cut double-stranded RNA molecules into 12-long siRNAs. -15 base pairs. The siRNA molecules produced by either enzyme have 2 or 3 nucleotide overhangs at the 3' end, and 5' phosphate and 3' hydroxyl ends. The siRNA molecules generated by the RNase III enzyme are the same as those produced by the DICER enzymes in the eukaryotic RNAi pathway, and are therefore attacked and degraded by the cell's inherent RNA degradation mechanism, once they they are unfolded, separated into single-stranded RNA, and hybridize with the sequences transcribed by the target gene. This process results in the effective degradation or removal of the RNA sequence encoded by the nucleotide sequence of the target gene in the insect. The result is the silencing of a particular target nucleotide sequence within the insect. Detailed descriptions of the enzymatic processes can be found in Hannon (2002).

A nucleotide sequence of the present invention can be recorded on a medium that can be read by a computer. As used, “a computer-readable medium” means any tangible medium of expression that can be read and easily accessed by a computer. Such media include, without limitation: magnetic storage media, such as 31/2 disks, hard drives, storage media, and magnetic tape; optical storage media such as CD-ROMs; electrical storage media such as RAM and ROM; optical character recognition format computer files, and hybrids of these categories such as magnetic/optical storage media. Those of skill in the art will readily appreciate that any of the presently computer-readable media can be used to create a construct comprising a computer-readable media having a recorded nucleotide sequence hereof. invention.

As used in this documentation, "recording" refers to a process for storing information on a medium that can be read by a computer. Those of skill in the art will readily adopt presently known methods for recording information on computer-readable media to generate media comprising the nucleotide sequence information of the present invention. A variety of data storage structures are available for those skilled in the art to create a medium that can be read by a computer having a recorded nucleotide sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processing programs and formats can be used to store the nucleotide sequence information of the present invention on a medium that can be read by a computer. The sequence information may be represented in a word processor text file in a format provided by commercially available software, such as WordPerfect and Microsoft Word, or it may be represented in the form of an ASCII text file, stored in a database application, such as DB2, Sybase, Oracle or the like. Those of skill in the art will be able to readily adapt any number of data processor structure formats (eg, text or database files) to obtain a medium that can be read by a computer having the sequence information recorded. nucleotides of the present invention.

Computer analysis software is publicly available that allows those skilled in the art to access sequence information provided on a medium that can be read by a computer. Software implementing the BLAST (Altschul et al., (1990) and BLAZE (Brutlag, (1993)) search algorithms on a Sybase system can be used to identify open reading frames (ORFs) within sequences, such as Unigenes and ESTs that are provided herein, that display homology to ORFs or proteins from other organisms. These ORFs are protein-encoding fragments within the sequences of the present invention, and are useful for producing proteins of commercial importance, such as enzymes used in amino acid biosynthesis, metabolism, transcription, translation, RNA processing. , nucleic acid and protein degradation, protein modification and DNA replication, restriction, modification, recombination and repair.

Furthermore, the present invention provides systems, particularly computer-based systems, that contain the sequence information described in the present documentation. These systems are designed to identify commercially important fragments of the nucleic acid molecule of the present invention. As used herein, "a computer-based system" designates hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimal hardware media for the computer-based systems of the present invention comprise a central processing unit (CPU), input media, output media, and data storage media. Those of skill in the art will readily appreciate that any currently available computer based system will be suitable for use in the present invention.

As used herein, "a target structural motif" or "a target motif" designates any rationally selected sequence or combination of sequences where the sequence(s) are selected on the basis of a three-dimensional configuration that is formed by fold the white motif. There are a variety of target motifs known in the art. Target protein motifs include, without limitation, active enzyme sites and signal sequences. Target nucleic acid motifs include, without limitation, promoter sequences, cis elements, buckle structures, and inducible expression elements (protein binding sequences).

B. Recombinant Vectors and Host Cell Transformation

A recombinant DNA vector can, for example, be a closed circular or linear plasmid. The vector system can be a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA that it is desired to introduce into the genome of the bacterial host. Also, a bacterial vector can be an expression vector. Nucleic acid molecules as set out in SEQ ID NO:1 to SEQ ID NO:906 or fragments or complements thereof may, for example, be appropriately inserted into a vector under the control of an appropriate promoter functioning in one or more microbial hosts to drive expression of an associated coding sequence or other DNA sequence. There are many vectors available for this purpose, and the selection of the appropriate vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (DNA amplification or DNA expression) and the particular host cell with which it is compatible. Components of the vector for bacterial transformation generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selection marker genes, and an inducible promoter that allows expression of exogenous DNA.

Expression and cloning vectors generally contain a selection gene, also known as a selection marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example, ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) provide critical nutrients that are not available in normal media. complexes, for example, the gene encoding Bacilli D-alanine racemase. Those cells that are successfully transformed with a heterologous protein or fragment thereof produce a protein that confers drug resistance, thus the cells survive the selection regimen.

An expression vector for producing an mRNA may also contain an inducible promoter that is recognized by the host bacterial organism and linked to the nucleic acid encoding, for example, the nucleic acid molecule encoding the D. v. mRNA. virgifera or the fragment thereof of interest. Inducible promoters suitable for use with bacterial hosts include the λ-lactamase promoter, P promotersL And pR. from E. coli λ phage, and the E. coli galactose promoter, arabinose promoter, alkaline phosphatase promoter, tryptophan (trp) promoter, and lactose operon promoter, and variations thereof, and hybrid promoters such as the tac promoter. However, there are other suitable inducible bacterial promoters.

The term "operably linked," as used in reference to a regulatory sequence and a structural nucleotide sequence, denotes that the regulatory sequence causes regulated expression of the structural nucleotide sequence linked thereto. Regulatory sequences or "control elements" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-translated sequences) of a structural nucleotide sequence, and which includes at the time and the level or extent of RNA transcription, processing, or stability, or translation of the associated structural nucleotide sequence. Regulatory sequences can include promoters, translational leader sequences, introns, enhancers, stem-loop structures, repressor binding sequences, and polyadenylation recognition sequences and the like.

Alternatively, the expression constructs can be integrated with an integration vector into the bacterial genome. Integration vectors typically contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. The integrations are the result of recombinations between the homologous DNA in the vector and the bacterial chromosome. For example, integration vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP 0 127328). Integration vectors can also be composed of bacteriophage or transposon sequences. Suicide vectors are also known in the art.

Construction of appropriate vectors containing one or more of the components listed above employs standard recombinant DNA procedures. The isolated plasmids or DNA fragments are separated, excised and assembled in a desired shape to generate the necessary plasmids. Examples of available bacterial expression vectors include, without limitation, multifunctional cloning and expression vectors of E. coli, such as Bluescript™ (Stratagene, La Jolla, CA), to which, for example, a protein from D. v. virgifera, or a fragment thereof, in frame with the amino terminal sequences and subsequent 7 residues of β-galactosidase, in order to produce a hybrid protein; pIN vectors (Van Heeke and Schuster, 1989); and the like.

A recombinant yeast construct can typically include one or more of the following: a promoter sequence, a fusion partner sequence, a leader sequence, a transcription termination sequence, a selection marker. These elements can be combined into an expression cassette that can be maintained in a replicon, such as an extrachromosomal element (eg, plasmids) capable of being stably maintained in a host, such as yeast or bacteria. The replicon can have two replication systems, which will allow it to be maintained, for example, to be expressed in yeast and to be cloned and amplified in a prokaryotic host. Examples of such bacteria-yeast shuttle vectors include YEp24 (Botstein et al., 1979), pCI/1 (Brake et al., 1984), and YRp17 (Stinchcomb et al., 1982). Furthermore, a replicon can be a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and typically from about 10 to about 150. A host containing a plasmid with a large number of copies will preferably have at least about 10, and more preferably at least about 20.

Useful yeast promoter sequences may be derived from genes encoding enzymes in the metabolic pathway. Examples of these genes include alcohol dehydrogenase (ADH) (EP 0 284044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglyceratomutase, and pyruvate. kinase (PyK) (EP 0 3215447). The yeast PHO5 gene, which encodes an acid phosphatase, also provides useful promoter sequences (Myanohara et al., 1983). In addition, non-naturally occurring synthetic promoters may also function as yeast promoters. Examples of these hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (US Pat. No.9 4876197 and 4880734). Examples of transcriptional terminator sequences and other termination sequences recognized by yeast, such as those encoding glycolytic enzymes, are known to those of skill in the art.

Alternatively, the expression constructs can be integrated with an integration vector into the yeast genome. Integration vectors typically contain at least one sequence homologous to a bacterial yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences surrounding the expression construct. The integrations appear as a result of recombinations between homologous DNAs in the vector and in the yeast chromosome (Orr-Weaver et al., 1983). An integrating vector can be targeted to a specific locus in yeast by selecting the appropriate homologous sequence to include in the vector. See Orr-Weaver et al., supra. One or more expression constructs can be integrated, possibly affecting the levels of recombinant protein produced (Riñe et al., 1983).

The present invention also contemplates the transformation of a nucleotide sequence of the present invention in a plant to achieve expression levels capable of inhibiting pests of one or more double-stranded RNA molecules. A transformation vector can be readily prepared using methods available in the art. The transformation vector comprises one or more nucleotide sequences that are capable of being transcribed into an RNA molecule, and that are substantially homologous and/or complementary to one or more nucleotide sequences encoded by the insect genome, such that, upon entering the RNA the downregulation of at least one of the respective nucleotide sequences of the insect genome is verified.

The transformation vector can be referred to as a dsDNA construct and can also be defined as a recombinant molecule, an insect control agent, a genetic molecule, or a chimeric genetic construct. A chimeric construct of the present invention may comprise, for example, nucleotide sequences encoding one or more antisense transcripts, one or more in-frame transcripts, one or more of each of the aforementioned sequences, where all or part of a transcript thereof is homologous to all or part of a DNA molecule comprising an RNA sequence encoded by a nucleotide sequence in the genome of an insect.

In one embodiment, the plant transformation vector comprises an isolated and purified DNA molecule, comprising a promoter operatively linked to one or more nucleotide sequences of the present invention. The nucleotide sequence is chosen from the group consisting of SEQ ID NO:1 through SEQ ID NO:906 as set forth in the sequence listing. The nucleotide sequence includes a segment encoding all or part of an RNA present in a target pest RNA transcript, and may comprise inverted repeats of all or part of a target pest RNA. The DNA molecule comprising the expression vector may also contain the sequence of a functional intron located upstream of the coding sequence or even within the coding sequence, and may also contain a non-translated (5') leader sequence (i.e. , a UTR or 5'-UTR) located between the promoter and the translation start point.

A plant transformation vector can contain sequences from more than one gene, allowing more than one double-stranded RNA to be produced to inhibit the expression of two or more genes in the cells of a target pest. Those skilled in the art will readily appreciate that DNA segments, the sequence of which corresponds to that present in different genes, can be combined into a single composite DNA segment, in order to express it in a transgenic plant. Alternatively, a plasmid of the present invention, which already contains at least one DNA segment, can be modified by consecutive insertion of additional DNA segments between the enhancer, promoter, and terminator sequences. In the insect control agent of the present invention designed to inhibit multiple genes, the genes to be inhibited can be obtained from the same insect species in order to improve the effectiveness of the insect control agent. In certain embodiments, the genes may be derived from different insects, in order to broaden the range of insects against which the agent may be effective. When it is desired to delete several genes, or when several genes are selected for expression and suppression in combination, a polycistronic DNA element can be made as illustrated and described in Fillatti, US Application Publication No.9 2004-0029283.

Promoters that function in different plant species are also known in the art. Promoters useful for expressing polypeptides in plants include those that are inducible, viral, synthetic, or constitutive, as described in Odell et al. (1985), and/or promoters that are temporally, spatially, and spatiotemporally regulated. Preferred promoters include the improved CaMV35S promoters and the FMV35S promoter. For the purpose of the present invention, eg to obtain optimal control of the root-feeding species, it may be preferable to obtain higher levels of expression of these genes in plant roots. Several promoters with enhanced expression in the root have been identified and are known in the art (Lu et al., 2000, US Pat. No.9 5837848 and 6489542).

A vector or recombinant DNA construct of the present invention will typically comprise a selection marker that confers on plant cells a selectable phenotype. Selection markers can also be used to select for plants or plant cells that contain the exogenous nucleic acids encoding the polypeptides or proteins of the present invention. The marker can code for biocide resistance, antibiotic resistance (eg, kanamycin, G418 bleomycin, hygromycin, etc.), or herbicide resistance (eg, glyphosate, etc.). Examples of selection markers include, without limitation, a neo gene, which encodes kanamycin resistance and can be selected for using kanamycin, G418, etc.; a bar gene, which encodes resistance to bialaphos; a mutant EPSP synthetase gene, encoding glyphosate resistance; a nitrilase gene, which confers resistance to bromoxynil; a mutant acetolactate synthetase (ALS) gene, which confers resistance to imidazolinone or sulfonylurea; and a DFHR gene with resistance to methotrexate. Examples of such selection markers are illustrated in US Patents 5,550,318; 5,633,435; 5,780,708 and 6,118,047.

A vector or recombinant construct of the present invention may also include a test marker gene. Assay markers can be used to monitor expression. Examples of marker genes for analysis include a β-glucuronidase or uidA (GUS) gene encoding an enzyme for which various chromogenic substrates are known (Jefferson, 1987; Jefferson et al., 1987); an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., 1988); a β-lactamase gene (Sutcliffe et al., 1978), a gene encoding an enzyme for which various chromogenic substrates are known (eg PADAC, a chromogenic cephalosporin); an Iuciferase gene (Ow et al., 1986) an xylE gene (Zukowsky et al., 1983) encoding a catechol dioxygenase that can convert chromogenic catechols; an aamylase gene (Ikatu et al., 1990); a tyrosine gene (Katz et al., 1983) encoding an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which is in turn condensed to melanin; an α-galactosidase, which catalyzes a chromogenic substrate of α-galactose.

Preferred plant transformation vectors include those derived from an Agrobacterium tumefaciens Ti plasmid (eg US Patent Nos. 4,536,475, 4,693,977, 4,886,937, 5,501,967 and EP 0 122 791 ). Agrobacterium rhizogenes (or "Ri") plasmids are also useful and are known in the art. Other known plant transformation vectors include those set forth, for example, in Herrera-Estrella (1983); Bevan (1983), Klee (1985) and EP 0 120 516.

In general, it is preferable to introduce functional recombinant DNA at a non-specific location in a plant genome. In special cases, it may be useful to insert a recombinant DNA construct by integration at specific sites. There are several specific recombination systems known to work in plants, including cre-lox, which is described in US Pat.yes 4,959,317, and FLP-FRT, which is described in US Patent No.yes5527695.

Appropriate methods for transformation of host cells for use with the present invention are believed to include virtually any method by which DNA can be introduced into a cell, such as direct delivery of DNA such as by PEG-mediated transformation of protoplasts (Omirulleh eía /., 1993), DNA uptake by desiccation/inhibition (Potrykus et al., 1985), by electroporation (US Pat. No. 5,384,253), by agitation with silicon carbide fibers (Kaeppler et al., 1990; US Patent No. 5,302,523; and US Patent No. 5,464,765), by transformation mediated by Agrobacterium (US Patent No. 5,591,616 and US Patent No. 5,563,055) and by acceleration of DNA-coated particles (US Patent No. 5,550 .318, US Patent No. 5,538,877, and US Patent No. 5,538,880), etc. Through the application of techniques such as these, cells of virtually any species can be stably transformed. In the case of multicellular species, the transgenic cells can be regenerated into transgenic organisms.

Methods for the creation of transgenic plants and expression of heterologous nucleic acids in particular plants are known and can be used with the nucleic acids provided herein to prepare transgenic plants that exhibit decreased susceptibility to ingestion by a pest organism. targets such as corn rootworms. Vectors for plant transformation can be prepared, for example, by inserting the double-stranded RNA producing nucleic acids set forth herein into plant transformation vectors and introducing these into plants. A known vector system has been produced by modifying the natural gene transfer system of Agrobacterium tumefaciens. The native system comprises large Ti (tumor inducing) plasmids containing a large segment, known as T-DNA, which is transferred to transformed plants. Another segment of the Ti plasmid, the vir region, is responsible for the transfer of T-DNA. The T-DNA region is bordered by terminal repeats. In the modified binary vectors the tumor inducing genes have been removed and the functions of the vir region are used to transfer exogenous DNA bordered by the T-DNA border sequences. The T region may also contain a selection marker for efficient recovery of transgenic plants and cells, and a multiple cloning site for inserting sequences to be transferred such as a double-stranded RNA encoding a nucleic acid.

A transgenic plant formed using Agrobacterium transformation methods typically contains a single single recombinant DNA sequence inserted into a chromosome, and is known as a transgenic event. These transgenic plants can be referred to as heterozygous for the inserted foreign sequence. A transgenic plant homozygous with respect to a transgene can be obtained by self-fertilization of a segregating transgenic plant to produce F1 seeds. One quarter of the F1 seeds produced will be homozygous for the transgene. Germination of F1 seeds results in plants that can be assessed for heterozygosity or homozygosity, typically using an SNP assay or a thermal amplification assay that allows one to distinguish between heterozygotes and homozygotes (ie, a zygosity assay).

C. Expression of Nucleic Acids and Suppression of the Target Gene

The present invention provides, by way of example, a transformed host organism or symbiont of the desired pest, transformed plant cells, and transformed plants and their progeny. Transformed plant cells and transformed plants can be engineered to express one or more of the double-stranded RNA or siRNA sequences described herein to provide an anti-pest protection effect. These sequences can be used for gene suppression in a pest organism, thereby reducing predation by the pest on a protected transformed host or symbiont organism. As used herein the words "gene suppression" refer to any of the well-known methods of reducing levels of gene transcription to mRNA and/or subsequent mRNA translation.

Gene deletion also denotes the reduction of protein expression from a gene or coding sequence, including post-transcriptional deletion and gene transcriptional deletion. Post-transcriptional deletion is mediated by homology between all or part of an mRNA transcribed from a gene or coding sequence to be deleted, and the corresponding double-stranded RNA used for deletion, and refers to the substantial and measurable reduction in the amount of mRNA available for binding to ribosomes. Transcribed RNA can be in the sense of reading frame, so the effect is known as co-suppression, in the antisense orientation, so the effect is known as in-frame deletion, or in both orientations, which which allows the production of a double-stranded RNA that exerts an effect known as RNA interference (RNAi).

Transcriptional suppression is mediated by the presence in the cell of a double-stranded RNA suppression agent that exhibits substantial sequence identity to or the complement of a promoter DNA sequence, exhibiting an effect known as trans suppression. of the promoter. Genetic deletion can be effective against a native plant gene associated with a trait, for example, to provide plants with reduced levels of a protein encoded by the native gene or with enhanced or reduced levels of an affected metabolite. Gene suppression may also be effective against target genes in plant pests that may ingest or come into contact with plant material containing gene suppression agents specifically designed to inhibit or suppress the expression of one or more homologous or complementary sequences in the cells of the plant. plague. Post-transcriptional suppression of genes by antisense or in-frame RNA to regulate gene expression in plant cells is described in US Pat.5 5107065, 5759829, 5283184 and 5231020. The use of double-stranded RNA to suppress genes in plants is described in WO 99/53050, WO 99/49029, US Patent Application Publication No.9 2003/0175965 and 2003/0061626, US Patent Application No. 10/465800, and US Patent Nos.9 6506559 and 6326193.

One beneficial method for effecting post-transcriptional gene suppression in plants employs antisense and sense-oriented transcript RNA, which is stabilized, for example, as buckle and stem-and-loop structures. A preferred DNA construct for effecting post-transcriptional gene suppression is one in which a first segment encodes an RNA exhibiting an antisense orientation that exhibits substantial identity with a segment of a target gene to be suppressed, which associates with a second segment in sense orientation with the reading frame encoding an RNA that exhibits substantial complementarity with the first segment. Said construction forms a stem-and-loop structure from the hybridization of the first segment with the second segment, and a loop structure from the nucleotide sequences that join both segments (see W094/01550, W098/05770 USA 2002/0048814 and USA 2003/0018993).

According to one embodiment of the present invention, there is provided a nucleotide sequence the expression of which in vitro results in the transcription of a stabilized RNA sequence that is substantially homologous to a target gene RNA molecule, in an insect comprising a RNA sequence encoded by a nucleotide sequence within the insect genome. Accordingly, once the stabilized RNA sequence incorporated in a diet or sprayed on the surface of a plant is ingested by the insect, downregulation of the nucleotide sequence corresponding to the target gene is effected in the cells of a target insect.

Inhibition of a target gene using the stabilized double-stranded RNA technology of the present invention exhibits sequence specificity, since nucleotide sequences corresponding to the double-stranded RNA region are the targets of genetic inhibition. Inhibition of RNA containing a nucleotide sequence identical to a portion of the target gene is preferred. RNA sequences with single point insertions, deletions and mutations relative to the target sequence have also been found to be effective for inhibition. In practicing the present invention, it is preferable that the inhibitory dsRNA and the target gene portion share at least about 80% sequence identity, or about 90% sequence identity, or about 95% identity. of sequence, or approximately 99% sequence identity, or even approximately 100% sequence identity. Alternatively, the RNA duplex region can be defined in functional terms as a nucleotide sequence capable of hybridizing to a portion of the target gene transcript. An incomplete sequence that exhibits greater homology makes up for the shorter length with greater homology. The length of the identical nucleotide sequences can be at least about 25, 50, 100, 200, 300, 400, 500, or at least about 1000 bases. Normally a sequence of greater than 20-100 nucleotides should be used, although a sequence of greater than about 200-300 nucleotides will be preferred, and a sequence of greater than about 500-1000 nucleotides will be especially preferred, depending on the size of the target gene. The invention has the advantage of being able to tolerate sequence variations that can be expected due to genetic mutation, strain polymorphism or evolutionary divergence. The introduced nucleic acid molecule may not need to be absolutely homologous, may not need to be full length, to the primary transcription product or the complete processed mRNA of the target gene. Accordingly, those of skill in the art will understand that, as described herein, 100% sequence identity between the RNA and the target gene is not required to practice the present invention.

Inhibition of target gene expression can be quantified by measuring endogenous target RNA or proteins produced by translation of the target RNA, and the consequences of inhibition can be confirmed by examining the external properties of the cell or organism. Procedures for quantifying RNA and protein are well known to those of skill in the art. There are multiple selection markers available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, spectinomycin, rifampicin, and tetracycline, and the like.

In some embodiments, gene expression is inhibited by at least 10%, preferably at least 33%, more preferably at least 50%, and even more preferably at least 80%. In particularly preferred embodiments of the invention, gene expression is inhibited by at least 80%, more preferably at least 90%, more preferably at least 95% or at least 99%, in the insect cells, such that a significant inhibition. Significant inhibition designates inhibition sufficient to result in a detectable phenotype (eg, larval growth arrest, paralysis or mortality, etc.) or a detectable decrease in RNA and/or protein corresponding to the desired target gene. inhibit. Although in some embodiments of the invention Inhibition occurs in substantially all cells of the insect, in other preferred embodiments Inhibition occurs in only a subset of cells expressing the gene. For example, if the gene to be inhibited has an essential role in the alimentary tract of the insect, inhibition of the gene in these cells is sufficient to have a detrimental effect on the insect.

Double-stranded RNA molecules can be synthesized in vivo or in vitro. Double-stranded RNA can be formed from one self-complementary RNA strand or from two complementary RNA strands. The endogenous RNA polymerase of the cell can mediate transcription in vivo, a cloned RNA polymerase can be used to effect transcription in vivo or in vitro. Inhibition can be directed by specific transcription in an organ, tissue, or cell type, stimulation of an environmental condition (eg, infection, stress, temperature, chemical inducers), and/or modification of transcription in a stage of development or a certain age. The RNA strands may or may not be polyadenylated; the RNA strands may or may not be capable of being translated into a polypeptide in the translation apparatus of the cell.

Those of skill in the art can produce an RNA, dsRNA, siRNA or miRNA of the present invention by chemical or enzymatic means, using manual or automated reactions, or in vivo in another organism. RNA can also be produced by partial or total organic synthesis; any modified ribonucleotide can be introduced using in vitro enzymatic or organic synthesis. The RNA can be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (eg, T3, T7, SP6). The use and production of an expression construct are known in the art (see, for example, WO 97/32016; US Pat. No.9 5593874, 5698425, 5712135, 5789214 and 5804693). If synthesized by chemical means or using in vitro enzymatic synthesis, the RNA can be purified before being introduced into bulk. For example, RNA can be purified from a mixture by performing solvent or resin extraction, precipitation, electrophoresis, chromatography, or a combination of these. Alternatively, RNA with minimal or no purification can be used to avoid losses caused by sample processing. The RNA can be dried for storage, or it can be dissolved in an aqueous solution. The solution may contain buffers or salts to promote alignment and/or stabilization of the duplexes.

To transcribe a transgene or expression construct in vivo, a regulatory region (eg, a promoter, enhancer, silencer, and polyadenylation signal) can be used to transcribe the RNA strand(s). Then, in one embodiment, the nucleotide sequences that are used to produce RNA molecules may be operatively linked to one or more promoter sequences functional in a microorganism, fungus, or plant host cell. Under ideal circumstances, nucleotide sequences are placed under the control of an endogenous promoter that normally resides in the host genome. The nucleotide sequence of the present invention, under the control of an operably linked promoter sequence, may be further surrounded by additional sequences that advantageously affect transcription and/or the stability of the resulting transcript. These sequences are generally located upstream of the operably linked promoter and/or downstream of the 3' end of the expression construct, and may occur upstream of the promoter and downstream of the 3' end of the expression construct, although they are also contemplated. the use of one of these strings upstream alone.

As used herein, the term "insect control agent" or "gene suppression agent" refers to a particular RNA molecule comprising a first RNA segment and a second RNA segment, wherein the Complementarity between the first and second RNA fragments results in the ability of the two segments to hybridize in vivo and in vitro to form a double-stranded molecule. It may generally be preferable to include a third RNA segment that associates and stabilizes the first and second sequences so that the entire structure forms a stem-and-loop structure, or even more strongly hybridizing structures may form a stem-and-loop structure. knotted. Alternatively, a symmetrical loop can be formed without a third segment in which there is no designed loop, but for steric reasons a loop would create its own loop when the stem is long enough to stabilize itself. The first and second RNA segments will generally extend within the length of the RNA molecule and will be substantially inverted repeats of one another and will associate together via the third RNA segment. The first and second segments invariably correspond, and do not, respectively, to an in-frame sequence and an antisense structure, relative to the target mRNA transcribed from the target gene in the target insect pest that is deleted by ingestion of the molecule. of double-stranded RNA. The insect control agent may also be a substantially purified (or isolated) nucleic acid molecule, and more specifically may comprise nucleic acid molecules or fragments of nucleic acid molecules from a genomic DNA (gDNA) or cDNA library. Alternatively, the fragments may comprise smaller oligonucleotides, with between about 15 and about 250 nucleotide residues, and more preferably between about 15 and about 30 nucleotide residues.

As used herein, the term "genome" as applied to cells of an insect or host encompasses not only chromosomal DNA found in the nucleus, but also organelle DNA found within the subcellular components of the cell. Therefore, the DNAs of the present invention, introduced into plant cells, can be integrated into chromosomes or located in organelles. The term "genome", applied to bacteria, encompasses the chromosome and plasmids within a bacterial host cell. Accordingly, the DNAs of the present invention, introduced into bacterial host cells, can be integrated into chromosomes or located on plasmids.

As used herein, the term "pest" refers to insects, arachnids, crustaceans, fungi, bacteria, viruses, nematodes, flatworms, roundworms, spindleworms, hookworms, tapeworms, typanosomes, sguistosomes , parasitic flies, fleas, ticks, mites and lice, and the like, which invade the human environment and can ingest or come into contact with one or more cells, tissues or fluids produced by a pest host or symbiont transformed to express or coated with a double-stranded gene suppression agent, or capable of ingesting plant material containing the gene suppression agent. As used herein, a "pest resistance" trait that is characteristic of a transgenic plant, transgenic animal, host, or transgenic symbiont causes the plant, animal, host, or symbiont to be resistant to attack by a pest that is typically capable of inflicting damage or loss on the plant, animal, host, or symbiont. This resistance to the pest may arise from a natural mutation, or more typically from the incorporation of recombinant DNA that confers resistance to the pest. To impart insect resistance to a transgenic plant, a recombinant DNA can be transcribed, for example, into an RNA molecule that forms a double-stranded RNA molecule within the tissues or fluids of the recombinant plant. The double-stranded RNA molecule is composed in part of an RNA segment, which is identical to the corresponding RNA segment, encoded by a DNA sequence within a pest insect that prefers to feed on the recombinant plant. Expression of the gene within the desired insect pest is suppressed by the double-stranded RNA, and suppression of expression of the gene in the desired insect pest results in a plant exhibiting resistance to the insect. Fire et al. (US Patent No. 6506599) described inhibition of pest infestation generically, providing specific details for only a few nucleotide sequences that were effective in inhibiting gene function in nematodes of the species Caenorhabditis elegans. Similarly, Plaetinck et al. (US 2003/0061626) describe the use of double-stranded RNA to inhibit the gene function of a variety of pest nematodes. Mesa et al. (USA. 2003/0150017) describe the use of dsDNA sequences to transform host cells so that they express corresponding dsRNA sequences substantially identical to target sequences in specific pathogens, and particularly describe the construction of recombinant plants that express these sequences of dsDNA. double-stranded RNA, which can be ingested by various plant pests, This facilitates downregulation of a gene in the pest genome and improves plant resistance to pest infestation.

The present invention provides for the inhibition of gene expression of one or more target genes in a target pest using stabilized double-stranded RNA methods. The invention is particularly useful in the modulation of eukaryotic gene expression, in particular the modulation of the expression of genes present in pests that exhibit a pH level of their digestive systems that is between about 4.5 and about 9.5, more preferably between about 5.0 and about 8.0, and even more preferably between about 6.5 and about 7.5. For plant pests with a digestive system that exhibits pH levels outside these ranges, the use of delivery methods that do not require the ingestion of double-stranded RNA molecules may be desired.

The modulating effect of double-stranded RNA can be applied to a variety of genes expressed in pests, including, for example, endogenous genes responsible for cell metabolism or cell transformation, including homeostasis genes, transcription factors, and other genes that encode polypeptides involved in cell metabolism.

As used herein, the phrase "inhibition of gene expression or "inhibition of a target gene in insect cells" refers to the absence (or observable decrease) in the level of protein products and/or or mRNA of the target gene. Specificity refers to the ability to inhibit the target gene without effect on other genes in the cell, and without effect on any gene within the cell that produces the double-stranded RNA molecule. Inhibition of the gene expression of the target gene in the insect pest may result in new phenotypic traits in the insect pest.

The present invention provides in part a delivery system for the administration, to insects, of insect control agents, comprising exposing the latter to a diet containing the insect control agents of the present invention. According to one embodiment, the stabilized double-stranded RNA or siRNA molecules can be incorporated into the insect's diet or can be placed on the diet for consumption by the insect. The present invention also provides in part a delivery system for administering the insect control agents to insects, comprising exposing them to a microorganism or a host, such as a plant, containing the insect control agents of the present invention, which is followed by ingestion of the microorganism or host cells, or the contents of the cells. According to another embodiment, the present invention involves the generation of a transgenic plant cell or plant containing a recombinant DNA construct that transcribes the stabilized double-stranded RNA molecules of the present invention. As used herein, the phrase "generating a transgenic plant cell or plant" refers to methods of employing recombinant DNA technologies available in the art (for example, according to Sambrook, et al., 1989 ) to construct a vector for transforming plants that allows transcription of the stabilized double-stranded RNA molecules of the present invention, to transform the plant cell or the plant, and to generate the plant cell or transgenic plant containing the transcribed stabilized double-stranded RNA molecules.

In yet another embodiment, non-pathogenic, attenuated strains of microorganisms can be used as a vehicle for insect control agents, and in this case, the microorganisms carrying these agents are also known as insect control agents. Microorganisms can be engineered to express a nucleotide sequence of a target gene so as to produce RNA molecules comprising RNA sequences homologous to or complementary to RNA sequences typically found within insect cells. Exposure of insects to microorganisms results in ingestion of the microorganisms and downregulation of target gene expression mediated directly or indirectly by RNA molecules, or fragments or derivatives thereof.

Alternatively, the present invention provides for exposure of an insect to the insect control agents of the present invention, incorporated into a spray mixer and applied to the surface of a host, such as a plant host. In one example of this embodiment, ingestion of the insect control agents by an insect allows the insect control agents to be delivered to the insect's gut, and subsequently to cells within the insect's body. In another embodiment, infection of the insect by the insect control agents through other means, such as by injection or by other physical methods, also allows administration of the insect control agents. In yet another embodiment, the RNA molecules themselves are encapsulated in a synthetic matrix, such as a polymer, and applied to the surface of a host, such as a plant. Ingestion of the host cells by an insect allows insect control agents to be delivered to the insect and results in downregulation of a target gene in the host.

It is contemplated that the compositions of the present invention may be incorporated into the seeds of a plant species, either as a product of recombinant gene expression incorporated into the genome of plant cells, or incorporated into a coating or treatment. for seeds that is applied to the seed before sowing it. The plant cell containing a recombinant gene is considered as a transgenic event in the present documentation.

It is believed that a pesticide seed treatment may provide significant advantages when combined with a transgenic event that provides protection against infestations of coleopteran pests that is within the preferred range of effectiveness against a target pest. Furthermore, it is believed that there are situations well known to those of skill in the art where it is advantageous to have these transgenic events within the preferred range of effectiveness.

The present invention provides in part a delivery system for delivering insect control agents to insects. The stabilized double-stranded RNA or siRNA molecules of the present invention can be introduced directly into the cells of an insect, or they can be introduced into an extracellular cavity, an interstitial space, the lymphatic system, the digestive system, into the insect circulation by oral ingestion or through other means as may be employed by those skilled in the art. Oral introduction methods can include direct mixing of the RNA with insect food, as well as modification approaches, where a food species will be modified to express dsRNA or siRNA, then fed to the insect. insect that you want to affect with it. In one embodiment, for example, the double-stranded RNA or siRNA molecules can be incorporated into, or placed on, the insect's diet. In another embodiment, the RNA can be sprayed on the surface of the plant. In yet another embodiment, the double-stranded RNA or siRNA can be expressed in microorganisms, and the microorganisms can be applied to the surface of a plant, or introduced into the root or stem through a physical means, such as an injection. In yet another embodiment, a plant can be genetically engineered to express the double-stranded RNA or siRNA in an amount sufficient to kill insects known to infect the plant.

Specifically, in practicing the present invention in WCR, double-stranded RNA or siRNA can be introduced into the interior of the insect, in the mesenteron, to achieve the desired inhibition of target genes. Double-stranded RNA or siRNA molecules can be incorporated into the diet or can be placed on the diet, as previously described, for later ingestion by insects. In either case, the double-stranded RNAs of the present invention will be provided in the diet of the target pest. The target pest of the present invention will exhibit a digestive tract pH between about 4.5 and about 9.5, or between about 5 and about 8.5, or between about 6 and about 8, or between about 6.5 and about 7.7, or about 7.0. The digestive tract of a target pest is defined herein as the location within the pest where food that is ingested by the target pest is exposed to an environment that is favorable for the uptake of double-stranded RNA molecules. of the present invention without undergoing a pH so extreme that it causes the dissociation of hydrogen bonds between the double strands of double-stranded RNA and the formation of single-stranded molecules.

It is also anticipated that double-stranded RNAs produced by chemical or enzymatic synthesis may be formulated in a manner consistent with common agricultural practices, and may be used as sprayable products to control insect infestations. Formulations can include appropriate adhesives and moisturizers necessary for efficient foliar coverage, as well as UV protectants to protect dsRNAs from UV radiation damage. These additives are commonly used in the bio-insecticide industry and are well known to those of skill in the art. These applications may be combined with other applications of sprayable insecticides, biologically based or not, to enhance plant protection against insect feeding damage.

The present inventors contemplate the use of the bacterial strains that produce insecticidal proteins to produce double-stranded RNA for the purpose of controlling insects. These strains may exhibit improved insect control properties. A variety of different bacterial hosts can be used to produce double-stranded RNA to control insects. Examples of bacteria may include E. coli, B. thuringiensis. Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Serratia entomophila and species related to Serratia sp., B. sphaericus, B. cereus, B. laterosporus, B. popilliae, Clostridium bifermentans and Clostridium species, or other bacteria that form gram positive spores. In certain embodiments, bacteria can be engineered to control pests such as mosquitoes.

The present invention also relates to recombinant DNA constructs that can be expressed in a microorganism. The exogenous nucleic acids, from which the RNA of interest is transcribed, can be introduced into a microbial host cell, such as a bacterial cell or a fungal cell, using methods known in the art.

The nucleotide sequences of the present invention can be introduced into a wide variety of prokaryotic and eukaryotic microorganism hosts in order to produce stabilized double-stranded RNA or siRNA molecules. The term "microorganism" includes prokaryotic and eukaryotic species of microbes, such as bacteria and algae. Fungi include yeasts and filamentous fungi, among others. Gram-negative and Gram-positive prokaryotes include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella and Proteus-, Bacillaceae-, Phyzobiaceae, such as Rhizobium; Spirillaceae, such as photobacteria, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae- Pseudomonadaceae, such as Pseudomonas and Acetobacter, Azotobacteraceae, Actinomycetales and Nitrobacteraceae. Among the eukaryotes are fungi, such as Phycomycetes and Ascomycetes, including yeasts, such as Saccharomyces and Schizosaccharomyces-, and Basidiomycetes, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.

For the purpose of protecting plants against insects, a large number of microorganisms that are known inhabitants of the phytoplane (the surface of plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide A variety of important cultures may also make desirable host cells for handling, propagating, storing, administering, and/or mutagenizing the described recombinant constructs. These microorganisms include bacteria, algae, and fungi. Microorganisms of particular interest are, such as bacteria, for example, the genera Bacillus (including the species and subspecies B. thuringiensis kurstaki HD-1, B. thuringiensis kurstaki HD-73, B. thuringiensis sotto, B. thuringiensis berliner, B. thuringiensis thuringiensis,

B. thuringiensis tolworthi, B. thuringiensis dendrolimus, B. thuringiensis alestí, B. thuringiensis galleriae, B. thuringiensis aizawai, B. thuringiensis subtoxicus, B. thuringiensis entomocidus, B. thuringiensis tenebríonis and B. thuringiensis san diego); Pseudomonas, Erwinia, Serraria, Klebsiella, Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophiiius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeasts, for example, genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such species of phytosera bacteria such as Pseudomonas syringae, Pseudomonas fluorescens, Serraria marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobacter sphaeroides, Xanthomonas campestris, Rhizobium meliori, Alcaligenes eutrophus, and Azotobacter vinlandii; and plant yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.

D. Transgenic Plants

The present invention provides seeds and plants having one or more transgenic events. Combinations of events are called "pooled" transgenic events. These pooled transgenic events may be events that are directed at the same target pest, or that may be directed at different target pests. In one embodiment, a seed that has the ability to express a nucleic acid provided herein also has the ability to express at least one additional insecticidal agent, including, but not limited to, an RNA molecule whose sequence is derived of the sequence of an RNA expressed in a target pest and which forms a double-stranded RNA structure when expressed in the seed or in the cells of a plant grown from the seed, wherein ingestion of one or more plant cells by the target pest results in suppression of RNA expression in the target pest cells.

In certain embodiments, a seed that has the ability to express a double-stranded RNA whose sequence is derived from a target pest also has a transgenic event that provides herbicide tolerance. A beneficial example of a herbicide tolerance gene provides resistance to glyphosate, N-(phosphonomethyl)glycine, including the isopropylamine salt form of said herbicide.

In the present method, the combination of the expression of an insecticidal amount of a double-stranded RNA within the cells of a transgenic seed or plant grown from the seed coupled with treatment of the seed or plant with certain chemical or protein pesticides to provide unexpected synergistic advantages, including unexpectedly superior efficacy for protection against damage to the resulting transgenic plant by the target pest. In particular embodiments, treatment of a transgenic seed that is capable of expressing certain constructs that form double-stranded RNA molecules, the sequence of which is derived from one or more sequences that are expressed in the corn rootworm, with between about 100 mg to approximately 400 mg of pesticide per 100 kg of seed provides unexpectedly superior protection against corn rootworm. Furthermore, such combinations are also believed to be effective in protecting emergent plants against preemption by other pests. The seeds of the present invention can also be used to lower the cost of pesticide use, since less pesticide can be used to obtain a required level of protection than when such methods are not used. Furthermore, because less pesticide is used and because it is applied prior to planting and without separate field application, the present method is believed to be consequently safer for the operator and the environment, and is potentially Less expensive than conventional methods.

By "synergistic" it is intended to encompass the synergistic effects of the combination on the pesticidal activity (or efficacy) of the combination of the transgenic event and the pesticide. However, it is not intended to limit such synergistic effects to pesticidal activity, but should also include advantages such as greater range of activity, advantageous activity profile in terms of type and amount of harm reduction, lower cost of the pesticide and its application, the lower distribution of pesticides in the environment, the lower exposure to the pesticide of the personnel who produce, handle and sow corn seeds, and other advantages known to those of skill in the art.

Pesticides are insecticides that are useful in compositions, in combination with the methods and compositions of the present invention, including seed treatments and coatings, as well as methods of using these compositions, which can be found, for example, in US Patent No.5 6551962, which is fully incorporated herein by reference.

Although it is believed that seed treatments can be applied to a transgenic seed in any physiological state, it may be preferred that the seed be in a state of sufficient durability that it is not damaged during the treatment process. Typically, the seed will be a seed that has been harvested in the field, removed from the transgenic plant, and separated from any other non-seed plant material. The seed will also preferably be physiologically stable so that the treatment does not cause any biological damage to the seed. In one embodiment, for example, the treatment can be applied to corn seeds that have been harvested, cleaned, and dried to a moisture content of less than about 15% by weight. In an alternative embodiment, the seed may be a seed that has been dried and then treated with water and/or other material, and then dried again before or during the pesticide treatment. Within the limitations described, it is believed that the treatment can be applied to the seed at any time between harvesting the seed and sowing the seed. As used herein, the term unsown seed includes seed in any period between harvesting the seed and sowing the seed in the soil, for the purpose of germinating and cultivating the plant. When it is said that the unsown seed is treated with the pesticide, this treatment does not include those practices where the pesticide is applied on the ground, instead of on the seed. For example, treatments such as band, T-band or furrow application of the pesticide simultaneously with seed sowing are not considered within the scope of the present invention.

The pesticide, or combination of pesticides, can be applied neat, ie without any diluent or other additional component present. However, the pesticide is typically applied to the seeds in the form of a pesticidal formulation. This formulation may contain one or more desirable additional components, including, without limitation, liquid diluents, binders that serve as a matrix for the pesticide, fillers to protect seeds during stress conditions, and plasticizers to improve flexibility, adhesion, and/or the dispersibility of the coating.

The present pesticides can be applied to a seed as a component of a seed coating. Seed coating methods and compositions that are known in the art are useful when modified by adding one of the embodiments of the pesticide combination of the present invention. Such coating methods and apparatus for their application are set forth in, for example, US Patent No. 5,918,413, 5,891,246, 5,554,445, 5,389,399, 5,107,787, 5,080,925, 4,759,945 and 4,465,017. Seed coating compositions are set forth, for example, in US Patent Nos. 5,939,356, 5,882,713, 5,876,739, 5,849,320, 5,834,447, 5,791,084, 5,661,103, 5,622,003, 5,580,544, 5,328,942, 5,300,127, 4,735,015, 4,634,587, 4,383,391, 4,372,080, 4,339,456, 4,272,417 and 4,245,432, among others.

Pesticides that are useful in coating are those pesticides that are described in the present documentation. The amount of pesticide used to treat the seed will vary depending on the type of seed and the type of active ingredients, but the treatment will comprise contacting the seeds with an amount of the pesticide combination that is pesticide effective. When the target pest comprises insects, the amount will be an insecticidal amount that is effective as an insecticide. As used herein, an insecticidally effective amount refers to the amount of insecticide capable of killing insect pests in the larval or pupal stage of growth, or capable of consistently reducing or delaying the amount of damage produced by insect pests. .

In general, the amount of pesticide applied to the seed in the treatment will vary between about 10 g and about 2000 g of pesticide active ingredient per 100 kg of seed weight. Preferably, the amount of pesticide will be in the range of between about 50 g and about 1000 g of active ingredient per 100 kg of seeds, more preferably in the range of between about 100 g and about 600 g of active ingredient per 100 kg of seeds. , and even more preferably in the range between about 200 g and about 500 g of active ingredient per 100 kg of seed weight. Alternatively, it has been found that it is preferable that the amount of pesticide is more than about 60 g of pesticide active ingredient per 100 kg of seeds, and more preferably more than about 80 g per 100 kg of seeds.

Pesticides used in treatment must not inhibit seed germination, and must be effective in protecting seeds and/or plants during the period in the life cycle of the target insect in which it causes injury to seeds or the plants. In general, the coating will be effective for between about 0 and 120 days after planting. The pesticides of the present invention can be applied to the seeds in the form of a coating.

The benefits provided by the present invention may include, but are not limited to: ease of introduction of dsRNA into insect cells, low concentration of dsRNA that can be used, stability of dsRNA , and the effectiveness of the inhibition. The ability to use a low concentration of a stabilized dsRNA allows one to avoid several drawbacks related to antisense interference. The present invention is not limited to in vitro use or to specific sequence compositions, to a particular set of target genes, to a particular portion of the nucleotide sequences of the target genes, or to a particular transgene or to a particular method of administration, unlike some of the available procedures known in the art, such as antisense and co-suppression methods. Furthermore, genetic manipulation is possible in organisms that are not classical genetic models.

In the practice of the present invention, selections may be made to ensure that the presence of the nucleotide sequences that are transcribed from the recombinant construct are not deleterious to non-pest cells. This can be achieved by targeting genes that exhibit a low degree of sequence identity to corresponding genes in a plant or vertebrate animal. Preferably, the degree of sequence identity is less than about 80%. More preferably, the degree of sequence identity is less than about 70%. More preferably, the degree of sequence identity is less than about 60%.

In addition to direct transformation of a plant with a recombinant DNA construct, transgenic plants can be prepared by crossing a first plant with a recombinant DNA construct with a second plant lacking the construct. For example, recombinant DNA for gene suppression can be introduced into a first plant line that can be transformed to produce a transgenic plant, which can be crossed with a second plant line in order to introduce the recombinant DNA for gene suppression into the second plant line. .

In practice, the present invention may be combined with other traits for controlling insects on a plant, in order to obtain traits for improving control of insect infestations. Combining insect control traits employing different modes of action may provide insect-protected transgenic plants with greater durability than plants containing a single insect control trait, due to the lower likelihood that resistance will develop in the insect. field.

The mechanism of the insecticidal activity of the crystal proteins of 8. thuringiensis has been studied extensively in the past decade. Crystal proteins have been shown to be toxic to the larval form of the insect only after ingestion of the protein. In Lepidopteran larvae, an alkaline pH and proteolytic enzymes in the insect's mesenterum solubilize proteins, allowing the release of components that are toxic to the insect. These toxic components destroy the cells of the mesenterum, which causes the insect to stop feeding and eventually leads to its death. For this reason, toxins from 8. thuringiensis have proven to be effective and environmentally safe insecticides to deal with various insect pests. Coleoptera and Hemiptera, and probably Diptera, Lygus, and other boring and sucking insects, have a mesenteron pH that is slightly acidic, so Bt toxins that are effective against Lepidopteran larvae are not effective against these pests. . This slightly acidic pH of the digestive tract of these insects is also believed to be more suitable for the compositions of the present invention, and without being bound by this particular theory, it is likely that the alkaline pH of the digestive tract of Lepidopteran larvae is a contributing factor for the failure of efficacy of previous attempts to display a double-stranded RNA (Fire et al. US Patent No. 6,506,559; Mesa et al. Patent Publication No. US2003/0150017; Rajagopal et al., 2002; Tabara et al., 1998). Therefore, it is believed that the double-stranded RNA methods described herein should preferably be used in compositions and in plants to control Coleoptera, Diptera, Hemiptera, Lygus, and boring and sucking insects. The methods and compositions set forth herein are particularly useful for targeting suppression genes in insects that exhibit a digestive tract Ph between about 4.5 and about 9.5, or between about 5.0 and about 9.0, or between about 5.5 and about 8.5, or between about 6.0 and about 8.0, or between about 6.5 and about 7.7, or between about 6.8 and about 7.6, or about 7.0. However, insects and other pest species that exhibit a digestive tract pH of between about 7.5 and about 11.5, or between about 8.0 and about 11.0, or between about 9.0 and about 10 .0, such as lepidopteran insect larvae, are also included within the scope of the present invention. This is particularly true when a specific double-stranded RNA to inhibit a gene in a lepidopteran larva is introduced into the larval diet, in combination with one or more Bt proteins, which, relative to the Bt protein, would commonly be toxic to the larvae. said lepidopteran larva when supplied at a level equal to or higher than the threshold. The presence of one or more Bt toxins toxic to the same insect species will effectively lower the pH of the mesenteron, providing a stable environment for the double-stranded RNA molecules to exert their target gene suppression effects on the insect pest.

It is anticipated that the combination of certain stabilized dsRNA constructs with one or more genes for insect control proteins will result in synergisms that will improve the insect control phenotype of a transgenic plant. Biological assays in insects using artificial diets or whole plant tissue can be used to define dose responses, in terms of mortality or growth inhibition of larvae, using double-stranded RNA and proteins to control insects. Those skilled in the art will be able to evaluate mixtures of double-stranded RNA molecules and proteins to control insects in biological assays, in order to identify combinations that are synergistic and desirable for introduction into plants with protection against insects (Tabashnik, 1992). Synergism between different insect control proteins in the removal of insect pests has been described (for a review, see Schnepf et al., 1998). Synergisms between certain dsRNAs and between certain dsRNAs and certain insect control proteins are anticipated to exist.

The invention also relates to commercial derivative products containing one or more of the sequences of the present invention, and those produced from a recombinant plant or seed containing one or more of the nucleotide sequences of the present invention are specifically contemplated. as embodiments of the present invention. A commercial primary product containing one or more of the sequences of the present invention includes, without limitation, whole or ground foods, oils, grains, or seeds of a plant, or any food product comprising any whole or ground foods, oils, or grains. from a recombinant plant, or a seed containing one or more of the sequences of the present invention. In fact, the detection of one or more of the sequences of the present invention in one or more primary products or commercial derived products contemplated in the present documentation is evidence that the primary product or commercial derived product is prepared from a plant. transgenic modified to express one or more of the nucleotide sequences of the present invention, with the purpose of controlling insect infestations using methods of gene suppression mediated by double-stranded RNA.

D. Obtaining Nucleic Acids

The present invention provides a method for obtaining a nucleic acid comprising a nucleotide sequence to produce a double-stranded RNA or siRNA. In one embodiment, said method comprises: (a) confronting a cDNA library gDNA with a hybridization probe comprising all or a portion of a nucleotide sequence or a homologue thereof from a target insect; (b) identifying a DNA clone that hybridizes with the hybridization probe; (c) isolating the DNA clone that was identified in step (b); and (d) sequencing the cDNA or gDNA fragment comprising the clone isolated in step (c) wherein the nucleic acid molecule sequenced all or a substantial portion of the RNA nucleic acid sequence or a homologue thereof.

In another embodiment, a method of the present invention for obtaining a nucleic acid fragment comprising a nucleotide sequence to produce a substantial portion of a double-stranded RNA or siRNA comprises: (a) synthesizing first and second oligonucleotide primers corresponding to a portion of the nucleotide sequences of a target insect; and (b) amplifying a cDNA or gDNA template into a cloning vector using the first and second oligonucleotide primers from step (a) wherein the amplified nucleic acid molecule transcribes a substantial portion of a double-stranded RNA or siRNA from the present invention.

In practicing the present invention, a target gene may be derived from a corn root caterpillar (CRW), such as WCR or SOR, or any insect species that causes damage to crop plants, and consequent crop losses. performance. The use of different criteria in the selection of preferred target genes is contemplated. The gene is one whose protein product has a high turnover rate, so that inhibition by dsRNA results in a rapid decline in protein levels. In certain embodiments, it is advantageous to select a gene for which a small drop in expression level results in detrimental effects to the insect. It is preferable to attack a wide range of insect species, so the selected gene should be highly conserved among these species. Similarly, in order to confer specificity, in certain embodiments of the invention a gene will be selected that contains poorly conserved regions between individual insect species, or between insects and other organisms. In certain embodiments, it may be desirable to select for a gene that has no known homologues in other organisms.

As used herein, the term "derived form" refers to a specific nucleotide sequence that can be obtained from a particular defined source or species, although it need not necessarily be from the same source or species.

In one embodiment, a gene that is expressed in the insect mesenterum is selected. The attack of genes expressed in the mesenterum obviates the need to disperse the double-stranded RNA within the insect. Target genes for use in the present invention may include, for example, those that share substantial homology to the nucleotide sequences of genes known to be expressed in the digestive tract that encode protein components of the membrane proton V-ATPase. vascular and plasmatic (Dow et al., 1997; Dow, 1999). This protein complex is the sole source of energy for epithelial ion transport, and is responsible for alkalinization of the lumen of the mesenteron. V-ATPase is also expressed in the Malpighian tubules, an extension of the hindgut that is involved in fluid balance and detoxification of foreign compounds, in a manner analogous to the mammalian kidney. In another embodiment, the V-ATPase can be Vha68-2, or a homolog or ortholog thereof (eg as found in SEQ ID NO:821).

In another embodiment, a gene that is essentially involved in the growth, development, and reproduction of an insect is selected. Examples of genes include, but are not limited to, a CHD3 gene, a β-tubulin gene, and a gene encoding a protein predicted to be involved in transport. The Drosophila melanogaster CHD3 gene encodes a protein with ATP-dependent DNA helicase activity, which is involved in the assembly/dismantling of chromatin in the nucleus. Similar sequences have been found in various organisms, such as Arabidopsis thaliana, Caenorhabditis elegans, and Saccharomyces cerevisiae. Genes of the beta-tubulin family encode proteins associated with microtubules that are components of the cellular cytoskeleton. Related sequences can be found in organisms as diverse as C. elegans and Manduca sexta. Proteins predicted to be subunits of the endosomal sorting complex required for transport (ESCRT)-III (Babst et al., 2002), for example Dv49, are found in various organisms including mammals, yeast, and insects such as D. virgifera. Another transport-related protein is the l'-coatomer protein, abbreviated D'Cop, which encodes a product that is involved in retrograde (Golgi to ER) transport. Similar or predicted sequences have been identified in C. elegans and D. virgifera, for example Dv248 (SEQ ID NO:819).

Other target genes for use in the present invention may include, for example, those that have important roles in viability, growth, development, reproduction, and infectivity. These target genes can be homeostasis genes, transcription factors and insect-specific genes, or lethal mutations in Drosophila. Target genes for use in the present invention may also be those from other organisms, eg, from a nematode (eg, C. elegans). Additionally, the nucleotide sequences for use in the present invention may also be derived from plant, viral, bacterial or fungal genes, the functions of which have been established in the literature and whose nucleotide sequences share substantial similarity with the target genes in the genome. of an insect In accordance with one aspect of the present invention for controlling WCR, the target sequences may be derived essentially from the WCR insect target. Some examples of target sequences from WCR cDNA libraries encoding D. virgifera proteins, or fragments thereof, that are homologous to known proteins can be found in the Sequence Listing. Nucleic acid molecules are known from D. virgifera that encode homologues of known proteins (Andersen et al., US Patent Application with NQ of Case 10/205189).

For the purposes of the present invention, double-stranded RNA or siRNA molecules can be obtained from CRW by means of polymerase chain reaction (PCR™) amplification of CRW target gene sequences derived from a corn root caterpillar gDNA or cDNA library, or portions thereof. WCR larvae can be prepared using methods known to those of skill in the art, and DNA/RNA can be extracted from them. Larvae of various sizes, ranging from first instar to fully developed CRW larvae, can be used for the purposes of DNA/RNA extraction of the present invention. Genomic DNA or cDNA libraries generated from WCR can be used to perform PCR™ amplification to produce double-stranded RNA or siRNA.

Target genes can then be amplified by PCR™, and sequenced using methods available in the art. Those of skill in the art will be able to modify the PCR™ conditions to ensure optimal formation of PCR™ products. The confirmed PCR™ product can be used as a template for in vitro transcription to generate sense and antisense RNA, with the minimal promoters included.

The present inventors contemplate that the nucleic acid sequences identified and isolated herein from any insect species in the insect kingdom may be used to control WCR and other target insects. In one aspect of the present invention, the nucleic acid may be derived from a coleopteran species. Specifically, the nucleic acid may be derived from leaf beetles belonging to the genus Diabrotica (Coleoptera, Chrysomelidae) and, more specifically, the nucleic acid molecules of the present invention may be derived from a species of the virgifera group. More specifically, the nucleic acid molecules of the present invention may be derived from Diabrotica virgifera virgifera LeConte, which is commonly known as WCR. The isolated nucleic acids may be useful, for example, to identify a target gene and construct a recombinant vector to produce the double-stranded RNAs or stabilized siRNAs of the present invention, to protect plants from WCR insect infestations.

Thus, in one embodiment, the present invention comprises isolated and purified nucleotide sequences from WCR or Lygus that may be used as insect control agents. Isolated and purified nucleotide sequences may comprise those set forth in the sequence listing.

WCR or other insect nucleic acids that can be used in the present invention may also comprise nucleic acid molecules isolated and substantially purified from

Unigenes and ESTs, or fragments of these nucleic acid molecules. EST nucleic acid molecules can encode significant portions or most of the polypeptides. Alternatively, the fragments may comprise smaller oligonucleotides, with between about 15 and about 250 nucleotide residues, and more preferably between about 15 and about 30 nucleotide residues. Alternatively, the nucleic acid molecules for use in the present invention may be from cDNA libraries of WCR, or any other pest beetle species.

Nucleic acid molecules and fragments thereof from WCR, or other pest beetle species can be used to obtain other nucleic acid molecules from other species for use in the present invention to produce desired siRNA and double-stranded RNA molecules. These nucleic acid molecules include the nucleic acid molecules that encode the complete coding sequence of a protein, and surrounding sequences of these molecules. Furthermore, these nucleic acid molecules include nucleic acid molecules that encode members of the same gene family. Such molecules can be readily obtained using the above-described nucleic acid molecules or fragments thereof to screen, for example, cDNA or gDNA libraries derived from D. v. virgifera or other beetles, or Lygus hesperus. Methods for forming these libraries are well known in the art.

As used herein, the phrase coding sequence, "structural nucleotide sequence" or "structural nucleic acid molecule" refers to a nucleotide sequence that is translated into a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5' end and a stop codon at the 3' end. A coding sequence can include, without limitation, genomic DNA, cDNA, EST, and recombinant nucleotide sequences.

The term "recombinant DNA" or "recombinant nucleotide sequence" refers to DNA containing a genetic modification, effected by manipulation by mutagenesis, restriction enzymes, and the like.

For many of the insects that are potential targets for control in accordance with the present invention, there may be limited information regarding the sequences of most genes or the phenotype resulting from mutation of particular genes. Then, the present inventors contemplate that the selection of appropriate genes from insect pests for use in the present invention may be effected using information available from the study of corresponding genes in a model organism, such as Drosophila, in another insect species, or even in a nematode species, in a fungus species, or in a plant species, where the genes have been characterized. In some cases, it will be possible to obtain the sequence of a corresponding gene from a target insect by searching databases, such as GenBank, using the name of the gene or sequence, for example, from Drosophila, another insect, a nematode, a fungus or a plant from which the target gene has been cloned. Once the sequence is obtained, PCR™ can be used to amplify an appropriately selected segment of the gene in the insect for use in the present invention.

In order to obtain a DNA segment of the corresponding gene in an insect species, PCR™ primers can be designed based on the sequence found in the WCR or in other insects from which the gene has been cloned. Primers are designed to amplify a segment of DNA of sufficient length for use in the present invention. DNA (genomic DNA or cDNA) is prepared from the insect species and the PCR™ primers are used to amplify the DNA segment. Amplification conditions are selected so that amplification occurs even if the primers do not exactly match the target sequence. Alternatively, the gene (or a portion thereof) can be cloned from a gDNA or cDNA library prepared from the pest insect species, using the WCR gene or another known insect gene as a probe. Techniques for performing PCR™ and cloning from libraries are known. Additional details are provided in the examples on the process by which DNA segments can be isolated from insect pest species, based on the sequence of genes previously cloned from WCR or another target insect species. Those of skill in the art will recognize that a variety of procedures can be used to isolate gene segments from insect pest species that correspond to genes previously isolated from other species.

When an insect is the target pest of the present invention, these pests include, without limitation: from the order Lepidoptera, for example, Acleris spp., Adoxophyes spp., Aegeria spp., Agrotis spp., Alabama argillaceae, Amylois spp., Anticarsia gemmatalis, Archips spp., Argyrotaenia spp., Autographa spp., Busseola fusca, Cadra cautella, Carposina nipponensis, Chilo spp., Choristoneura spp., Clysia ambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp., Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydia spp., Diatraea spp., Diparopsis castanea, Earias spp. , Ephestia spp., Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis, Hyphantria cunea, Keifería lycopersicella, Leucoptera scitella, Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp., Malacosoma spp., Mamestra brassicae, Manduca sexta, Operophtera spp., Ostrínia Nubilalis, Pammene spp., Pandemis spp., Panolis flammea, Pectinophora gossypiella, Phthorímaea operculella, Pierisrapae, Pierisspp., Plutella xylostella, Prays spp., Scirpophaga spp., Sesamia spp., Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea spp., Tortrix spp., Trichoplusiani and Yponomeuta spp., of the order Coleoptera, for example, Agriotes spp. , Anthonomus spp., Afamaría linearis, Chaetocnema tibialis, Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinus spp., Popitia spp., Psytiodes spp., Rhizopertha spp., Scarabeidae, Sitophilus spp., Sifatroga spp., Tenebrío spp., Tríbolium spp. and Trogoderma spp., of the order Orthoptera, for example, Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea maderae, Locusta spp., Periplaneta ssp. and Schistocerca spp.;

from the order Isoptera, for example Reticulitemes ssp;

from the order Psocoptera, for example, Liposcelis spp.;

from the order Anoplura, for example Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigus spp. and Phylloxera spp.;

from the order Mallophaga, for example, Damalinea spp. and Trichodectes spp.;

from the order Thysanoptera, for example, Franklinella spp., Hercinothríps spp., Taeniothríps spp., Thríps palmi, Thríps tabaci and Scirtothríps aurantir, from the order Heteroptera, for example, Cimex spp., Distantiella theobroma, Dysdercus spp., Euchistus spp., Eurygaster spp., Leptocorísa spp., Nezara spp., Piesma spp., Rhodnius spp., Sahlbergella singularís, Scotinophara spp., Triatoma spp., Miridae family spp. such as Lygus hesperus and Lygus lineoloris, Lygaeidae family spp. such as Blissus leucopterus and Pentatomidae family spp.;

from the order Homoptera, for example, Aleurothríxus floccosus, Aleyrodes brassicae, Aonidiella spp., Aphididae, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplaster spp., Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccus hesperidum, Empoasca spp., Eriosoma larige, Erythroneura spp., Gascardia spp., Laodelphax spp., Lacanium corni, Lepidosaphes spp., Macrosiphus spp., Myzus spp., Nehotettix spp., Nilaparvata spp., Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp. , Pseudococcus spp., Psylla spp., Pulvinaria aethiopica, Quadraspidiotus spp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., Schizaphis spp., Sitobion spp., Trialeurodes vaporariorum, Trioza erytreae and Unaspis citrr, of the order Hymenoptera, for example , Acromyrmex, Atta spp., Cephus spp., Diprion spp., Diprionidae, Gilpinia politoma, Hoplocampa spp., Lasius spp., Monomorium pharaonis, Neodiprion spp, Solenopsis spp. and Vespa ssp.;

from the order Diptera, for example, Aedes spp., Antherigona soccata, Bibio hortulanus, Calliphora erythrocephala, Ceratitis spp., Chrysomyia spp., Culex spp., Cuterebra spp., Dacus spp., Drosophila melanogaster, Fannia spp., Gastrophilus spp. , Glossína spp., Hypoderma spp., Hyppobosca spp., Liriomysa spp., Lucilia spp., Melanagromyza spp., Musca ssp., Oestrus spp., Orseolia spp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxys spp., Tabanus spp., Tannia spp. and Típula spp., of the order Siphonaptera, for example, Ceratophyllus spp. und Xenopsylla cheopisy of the order Thysanura, for example, Lepisma saccharina.

The present invention has been found to be particularly effective when the pest insect is Diabrotica spp., and especially when the pest is Diabrotica virgifera virgifera (western corn root caterpillar, WCR), Diabrotica barben (western corn root caterpillar, NCR), Diabrotica virgifera zea (Mexican corn root caterpillar, MCR), Diabrotica balteata (Brazilian corn root caterpillar (BZR) or Brazilian corn root caterpillar (BCR) complex), consisting of Diabrotica viridula and Diabrotica speciosa), or Diabrotica undecimpunctata howardi (southern corn root caterpillar, SCR).

EXAMPLES

The inventors herein have identified means to control ingestion by coleopteran pests by providing double-stranded nucleic acid molecules to the pests' diet. Surprisingly, the inventors have discovered double-stranded nucleic acid molecules that function upon ingestion by the pest to inhibit a biological function in the pest, resulting in one or more of the following attributes: reduced ingestion by the pest, reduced viability of the pest, death of the pest, inhibition of development and differentiation of the pest, absence or decreased capacity for sexual reproduction of the pest, muscle formation, formation of juvenile hormone, juvenile hormone regulation, ion regulation and transport, cell membrane maintenance, amino acid biosynthesis, amino acid degradation, sperm formation, pheromone synthesis, pheromone sensing, antennae formation, wing formation, leg formation, development and differentiation, egg formation, larval maturation, digestive enzyme formation, hemolymph synthesis, hemolymph maintenance, neurotransmission, cell division, energy metabolism, respiration , apoptosis, or any other component of a eukaryotic cell cytoskeleton structure, such as, for example, actins and tubulins. Any or any combination of these attributes can result in effective inhibition of infestation by the pest and, in the case of a plant pest, inhibition of infestation of the plants. For example, when used as a diet composition containing a sufficient amount to inhibit a pest of one or more double-stranded ribonucleic acid molecules administered to the plant as a topical, as a treatment, as a soil application about the plant, or when produced by a plant from a recombinant DNA molecule present within the cells of a plant, plant pest infestation is unexpectedly and drastically reduced. The examples given herein are illustrative of the invention when applied to a single pest. However, the specialist will understand that the methods, The formulas and ideas presented in the examples are not intended to be limiting and are applicable to all coleopteran pest species that may consume food sources that can be formulated to contain a sufficient amount of a pest inhibitor agent consisting of at least one or more of the exemplary double-stranded RNA molecules described herein for suppressing some essential characteristic or function of the pest.

EXAMPLE 1

Identification of Target Nucleotide Sequences for the Preparation of Double-stranded RNA Useful for Controlling Corn Caterpillars.

Maize caterpillar cDNA libraries (LIB149, LIB 150, LIB3027, LIB3373) were constructed from whole larvae, pupae, and from dissected mid-GI tract sections, and nucleotide sequence information was obtained (see Andersen et al. al., US Patent Application Serial No. 10/205,189 filed July 24, 2002, which is specifically incorporated herein by reference in its entirety). In addition, cDNA libraries were constructed from whole larvae at different stages of development and at different times within each stage of development in order to maximize the number of different EST sequences from Diabrotlca species. Libraries LIB5444 and LIB5462 were constructed, respectively, from pools of mRNA obtained from the first (1 gram) and third (2.9 gram) instars of western corn rootworm larvae. Harvested insects were snap frozen by immersion in liquid nitrogen. Insects were ground in a mortar and then preserved -20 Ό or less by cooling on dry ice and/or by adding liquid nitrogen to the mortar until the tissue had been ground into a fine powder. RNA was extracted using TRIzol® reagent (Invitrogen) according to the supplier's instructions. Poly A+ RNA was isolated from total RNA preparations using DYNABEADS Oligo dT (Invitrogen) according to the supplier's instructions. A cDNA library was constructed from the Poly A+ RNA using the SuperScript™ Plasmid System (Invitrogen). The cDNA was size fractionated using chromatography. The fourth and fifth fractions were collected and ligated into the vector pSPORTI (Life Technologies Inc., Gaithersburg MD) between the Sa/1 and Nof\ restriction endonuclease recognition sites, and then transformed into DH10B electro-competent cells. of E. coli by electroporation. The library of first instar larvae yielded approximately 420,000 colony-forming units. The library of larvae in the third instar allowed to obtain approximately 2.78 x 106 colony forming units. LIB149, LIB150 colonies were washed from the plates, briefly mixed until homogeneous by vortexing and pooled in Tris-EDTA buffer. Half of the wash was taken to 10% glycerol, aliquoted into cñovials, and then stored at -70 °C. The other half was used to produce plasmid DNA using a Quiagen midi-prep purification column, or an equivalent thereof. Purified plasmid DNA was aliquoted into microcentrifuge tubes and then stored at -20°C.

Single colonies of the Diabrotica virgifera cDNA libraries LIB5444 and LIB5462 were amplified in high viscosity medium. Approximately 200,000 colony-forming units of LIB5444 and 600,000 colony-forming units of LIB5462 were mixed on a separate shake plate in 500 ml of LB medium containing 0.3% SeaPrep® agarose and 50 mg/l carbenecillin at 37°C and then were mixed. rapidly cooled in an ice/water bath for 1 hour to obtain a uniform suspension of bacterial colonies. The inoculated libraries were then grown at 30°C for 42 hours. After incubation, the cells were mixed for 5 minutes on a shake plate. The medium was then transferred to two 250 ml centrifuge bottles. Bacterial cells were pelleted at 10,000 xg for 10 minutes. The medium was removed from the bottles and the cells were resuspended in a total of 20 ml of LB medium with 50 mg/l carbenecillin. Dimethyl sulfoxide was added up to 10% to preserve cells during freezing. Both libraries were amplified to a final titer of 108 colony forming units per milliliter. Samples of the Diabrotica virgifera cDNA libraries LIB5444 and LIB5462 were pooled and adjusted to a DNA concentration of approximately 1.25 micrograms per microliter in sterile distilled, deionized water and aliquoted into twenty-five cryovials, each cryovial containing approximately 8.75 micrograms of DNA. These samples were deposited by Applicants/Inventors at the American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, Virginia, USA ZIP 20110-2209 on June 10, 2004, and were designated LIB5444/ 62. The ATCC provided a deposit receipt to the Applicant, assigning it the No.gATCC Warehouse Access, PTA-6072.

High molecular weight corn rootworm cDNA libraries, ie, LIB5496 and LIB5498, were prepared essentially as described above for the production of corn rootworm cDNA libraries. LIB5496 and LIB5498 libraries were constructed, respectively, from mRNA pools obtained from the first (1 gram) and second and third (1 gram) instars of western corn rootworm larvae. Briefly, the insects were snap frozen in liquid nitrogen. The frozen insects were reduced to a fine powder by grinding in a mortar. RNA was extracted using TRIzol® reagent (Invitrogen) according to the supplier's instructions. Poly A+ RNA was isolated from the total RNA preparation using DYNABEADS Oligo dT (Invitrogen). A high molecular weight cDNA library was obtained from 20 micrograms of poly A+ RNA using the SuperScript™ Plasmid System (Invitrogen). The cDNA was size fractionated on a 1% TAE agarose gel, and cDNA in the 1 Kb to 10 Kb range was collected, ligated into the pSPORT 1 vector between the Sa/1 and Nott restriction sites and then ligated. transformed into competent E. coli DH10B cells by electroporation. The LIB5496 library allowed to obtain a total title of approximately 3.5 x 106 colony forming units. The LIB5498 library allowed a total title of approximately 1.0 x 10 to be obtained.6 colony forming units. Colonies of the high molecular weight corn rootworm cDNA libraries LIB5496 and LIB5498 were individually amplified in high viscosity medium. Approximately 600,000 colony-forming units of LIB5496 and LIB5498 were mixed on a separate shake plate in 500 mL of LB medium containing 0.3% SeaPrep® agarose and 50 mg/l carbenecillin at 37°C and then rapidly cooled on a ice/water bath for 1 hour to obtain a uniform suspension of bacterial colonies. Libraries were then grown at 30°C for 42 hours. After incubation, the cells were mixed for 5 minutes on a shake plate. The medium was then transferred to two 250 ml centrifuge bottles. Bacterial cells were pelleted at 10,000 xg for 10 minutes. The medium was removed from the bottles and the cells were resuspended in a total of 20 mL of LB medium with 50 mg/L carbenecillin.

Dimethyl sulfoxide was added up to 10% to preserve cells during freezing. Both libraries were amplified to a final titer of 108 colony forming units per milliliter. Information on the inserted cDNA sequence was obtained from corn rootworm species-specific plasmid libraries.

The rootworm libraries of Andersen et al. together with additional sequences from libraries LIB5444 and LIB5462 initially produced approximately 18,415 individual EST sequences, consisting of approximately 1.0 x 107 nucleotide residues. The average length of an EST sequence was approximately 586 nucleotide residues. These EST sequences were subjected to bioinformatics algorithms which resulted in the assembly of contig sequences, herein referred to as UNIGENE sequences, and individual EST sequences that could not be compiled by overlapping identity with other EST sequences, herein referred to as singletons. The LIB5444 and LIB5462 libraries were then sequenced in greater detail, resulting in additional individual EST sequences. EST The EST sequences obtained from the libraries, ie, LIB149, LIB150, LIB3027, LIB3373, LIB5444, LIB5462, LIB5496 and LIB5503 were selected for further investigation in feeding bioassays as stated below and the corresponding sequences are given in the listing. of sequences..

EST sequences isolated from CRW cDNA libraries were assembled, where possible, into UNIGENE kits and these assembled Unigene sequences are included in the sequence listing. A UNIGENE is a gene-oriented cluster formed from the overlap of individual EST sequences within regions of sequence identity to form a larger sequence. Pontius et al. (2003). Each nucleotide sequence shown in the sequence listing was analyzed for the presence of open reading frames. Amino acid sequence information deduced from the open reading frames was compared with known amino acid sequence information available in public databases in order to infer the extent of amino acid sequence identity or similarity with known amino acid sequences. The biological function, if any, associated with the amino acid sequences was annotated in public databases for the amino acid sequences deduced from the nucleotide sequence information of the cDNA libraries. The annotations provided information that suggested the function of the protein that could be expressed from a particular gene that gave rise to a particular cDNA sequence, but was not determinative of outcome. Based on this information suggested in the annotations, certain cDNA sequences were characterized as encoding a protein that was most likely involved in some biological function within corn rootworm cells that was either essential for the life or was necessary to ensure the health and vitality of a cell or would be compromised in cellular integrity, cell maintenance, reproductive capacity and the like.

The sequences that were chosen for further investigation were used for the construction of double-stranded RNA molecules for incorporation into the CRW diet. Primer pairs for thermal amplification were designed based on the starting cDNA and EST sequences to obtain the sequences used in the feeding assays. Primer pairs were constructed as either a pair of nucleotide sequences, where each member of a primer pair exhibited perfect complementarity to a sequence oriented in-frame or antisense. Some primer pair sequences were constructed such that each member of the pair exhibited a sequence with a promoter for phage T7 RNA polymerase at its 5' end. Preferably a first high fidelity amplification reaction was carried out using a first pair of T7 promoterless primers to generate a first amplicon using CRW genomic DNA as a template. A cDNA or mRNA sequence is preferably used as a template for the synthesis of a dsRNA molecule to be used in the present invention because it is recognized in the art that eukaryotic genomic sequences contain sequences that are not present in the dsRNA molecule. mature RNA. A sample of the first amplicon generated in said first higher fidelity amplification reaction was then used as a template in a second thermal amplification reaction with a second pair of primers containing the T7 promoter sequence to produce a second amplicon containing said T7 promoter near or included in the 5' end of each strand of the second amplicon. The complete nucleotide sequence of the second amplicon in both directions was obtained and compared to the nucleotide sequence reported for the cDNA, and discrepancies between the two sequences, if any, were recorded. In general, the sequences that were prepared using genomic DNA as a template were inconsistent with their further use as dsRNA molecules to obtain significant levels of deletion due to variations within the genomic sequences that were not observed in the mRNA or cDNA sequences.

An in vitro transcription reaction typically contained between about 1 and about 2 micrograms of linearized DNA template, 10X concentrated T7 polymerase reaction buffer, the ribonucleotides ATP, CTP, GTP, and UTP at a final concentration between 50 and 100 mM each and 1 unit of T7 RNA polymerase enzyme. The RNA polymerase reaction was incubated at approximately 37°C, depending on the optimum temperature of the RNA polymerase used according to the supplier's instructions, for a period of time ranging from several minutes to several hours. In general, reactions took between about 2 and about 6 hours for transcription of template sequences up to about 400 nucleotides in length, and up to 20 hours for transcription of template sequences greater than about 400 nucleotides in length. Heating the reaction at 65 Ό for fifteen minutes terminates RNA transcription. The RNA transcripts were ethanol precipitated, washed, air dried, and resuspended in RNase-free water to a concentration of approximately 1 microgram per microliter. Most of the transcripts taking advantage of the T7 promoter opposition strategy described above produced dsRNA in the in vitro transcription reaction, however, a higher yield of dsRNA was obtained by heating the purified RNA to 65°C and subsequent slow cooling to room temperature to ensure proper alignment of the sense and antisense oriented RNA segments. The double-stranded RNA products were then incubated with DNase I and RNase at 37°C for one hour to remove any DNA or single-stranded RNA present in the mixture. Double-stranded RNA products were purified on a column according to the supplier's instructions (AMBION MEGAscrlpt® RNAi Element Set) and resuspended in 10 mM Tris-HCI buffer (pH 7.5) or RNase-free water to a concentration between 0.1 and 1.0 micrograms per microliter.

The following nucleotide sequences were first derived as cDNA sequences identified in a corn rootworm gut cDNA library (Andersen et al., ibid) and then adapted for use in the construction of RNA molecules. double-stranded for the evaluation of the efficacy of inhibition of biological function in a pest by feeding double-stranded RNA molecules in the diet of said pest.

A. Chd3 Homologous Sequences

CHD genes have been identified in numerous eukaryotes and the corresponding proteins have been proposed to function as chromatin remodeling factors. The term CHD derives from the three domains of sequence homology found in CHD proteins: a chromo (chromatin organization modifier) domain, a SNF2-related helicase/ATPase domain, and a DNA-binding domain, where each is believed to confer a distinct chromatin-related activity. CHD proteins are separated into two categories based on the presence or absence of another domain with sequence homology, a PHD zinc finger domain, typically associated with chromatin-related activity. CHD3-related proteins possess a PHD zinc finger domain, but CHD1-related proteins do not. Experimental observations have suggested a role for CHD3 proteins in the repression of transcription, and it has been shown that in some species they are a component of a complex containing a histone deacetylase as a subunit. Histone deacetylation correlates with inactivation of transcription, and it is for this reason that CHD3 proteins have been implicated in a role as transcriptional repressors by virtue of being components of a histone deacetylase complex (Ogas et al., 1999). Therefore, suppression of CHD3 protein synthesis may be a useful target for double-stranded RNA-mediated inhibition of coleopteran pests.

B. · Homologous sequences of beta-tubulin

Tubulin proteins are important structural components of many cellular structures in all eukaryotic cells and mainly in the formation of microtubules. Inhibition of microtubule formation in cells results in catastrophic effects including interference with mitotic spindle formation, blockade of cell division, and the like. Thus, suppression of tubulin protein formation may be a useful target for double-stranded RNA-mediated inhibition.

C. 40 kDa V-ATPase homologous sequences

Energy metabolism within subcellular organelles in eukaryotic systems is an essential function. Vacuolar ATP synthetases are involved in maintaining sufficient levels of ATP within the vacuoles. Thus, vacuolar ATP synthetases may be useful targets for double-stranded RNA-mediated inhibition.

D. Sequence Homologous to EF1a

Transcription elongation and transcription termination factors are essential for metabolism and may be advantageous targets for double-stranded RNA-mediated inhibition.

E. Homologous sequences of p28 of the 26S proteasome subunit

The 26S proteasome is a large, ATP-dependent, multi-subunit protease that is highly conserved in all eukaryotes. It has a general role in the selective removal of several short-lived proteins that first bind covalently to ubiquitin and are then degraded by the 26S proteasome complex. The ubiquitin pathway plays an important role in the control of the cell cycle through the specific degradation of a number of regulatory proteins including mitotic cyclins and inhibitors of cyclin-dependent kinases such as p27 in mammalian cells. Therefore, suppression of the synthesis of the 26S proteasome and suppression of the synthesis of its component subunits may constitute preferred targets for double-stranded RNA-mediated inhibition. (Smith et al., 1997).

F. · Homologous sequences of the epoxide hydrolase of the juvenile hormone

Insect juvenile hormone controls and regulates a variety of biological processes necessary within the insect life cycle including, but not necessarily limited to, metamorphosis, reproduction, and diapause. Peak concentrations of juvenile hormone (JH) are required, at the appropriate time, in the hemolymph of the larval form of an insect pest, particularly Lepidopteran and Coleopteran larvae, and must then be broken down in order to terminate the effects of the hormonal response. Enzymes involved in lowering juvenile hormone concentration are effective through two primary pathways of metabolic degradation. One pathway involves a juvenile hormone esterase (JHE), which hydrolyzes the methyl ester to give the corresponding acid. The second pathway uses juvenile hormone epoxide hydrolase (JHEH) to achieve hydrolysis of the epoxide, resulting in the formation of the diol. The contribution of JHE in JH degradation is well elucidated and has been found to not vary between Lepidopteran and Coleopteran species. Inhibition of JH esterase has been associated with severe morphological changes including, but not limited to, larval wandering, delayed pupation, and development of intermediate malformations. In contrast, the contribution of JHEH to JH metabolism is less understood and has been shown to vary between species, although recent studies point to suggest that JHEH may be the primary route of JH metabolism (Brandon J. Fetterolf, PhD Thesis, North Carolina State University (February 10, 2002) Synthesis and Analysis of Mechanism Based Inhibitors of Juvenile Hormone Epoxide Hydrolase from Insect Trichoplusia ni). In either case, disruption of the degradation of any of the JH pathways using gene knockout technology could constitute an effective target for double-stranded RNA-mediated pest inhibition.

G. · Swelling-dependent chloride channel protein homologous sequences

Swelling chloride-gated channel proteins have been proposed to play a critical role in osmotic regulation in eukaryotic animal cell systems. Therefore, a nucleotide sequence capable of expressing an amino acid sequence that was homologous to previously identified swelling-dependent chloride channel proteins may be useful as a target for RNA inhibition in a pest.

H · Homologous sequences of the protein glucose-6-phosphate 1-dehydro genase

The protein glucose-6-phosphate 1-dehydrogenase (G6PD) catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate by concomitantly reducing the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) to NADPH. NADPH is known in the art as a necessary cofactor in many biosynthesis reactions in eukaryotes and is known to maintain glutathione in its reduced form. Reduced glutathione acts as a sequestering agent for deleterious oxidative metabolites in eukaryotic cells, and with the assistance of the enzyme glutathione peroxidase, converts deleterious hydrogen peroxide to water (Beutler et al., 1991). Thus, G6PD may constitute a preferred target for double-stranded RNA-mediated inhibition of a coleopteran pest.

I. Sequences Homologous to the Act42A Protein

Actin is a ubiquitous and highly conserved eukaryotic protein that is required for cell mobility and locomotion (Lovato et al., 2001). Numerous CRW cDNA sequences were identified that are likely to encode actin or proteins that have an amino acid sequence structure related to actin proteins. Therefore, genes encoding actin homologues in pest cells may be useful targets for double-stranded RNA-mediated inhibition.

J Sequences homologous to ADP ribosylation factor 1

Ribosylation factors have been shown to be essential in cell function because they play integral roles in the DNA damage repair process, in carcinogenesis, in cell death, and in genomic stability. Thus, it would be useful to possess the ability to selectively disrupt the transcription of ADP-ribosylation factors in pest Coleopteran species, using double-stranded RNA-mediated inhibition.

K Sequences homologous to the Transcription Factor IIB protein

Transcription elongation and transcription termination factors are, as noted above, essential for metabolism and may be advantageous targets in double-stranded RNA-mediated inhibition to control or eliminate infestation by Coleopteran pests.

L Chitinase homologous sequences

Chitin is a β(1->4) homopolymer of N-acetylglucosamine and is found in the exoskeletons of insects. Chitin is formed from UDP-N-acetylglucosamine in a reaction catalyzed by chitin synthetase. Chitin is a structural homopolymeric polysaccharide, and there are many enzymatic steps involved in the construction of this highly branched, cross-linked structure. Chitin gives insects shape, rigidity, and support and provides them with the scaffolding to which internal organs, such as muscles, are attached. Chitin must in turn be degraded to some extent to mediate the steps involved in the insect molting process. Therefore, it is believed that double-stranded RNA-mediated inhibition of the proteins of these pathways would be useful as a means of controlling infestation of Coleopteran pests.

M Sequences homologous to an enzyme that conjugates ubiquitin

The ubiquitin pathway plays an important role in the control of the cell cycle through the specific degradation of a number of regulatory proteins including mitotic cyclins and inhibitors of cyclin-dependent kinases such as p27 in mammalian cells. Therefore, genes encoding ubiquitin and associated components may be preferred targets for double-stranded RNA-mediated inhibition. (Smith et al., 1997). The ubiquitin-dependent proteolytic pathway is one of the main routes by which intracellular proteins are selectively destroyed in eukaryotes. The conjugation of ubiquitin to substrate proteins is mediated by a markedly diverse collection of enzymes. The process of proteolytic targeting of a protein may also be regulated in the steps that occur between ubiquitination of the substrate and its degradation to peptides by the 26S multisubunit protease. The complexity of the ubiquitin system suggests a central role for protein turnover in the regulation of eukaryotic cells, and implicates other proteins in the pathway, including ubiquitin-activating enzyme, ubiquitin-conjugating enzyme , to ubiquitin protein ligase, and to component subunits of the 26S proteasome. Therefore, it is believed that double-stranded RNA-mediated inhibition of the proteins of this pathway would be useful as a means of controlling infestation of Coleopteran pests.

N Sequences homologous to glyceraldehyde-3-phosphate dehydrogenase

The glycolytic pathway is an essential pathway in most organisms and is involved in the production of metabolic energy from the breakdown of glucose. An important enzyme in the second step of the glycolytic pathway is glyceraldehyde-3-phosphate dehydrogenase (G3PDH), which, in the presence of NAD+ and inorganic phosphate, catalyzes the oxidation of glyceraldehyde 3-phosphate to 1.3bisphosphoglycerate along with the formation of NADH. The important event of this reaction is the storage of energy through the formation of NADH. Genes encoding enzymes associated with the glycolytic pathway, and particularly genes encoding enzymes involved in useful steps for the formation of energy reserves, may be particularly advantageous targets for double-stranded RNA-mediated inhibition in pests consisting of Coleopteran species. .

O Sequences Homologous to Ubiquitin B

As previously described, the ubiquitin pathway for protein degradation plays an important role in cell cycle control through the specific degradation of a number of regulatory proteins including mitotic cyclins and inhibitors of cyclin-dependent kinases. such as p27 from mammalian cells. Therefore, genes encoding ubiquitin and associated components may be preferred targets for double-stranded RNA-mediated inhibition. (Smith et al., 1997).

P Homologs of Juvenile Hormone Esterase

As indicated above, insect juvenile hormone (JH) controls and regulates a variety of biological processes necessary within the insect life cycle including but not necessarily limited to metamorphosis, reproduction, and diapause. Interruption of HJ synthesis or degradation pathways, using gene suppression technology, could be an effective target for double-stranded RNA-mediated pest inhibition.

Q · Alpha-tubulin homologous sequences

Eukaryotic cells generally use the structural elements of the cytoskeleton that are important, not only as a mechanical scaffold, but also as sustaining the shape of the cell. Microfilaments, which are semi-flexible, give the cell mobility, help it divide during mitosis (cytokinesis) and, in vertebrate and invertebrate animals, are responsible for muscle contraction. Microtubules, which are relatively rigid and are made of alpha and beta tubulin proteins, play an important role in acting as a kind of highway for the transport of vesicles and organelles, and in the separation of chromosomes during mitosis (karyokinesis). Intermediate filaments, which are flexible, provide at least one additional strength to the overall cell structure. The cytoskeleton is also known to be involved in signaling through the cell cytoplasm. Taking these functions into account, it is believed that any disruption of the cytoskeleton or even slight changes in its integrity can cause pathological consequences to a cell.

R Transport Related Sequences

As indicated above, the sorting and transport of different molecules within a cell, including to the appropriate organelles, as well as their secretion is an important physiological function. Such sorting pathways may include those that fall on the endosomal sorting complex required for transport (ESCRT), on the l-lll complexes, among others. Therefore, transport-related functions of polypeptides and other molecules may also be a preferred target for double-stranded RNA-mediated inhibition.

Example 2

Insect feeding bioassays

Samples of siRNA or double-stranded RNA (dsRNA) were subjected to bioassays with a selected number of target pests. dsRNA was prepared from the identified sequences according to Example 1, using a complete contig sequence in the case of SEQ ID N91-6, or a sequence amplified from the constructed contig using the primer pairs indicated in the sequence listing. Varying doses of dsRNA were applied to the artificial corn rootworm diet according to the following procedure. Diabrotica virgifera virgifera (WCR) eggs were obtained from Crop Characteristics, Inc., Farmington, Minnesota. Non-diapausing WCR eggs were incubated on soil for approximately 13 days to 249C, with 60% relative humidity, in total darkness. On day 13, the soil containing the WCR eggs was placed between N mesh sieves.yes 30 and Nyes 60, and the eggs were washed from the soil using a high-pressure garden hose. Eggs were surface disinfected by soaking in LYSOL for three minutes, then washed three times with sterile water, washed once with 10% formalin solution, and then washed three more times with sterile water. Eggs thus treated were placed on sterile coffee filters and hatched overnight at 279C, with 60% relative humidity, in total darkness.

To prepare dsRNAs, amplicons of selected sequences were cloned into a plasmid vector capable of replication in E. coti, and sufficient amounts of plasmid DNA were recovered to allow in vitro transcription with T7 RNA polymerase, from inserted convergent T7 promoters. at the ends of the cloned fragment. Double-stranded RNA was produced and bioassayed; where one RNA segment comprised the sequence detailed in the sequence listing, and the other DNA segment was substantially the reverse complement of the nucleotide sequence, with uridine instead of thymidine. A sample of double-stranded RNA (dsRNA) was treated with DICER or RNase III to produce sufficient amounts of small interfering RNA (siRNA). Samples containing siRNA or dsRNA were applied to the CRW diets in the bioassay described above, and the larvae were allowed to feed as described below.

A sample of dsRNA was added directly to each well containing the insect diet listed above, or modified prior to addition to the diet. Double-stranded RNA modification was performed according to the instructions for RNase III (AMBION CORPORATION, Austin, Texas) or DICER (STRATAGENE, La Jolla, California) provided by the manufacturer. Digestion of double-stranded RNA with RNase III produced twenty-one and twenty-two nucleotide pairs, containing 5' phosphorylated ends and 3' hydroxylated ends with 2-3 base overlaps, similar to the approximately 21-26 base pairs of the pairs. of small interfering RNA fragments (siRNA) produced with the enzyme dicer in the eucañotes pathway identified by Hamilton ei. to the. (1999) and Elbashir eí. to the. (2001a). This set of small interfering RNA duplexes was subjected to further purification, and the sample was characterized by polyacrylamide gel electrophoresis, to determine the integrity and efficiency of duplex formation. Afterwards, the purity and quantity of the samples were determined by spectrometry at a wavelength of 250 nanometers, and unused samples were retained for further use by storing them at -202c.

An insect diet was prepared essentially according to Pleau et al. (2002), with the following modifications. 9.4 grams of SERVA agar was placed in 540 milliliters of purified water, and agitated until exhaustive distribution of the agar was achieved. The water/agar mixture was heated to the boil to dissolve the agar completely, and then poured into a WARING blender. The mixer was kept at low speed while adding 62.7 grams of BIO-SERV DIET mix (F9757), 3.75 grams of freeze-dried corn root, 1.25 milliliters of green food coloring, and 0.6 milliliters of formalin. to the hot agar mixture. The pH of the mixture was then adjusted to 9.0 with the addition of 10% potassium hydroxide stock solution. The volume of liquid diet, approximately 600 milliliters, was mixed continuously at high speed, and maintained at a temperature between approximately 48QC and about 609C using a NALGENE-coated magnetic stir bar, sterilized on a hot magnetic stir plate, while 200 microliter aliquots were placed in each well of FALCON 96-well round bottom microtiter plates. The diet on the plates was allowed to solidify and air dried under a sterile laminar flow hood for approximately ten minutes.

Thirty (30) microliter volumes of test samples, containing control reagents or double-stranded RNA, in varying amounts, were used to coat the surface of the insect diet in each well, using an automated micropipetter. The insect diet was allowed to stand under a sterile laminar flow hood for up to half an hour after applying the test sample, to allow the reagents to diffuse through the diet, and to dry its surface. A neonate WCR larva was deposited in each cavity with a fine brush. The plates were then sealed with MYLAR, and vented using an insect stick. 12-72 insect larvae per dose were evaluated, depending on the trial design. The bioassay plates were incubated at 279C, with 60% relative humidity, in total darkness, for 12-14 days. The number of larvae that survived per dose at 12-14 days was recorded. Larval mass was determined using a suitable microbalance for each surviving larva. Data were analyzed using JMP®4 statistical software (SAS Institute, 1995), and a full factorial ANOVA with Dunnet's test was performed to find the effects of treatment, compared to untreated control (P<0.05 ). A post hoc Tukey-Kramer test was also performed to compare all treatment pairs (P<0.05). Results of CRW larval feeding trials indicated significant growth inhibition and mortality, compared to controls, as explained below.

Example 3

Insect Feeding Bioassay Results

Sufficient artificial diet to rear corn rootworm was prepared by applying samples of double-stranded RNA sequences, identified as described in Example 1, with bioassays carried out as described in Example 2. Corn rootworm larvae were typically allowed to feed for twelve days and monitored for mortality and dieback, compared to rootworms fed only negative and positive control diets. The results of the studies confirmed significant (p<0.05) levels of larval fading and/or mortality when dsRNAs containing portions of sequences homologous to a variety of different gene classes were used. Sequences and vectors that provide significant fading and/or mortality, and SEQ ID N9 Corresponding sequences for the sequences, expressed as dsRNA, are provided in Tables 1-5 below.

Table 1: dsRNA Constructs Giving a Significant Fading and/or Mortality Effect in Insect Feeding Bioassays with Southern Corn Rootworms or Western Corn Rootworms

<td>Vector</td><td>Sequence expressed as dsRNA</td><td>SEQ ID No.and</td>

<td>RNAi-plC17553:001</td><td>Apple</td><td> 697</td>

<td>RNAi-pICl 7504:049</td><td>Complete V-ATPase EST</td><td> 695</td>

<td>RNAi-pICl 7504:050</td><td>V-ATPase Section 6.0</td><td> 711</td>

<td>RNA¡-plC19514:001</td><td>WCR enzyme LIB5496 mRNA- 028-A1-M1-A7</td><td> 696</td>

<td>RNAi-plC17552:003</td><td>Ll B5462-042-A1 -M1 -H10 Dv.6_CG9355 DUSKY CONSTITUENT CUTICLE STRUCTURAL</td><td> 700</td>

<td>RNAi-pICl 7546:003</td><td>LIB5462-091-A1-M1-G3 Dv.1_CG6217 KNICKKOPF UNK</td><td> 701</td>

<td>RNAi-pMON78412:002</td><td>Dv.7 CG3416; probable ortholog of Mov34:CG3416</td><td> 10</td>

<td>RNAi-pMON96172:002</td><td>Dv.8_CG1088; probable vacuolar H+ATPase E subunit: ortholog of CG1088</td><td> 14</td>

<td>RNAi-pMON96168:002</td><td>Dv.9_CG2331; probable activity of ATPase: CG2331 ortholog</td><td> 18</td>

<td>RNAi-pMON78424:002</td><td>Dv.10_CG6141; probable protein ribosomal L9: ortholog of CG6141</td><td> 22</td>

<td>RNAi-pMON78444:002</td><td>Dv.12_CG1341; probable proteasome regulatory particle, rpt1: ortholog of CG1341</td><td> 30</td>

<td>RNAi-pMON78416:002</td><td>Dv.13_CG11276; probable S4 ribosomal protein: ortholog of CG11276</td><td> 34</td>

<td>RNAi-pMON78434:001</td><td>Dv.14_CG17927_2; probable myosin heavy chain: ortholog of CG17927</td><td> 38</td>

<td>RNAi-pMON78439:001</td><td>Dv.16 CG5394; likely glutamyl-prolyl- tRNA synthetase: CG5394 ortholog</td><td> 46</td>

<td>RNAi-pMON78438:001</td><td>Dv.17_CG10149; probable p44.5 proteasome subunit, rpn6: from ortholog CG10149</td><td> 50</td>

<td>RNAi-pMON78435:002</td><td>Dv.18_CG1404; probable monomeric GTPase small RNA: ortholog of CG1404</td><td> 54</td>

<td>RNAi-pMON78449:002</td><td>Dv.19_CG18174; probable proteasome regulatory particle, lid subcomplex, rpn 11: CG18174 ortholog</td><td> 58</td>

<td>RNAi-pMON78419:001</td><td>Dv.20_CG3180_1; probable RNA RNA-directed polymerase II: CG3180 ortholog</td><td> 62</td>

<td>RNAi-pMON78440:002</td><td>Dv20_CG3180_2; probable RNA RNA-directed polymerase II: CG3180 ortholog</td><td> 706</td>

<td>RNAi-pMON78420:001</td><td>Dv.21_CG3320; probable Rabbi: orthologue by CG3320</td><td> 70</td>

<td>RNAi-pMON78410:002</td><td>Dv.22_CG3395; probable protein ribosomal S9: ortholog of CG3395</td><td> 74</td>

<td>RNAi-pMON78422:001</td><td>Dv.23_CG7269; probable helicase: ortholog of CG7269</td><td> 78</td>

<td>RNAi-pMON78423:001</td><td>Dv.25_CG9012; probable clathrin heavy chain: CG9012 ortholog</td><td> 86</td>

<td>RNAi-pMON78414:001</td><td>Dv.26_CG9261; probable ATPase sodium/potassium exchanger: ortholog of CG9261</td><td> 90</td>

<td>RNAi-pMON78413:001</td><td>Dv.27_CG 12052; probable RNA polymerase II transcription factor: CG12052 ortholog</td><td> 94</td>

<td>RNAi-pMON78427:001</td><td>Dv.35_CG3762; probable Vha68-2: ortholog of CG3762</td><td> 126</td>

<td>RNAi-pMON97122:001</td><td>C1_Dv.35; probable Vha68-2: ortholog of CG3762; concatamer</td><td> 713</td>

<td>RNAi-pMON97127:001</td><td>C2_Dv.35; probable Vha68-2: ortholog of CG3762; concatamer</td><td> 714</td>

<td>RNAi-pMON97125:001</td><td>C3_Dv.35; probable Vha68-2: ortholog of CG3762; concatamer</td><td> 715</td>

<td>RNAi-pMON78441:001</td><td>Dv.39_CG9078; probable sphingolipid delta-4 desaturase; stearoyl-CoA 9desaturase: ortholog of CG9078</td><td> 142</td>

<td>RNAi-pMON97114:001</td><td>Dv.41 CG2637; Ketel of female sterility; participates in the import of proteins into the nucleus: ortholog of CG2637</td><td> 150</td>

<td>RNAi-pMON97140:001</td><td>Dv.44_CG1244; Probable nucleic acid binding activity: CG1244 ortholog</td><td> 162</td>

<td>RNAi-pMON78429:001</td><td>Dv.46_CG 10689; probable RNA helicase: CG 10689 ortholog</td><td> 170</td>

<td>RNAi-pMON78432:001</td><td>Dv.48_CG33196; probable transmembrane receptor protein, tyrosine kinase: ortholog of CG33196</td><td> 178</td>

<td>RNAi-pMON78428:001</td><td>Dv.49_CG8055_1; probable binding, vehicle activity: CG8055 ortholog 1</td><td> 182</td>

<td>RNAi-pMON78428:003</td><td>Dv.49_CG8055_1; probable binding, vehicle activity: CG8055_1 ortholog, free region selected from DV.49</td><td> 704</td>

<td>RNAi-pMON78426:001</td><td>Dv.50_CG10110_1; probable cleavage and polyadenylation specificity factor: CG10110 ortholog 1</td><td> 186</td>

<td>RNAi-pMON96185:001</td><td>Dv.55_CG5931; probable cutting factor activity, RNA helicase activity: CG5931 ortholog</td><td> 202</td>

<td>RNAi-pMON78442:001</td><td>Dv.57_CG2968; probable hydrogen-exporting ATPase: ortholog of CG2968</td><td> 206</td>

<td>RNAi-pMON78431:001</td><td>Dv.58 CG1751; probable peptidase of signal: CG1751 ortholog</td><td> 210</td>

<td>RNAi-pMON96177:001</td><td>Dv.61_CG3725_1; probable calcium ATPase: CG3725 ortholog</td><td> 222</td>

<td>RNAi-pMON96183:001</td><td>Dv.62_CG3612; probable bellwether: CG3612 ortholog</td><td> 230</td>

<td>RNAi-pMON96180:002</td><td>Dv.65_CG7033; probable chaperone activity: CG7033 ortholog</td><td> 242</td>

<td>RNAi-pMON96176:001</td><td>Dv.66_CG32019; likely bent: ortholog by CG32019</td><td> 246</td>

<td>RNAi-pMON96170:002</td><td>Dv.67_CG16916; probable endopeptidase activity: CG16916 ortholog</td><td> 250</td>

<td>RNAi-pMON96166:001</td><td>Dv.70_CG5771; probable Rab protein 11: ortholog of CG5771</td><td> 258</td>

<td>RNAi-pMON96179:001</td><td>Dv.72_CG6831; probable rhea: ortholog by CG6831</td><td> 266</td>

<td>RNAi-pMON96186:001</td><td>Dv.73 CG10119; probable Lamin C: ortholog of CG10119</td><td> 270</td>

<td>RNAi-pMON96160:002</td><td>Dv.74_CG6375; probable pitchoune: ortholog of CG6375</td><td> 274</td>

<td>RNAi-pMON97137:001</td><td>Dv.77_CG4214; probable Syntaxin 5; Involved in intracellular protein transport: CG4214 ortholog</td><td> 286</td>

<td>RNAi-pMON96167:001</td><td>Dv.82_CG8264; probable Bx42: ortholog by CG8264</td><td> 302</td>

<td>RNAi-pMON96171:001</td><td>Dv.83_CG11397; likely gluon: ortholog by CG11397</td><td> 306</td>

<td>RNAi-pMON96187:003</td><td>Dv.85_CG4494; probable protein-binding activity: CG4494 ortholog</td><td> 314</td>

<td>RNAi-pMON96174:001</td><td>Dv.86_CG5055; probable bazooka: CG5055 ortholog</td><td> 318</td>

<td>RNAi-pMON97126:001</td><td>Dv.88_CG8756; function unknown; contains chitin-binding domain: ortholog of CG8756</td><td> 326</td>

<td>RNAi-pMON97130:001</td><td>Dv.93_CG8515; probable structural constituent of cuticle; contains two chitin-binding domain: ortholog of CG8515</td><td> 342</td>

<td>RNAi-pMON97109:001</td><td>Dv.99 CG2446; a stranger; lethal in Drosophila and low homology with humans: CG2446 ortholog</td><td> 366</td>

<td>RNAi-pMON97111:001</td><td>Dv.105_CG1250_1; GTPase activator, involved in intracellular protein transport: CG1250 ortholog</td><td> 390</td>

<td>RNAi-pMON97112:001</td><td>Dv.105_CG1250_2; GTPase activator, involved in intracellular protein transport: CG1250 ortholog</td><td> 394</td>

<td>RNAi-pMON97107:001</td><td>Dv.107_CG14813; COPI vesicle coating; participates in the transport of proteins from the Golgi to the ER: ortholog of CG14813</td><td> 398</td>

<td>RNAi-pMON97115:001</td><td>Dv.108_CG17248; n-synaptobrevin; involved in intracellular protein transport: ortholog of CG17248</td><td> 402</td>

<td>RNAi-pMON97133:001</td><td>Dv.113; function unknown; unique sequence of WCR</td><td> 422</td>

<td>RNAi-pMON97121:001</td><td>Dv.122_CG3164; probable ATP-binding transporter cassette activity: CG3164 ortholog</td><td> 454</td>

<td>RNAi-pMON97134:001</td><td>Dv.127; function unknown; no homology with humans</td><td> 470</td>

<td>RNAi-pMON97171:001</td><td>Dv.146; unknown function, WCR single sequence</td><td> 514</td>

<td>RNAi-pMON97166:001</td><td>Dv.147; unknown function, WCR single sequence</td><td> 518</td>

<td>RNAi-pMON97167:001</td><td>Dv.149; unknown function, WCR single sequence</td><td> 526</td>

<td>RNAi-pMON97169:001</td><td>Dv.155; unknown function, WCR single sequence</td><td> 550</td>

<td>RNAi-pMON97173:001</td><td>Dv.162; unknown function, WCR single sequence</td><td> 578</td>

<td>RNAi-pMON97170:001</td><td>Dv.170; unknown function, sequence</td><td> 610</td>

Table 2: dsRNA Constructs Causing Significant Fading Levels in Western Corn Rootworm (WCR) Larval Feeding Bioassays

<td>Vector</td><td>Sequence expressed as dsRNA</td>

<td>RNAi-plC17553:001</td><td>Apple</td>

<td>RNA¡-plC17504:049</td><td>Complete V-ATPase EST</td>

<td>RNAi-plC17504:050</td><td>V-ATPase Section 6.0</td>

<td>RNAi-plC16005:001</td><td>V-ATPase D subunit 1</td>

<td>RNAi-pICl 9514:001</td><td>WCR enzyme LIB5496-028-A1 -M1-A7 mRNA</td>

<td>RNAi-pMON78412:002</td><td>Dv.7 CG3416; probable Mov34: ortholog of CG3416</td>

<td>RNAi-pMON96172:002</td><td>Dv.8_CG1088; probable vacuolar H+ATPase E subunit: ortholog of CG1088</td>

<td>RNAi-pMON96168:002</td><td>Dv.9_CG2331; probable ATPase activity: ortholog of CG2331</td>

<td>RNAi-pMON78424:002</td><td>Dv.10_CG6141; probable L9 ribosomal protein: ortholog of CG6141</td>

<td>RNAi-pMON78444:002</td><td>Dv.12_CG1341; probable proteasome regulatory particle, rpt1: ortholog of CG1341</td>

<td>RNAi-pMON78416:002</td><td>Dv.13_CG11276; probable S4 ribosomal protein: ortholog by CG11276</td>

<td>RNAi-pMON78434:001</td><td>Dv.14_CG17927_2; probable myosin heavy chain: ortholog of CG17927</td>

<td>RNAi-pMON78439:001</td><td>Dv.16_CG5394; probable glutamyl-prolyl-tRNA synthetase: ortholog of CG5394</td>

<td>RNAi-pMON78435:002</td><td>Dv.18_CG1404; probable monomeric GTPase small RNA: ortholog of CG1404</td>

<td>RNAi-pMON78449:002</td><td>Dv.19_CG18174; probable regulatory particle of proteasome, lid subcomplex, rpn 11: CG18174 ortholog</td>

<td>RNAi-pMON78440:002</td><td>Dv20_CG3180_2; probable ANA polymerase II directed to RNA: CG3180 ortholog</td>

<td>RNAi-pMON78420:002</td><td>Dv.21 CG3320; likely Rabbi: ortholog of CG3320</td>

<td>RNAi-pMON78410:001</td><td>Dv.22_CG3395; probable ribosomal protein S9: ortholog of CG3395</td>

<td>RNAi-pMON78422:001</td><td>Dv.23 CG7269; probable helicase: CG7269 ortholog</td>

<td>RNAi-pMON78423:001</td><td>Dv.25_CG9012; probable clathrin heavy chain: CG9012 ortholog</td>

<td>RNAi-pMON78414:001</td><td>Dv.26_CG9261; probable sodium/potassium exchanger ATPase: ortholog of CG9261</td>

<td>RNAi-pMON78413:001</td><td>Dv.27_CG 12052; probable RNA polymerase II transcription factor: CG12052 ortholog</td>

<td>RNAi-pMON97122:001</td><td>C1_Dv.35; probable Vha68-2: ortholog of CG3762; concatamer</td>

<td>RNAi-pMON97127:001</td><td>C2_Dv.35; probable Vha68-2: ortholog of CG3762; concatamer</td>

<td>RNAi-pMON97125:001</td><td>C3_Dv.35; probable Vha68-2: ortholog of CG3762; concatamer</td>

<td>RNAi-pMON78427:007</td><td>Dv.35 CG3762; probable Vha68-2: ortholog of CG3762</td>

<td>RNAi-pMON78441:001</td><td>Dv.39 CG9078; probable sphingolipid delta-4 desaturase; stearoyl-CoA 9-desaturase: ortholog of CG9078</td>

<td>RNAi-pMON97114:001</td><td>Dv.41_CG2637; Ketel of female sterility; participates in the import of proteins into the nucleus: ortholog of CG2637</td>

<td>RNAi-pMON78429:001</td><td>Dv.46_CG 10689; probable RNA helicase: ortholog of CG10689</td>

<td>RNAi-pMON78432:001</td><td>Dv.48_CG33196; probable receptor protein transmembrane, tyrosine kinase: CG33196 ortholog</td>

<td>RNAi-pMON78428:001</td><td>Dv.49_CG8055_1; probable binding, vehicle activity: CG8055 ortholog 1</td>

<td>RNAi-pMON78428:003</td><td>Dv.49_CG8055_1; probable binding, vehicle activity: CG8055 ortholog 1, free region selected from DV.49</td>

<td>RNAi-pMON78426:001</td><td>Dv.50_CG10110_1; probable cut-off specificity factor and polyadenylation: ortholog of CG10110 1</td>

<td>RNAi-pMON96185:001</td><td>Dv.55_CG5931; probable cutting factor activity, RNA helicase activity: CG5931 ortholog</td>

<td>RNAi-pMON78442:001</td><td>Dv.57_CG2968; probable hydrogen-exporting ATPase: ortholog of CG2968</td>

<td>RNAi-pMON78431:001</td><td>Dv.58_CG1751; probable signal peptidase: ortholog of CG1751</td>

<td>RNAi-pMON96177:001</td><td>Dv.61 CG3725 1; probable calcium ATPase: ortholog of CG3725</td>

<td>RNAi-pMON96182:001</td><td>Dv.61_CG3725_2; probable calcium ATPase: ortholog of CG3725</td>

<td>RNAi-pMON96183:001</td><td>Dv.62 CG3612; probable bellweter: ortholog of CG3612</td>

<td>RNAi-pMON96180:002</td><td>Dv.65_CG7033; probable chaperone activity: ortholog by CG7033</td>

<td>RNAi-pMON96180:001</td><td>Dv.65 CG7033; probable chaperone activity: CG7033 ortholog</td>

<td>RNAi-pMON96176:001</td><td>Dv.66 CG32019; likely bent: ortholog of CG32019</td>

<td>RNAi-pMON96170:002</td><td>Dv.67_CG16916; probable endopeptidase activity: CG16916 ortholog</td>

<td>RNAi-pMON96166:001</td><td>Dv.70_CG5771; probable Rab 11 protein: ortholog of CG5771</td>

<td>RNAi-pMON96179:001</td><td>Dv.72 CG6831; probable rhea: ortholog of CG6831</td>

<td>RNAi-pMON96186:001</td><td>Dv.73 CG10119; probable Lamin C: ortholog of CG10119</td>

<td>RNAi-pMON96160:002</td><td>Dv.74 CG6375; probable pitchoune: ortholog of CG6375</td>

<td>RNAi-pMON96160:001</td><td>Dv.74 CG6375; probable pitchoune: ortholog of CG6375</td>

<td>RNAi-pMON97137:002</td><td>Dv.77 CG4214; probable Syntaxin 5; Involved in intracellular protein transport: CG4214 ortholog</td>

<td>RNAi-pMON96167:001</td><td>Dv.82 CG8264; probable Bx42: ortholog of CG8264</td>

<td>RNAi-pMON96171:001</td><td>Dv.83 CG11397; probable gluon: ortholog of CG11397</td>

<td>RNAi-pMON96187:003</td><td>Dv.85_CG4494; probable protein-binding activity: CG4494 ortholog</td>

<td>RNAi-pMON97126:001</td><td>Dv.88_CG8756; function unknown; contains chitin-binding domain: ortholog of CG8756</td>

<td>RNAi-pMON97109:001</td><td>Dv.99_CG2446; a stranger; lethal in Drosophila and low homology with humans: CG2446 ortholog</td>

<td>RNAi-pMON97111:001</td><td>Dv.105_CG1250_1; GTPase activator, involved in intracellular protein transport: CG1250 ortholog</td>

<td>RNAi-pMON97107:001</td><td>Dv.107_CG14813; COPI vesicle coating; participates in the transport of proteins from the Golgi to the ER: ortholog of CG14813</td>

<td>RNAi-pMON97115:001</td><td>Dv.108_CG17248; n-synaptobrevin; involved in intracellular protein transport: ortholog of CG17248</td>

<td>RNAi-pMON97121:001</td><td>Dv.122 CG3164; probable ATP-binding transporter cassette activity: CG3164 ortholog</td>

<td>RNAi-pMON97171:001</td><td>Dv.146; unknown function, WCR single sequence</td>

<td>RNAi-pMON97166:001</td><td>Dv.147; unknown function, WCR single sequence</td>

<td>RNAi-pMON97167:001</td><td>Dv.149; unknown function, WCR single sequence</td>

<td>RNAi-pMON97169:001</td><td>Dv.155; unknown function, WCR single sequence</td>

<td>RNAi-pMON97173:001</td><td>Dv.162; unknown function, WCR single sequence</td>

<td>RNAi-pMON97170:001</td><td>Dv.170; unknown function, WCR single sequence</td>

Table 3: dsRNA Constructs Causing Significant Mortality Levels in Western Corn Rootworm Larvae (WCR) Feeding Bioassays

<td>Vector</td><td>Sequence expressed as dsRNA</td>

<td>RNAi-plC17553:001</td><td>Apple</td>

<td>RNAi-pICI 7504:049</td><td>Complete V-ATPase EST</td>

<td>RNAi-pICl 7504:050</td><td>V-ATPase Section 6.0</td>

<td>RNAi-plC17504:054</td><td>Diabrotica virgifera V-ATPase subunit 2 sequence, full length EST sequence serving as positive control</td>

<td>RNAi-plC19514:001</td><td>WCR enzyme LIB5496-028-A1 -M1-A7 mRNA</td>

<td>RNAi-pMON78412:002</td><td>Dv.7 CG3416; probable Mov34: ortholog of CG3416</td>

<td>RNAi-pMON96172:001</td><td>Dv.8_CG1088; probable vacuolar H+ATPase E subunit: ortholog of CG1088</td>

<td>RNAi-pMON96168:001</td><td>Dv.9_CG2331; probable ATPase activity: ortholog of CG2331</td>

<td>RNAi-pMON78424:002</td><td>Dv.10_CG6141 ; probable L9 ribosomal protein: ortholog of CG6141</td>

<td>RNAi-pMON78444:001</td><td>Dv.12_CG1341; probable regulatory particle of proteasome, rpt1: ortholog of CG1341</td>

<td>RNAi-pMON78416:002</td><td>Dv.13_CG11276; probable S4 ribosomal protein: ortholog by CG11276</td>

<td>RNAi-pMON78434:001</td><td>Dv.14 CG17927 2; probable myosin heavy chain: ortholog of CG17927</td>

<td>RNAi-pMON78435:001</td><td>Dv.18_CG1404; probable monomeric GTPase small RNA: ortholog of CG1404</td>

<td>RNAi-pMON78449:001</td><td>Dv.19_CG18174; probable regulatory particle of proteasome, lid subcomplex, rpn 11: CG18174 ortholog</td>

<td>RNAi-pMON78440:001</td><td>Dv20_CG3180_2; probable ANA polymerase II directed to RNA: CG3180 ortholog</td>

<td>RNAi-pMON78420:001</td><td>Dv.21 CG3320; likely Rabbi: ortholog of CG3320</td>

<td>RNAi-pMON78410:001</td><td>Dv.22 CG3395; probable ribosomal protein S9: ortholog of CG3395</td>

<td>RNAi-pMON78422:001</td><td>Dv.23 CG7269; probable helicase: CG7269 ortholog</td>

<td>RNAi-pMON78414:006</td><td>DV.26 half thirst - 5' from EST</td>

<td>RNAi-pMON78414:001</td><td>Dv.26_CG9261; probable sodium/potassium exchanger ATPase: ortholog of CG9261</td>

<td>RNAi-pMON97122:001</td><td>C1_Dv.35; probable Vha68-2: ortholog of CG3762; concatamer</td>

<td>RNAi-pMON97127:001</td><td>C2_Dv.35; probable Vha68-2: ortholog of CG3762; concatamer</td>

<td>RNAi-pMON97125:001</td><td>C3_Dv.35; probable Vha68-2: ortholog of CG3762; concatamer</td>

<td>RNAi-pMON78427:001</td><td>Dv.35 CG3762; probable Vha68-2: ortholog of CG3762</td>

<td>RNAi-pMON97114:001</td><td>Dv.41_CG2637; Ketel of female sterility; participates in the import of proteins into the nucleus: ortholog of CG2637</td>

<td>RNAi-pMON97140:001</td><td>Dv.44_CG1244; Probable nucleic acid binding activity: CG1244 ortholog</td>

<td>RNAi-pMON78429:001</td><td>Dv.46_CG 10689; probable RNA helicase: ortholog of CG10689</td>

<td>RNAi-pMON78428:001</td><td>Dv.49_CG8055_1; probable binding, vehicle activity: CG8055 ortholog 1</td>

<td>RNAi-pMON78428:003</td><td>Dv.49_CG8055_1; probable binding, vehicle activity: CG8055 ortholog 1, free region selected from DV.49</td>

<td>RNAi-pMON78426:001</td><td>Dv.50_CG10110_1; probable cleavage and polyadenylation specificity factor: CG10110 ortholog 1</td>

<td>RNAi-pMON96185:001</td><td>Dv.55_CG5931; probable cutting factor activity, RNA helicase activity: CG5931 ortholog</td>

<td>RNAi-pMON78431:001</td><td>Dv.58_CG1751; probable signal peptidase: ortholog of CG1751</td>

<td>RNAi-pMON96177:001</td><td>Dv.61 CG3725 1; probable calcium ATPase: ortholog of</td>

<td>RNAi-pMON96182:001</td><td>Dv.61 CG3725 2; probable calcium ATPase: ortholog of CG3725</td>

<td>RNAi-pMON96180:001</td><td>Dv.65_CG7033; probable chaperone activity: CG7033 ortholog</td>

<td>RNAi-pMON96176:001</td><td>Dv.66 CG32019; likely bent: ortholog of CG32019</td>

<td>RNAi-pMON96170:001</td><td>Dv.67_CG16916; probable endopeptidase activity: CG16916 ortholog</td>

<td>RNAi-pMON96166:001</td><td>Dv.70 CG5771; probable Rab 11 protein: ortholog of CG5771</td>

<td>RNAi-pMON96160:001</td><td>Dv.74 CG6375; probable pitchoune: ortholog of CG6375</td>

<td>RNAi-pMON97137:001</td><td>Dv.77_CG4214; probable Syntaxin 5; Involved in intracellular protein transport: CG4214 ortholog</td>

<td>RNAi-pMON96167:001</td><td>Dv.82 CG8264; probable Bx42: ortholog of CG8264</td>

<td>RNAi-pMON96187:002</td><td>Dv.85_CG4494; probable protein-binding activity: CG4494 ortholog</td>

<td>RNAi-pMON97126:001</td><td>Dv.88_CG8756; function unknown; contains chitin-binding domain: ortholog of CG8756</td>

<td>RNAi-pMON97130:001</td><td>Dv.93_CG8515; probable structural constituent of the cuticle; contains chitin-binding domain: ortholog of CG8515</td>

<td>RNAi-pMON97111:001</td><td>Dv.105_CG1250_1; GTPase activator, involved in intracellular protein transport: CG1250 ortholog</td>

<td>RNAi-pMON97107:001</td><td>Dv.107CG14813; COPI vesicle coating; participates in the transport of proteins from the Golgi to the ER: ortholog of CG14813</td>

<td>RNAi-pMON97115:001</td><td>Dv.108_CG17248; n-synaptobrevin; involved in intracellular protein transport: ortholog of CG17248</td>

<td>RNAi-pMON97133:001</td><td>Dv.113; function unknown; unique sequence of WCR</td>

<td>RNAi-pMON97121:001</td><td>Dv.122_CG3164; probable cassette activity ATP-binding transporter: CG3164 ortholog</td>

<td>RNAi-pMON97134:001</td><td>Dv.127; function unknown; no homology with humans</td>

Table 4: dsRNA constructs causing significant fading levels in southern corn rootworm (SCR) larval feeding bioassays

<td>Vector</td><td>Sequence expressed as dsRNA</td>

<td>RNAi-pMON96172:001</td><td>Dv.8_CG1088; probable vacuolar H+ATPase E subunit: ortholog of CG1088</td>

<td>RNAi-pMON96168:001</td><td>Dv.9_CG2331; probable ATPase activity: ortholog of CG2331</td>

<td>RNAÍ-pMON96155:002</td><td>Dv.10_CG6141; probable L9 ribosomal protein: ortholog of CG6141</td>

<td>RNAÍ-pMON96158:002</td><td>Dv.11_CG2746; probable L19 ribosomal protein: ortholog of CG2746</td>

<td>RNAÍ-pMON78434:001</td><td>Dv.14_CG17927_2; probable myosin heavy chain: CG17927 ortholog</td>

<td>RNAÍ-pMON78435:001</td><td>Dv.18_CG1404; probable monomeric GTPase small RNA: ortholog of CG1404</td>

<td>RNAÍ-pMON78449:001</td><td>Dv.19_CG18174; probable regulatory particle of proteasome, lid subcomplex, rpn 11: CG18174 ortholog</td>

<td>RNAi-pMON78420:001</td><td>Dv.21 CG3320; likely Rabbi: ortholog of CG3320</td>

<td>RNAÍ-pMON78414:001</td><td>Dv.26_CG9261; probable sodium/potassium exchanger ATPase: ortholog of CG9261</td>

<td>RNAi-pMON97122:001</td><td>C1 Dv.35; probable Vha68-2: ortholog of CG3762; concatamer</td>

<td>RNAi-pMON97125:001</td><td>C3_Dv.35; probable Vha68-2: ortholog of CG3762; concatamer</td>

<td>RNAi-pMON78427:008</td><td>Dv.35 CG3762; probable Vha68-2: ortholog of CG3762.</td>

<td>RNAÍ-pMON78428:001</td><td>Dv.49_CG8055_1; probable binding, vehicle activity: CG8055 ortholog 1</td>

<td>RNAÍ-pMON78442:001</td><td>Dv.57_CG2968; probable hydrogen-exporting ATPase: ortholog of CG2968</td>

<td>RNAÍ-pMON96177:001</td><td>Dv.61_CG3725_1; probable calcium ATPase: ortholog of CG3725</td>

<td>RNAÍ-pMON96166:001</td><td>Dv.70_CG5771; probable Rab 11 protein: ortholog of CG5771</td>

<td>RNAÍ-pMON97126:001</td><td>Dv.88_CG8756; function unknown; contains chitin-binding domain: ortholog of CG8756</td>

<td>RNAÍ-pMON97111:001</td><td>Dv.105_CG1250_1; GTPase activator, involved in intracellular protein transport: CG1250 ortholog</td>

<td>RNAÍ-pMON97112:001</td><td>Dv.105_CG1250_2; GTPase activator, involved in intracellular protein transport: CG1250 ortholog</td>

<td>RNAÍ-pMON97107:001</td><td>Dv.107 CG14813; COPI vesicle coating; participates in the transport of proteins from the Golgi to the ER: ortholog of CG14813</td>

<td>RNAÍ-pMON97121:001</td><td>Dv.122 CG3164; probable ATP-binding transporter cassette activity: CG3164 ortholog</td>

Table 5: dsRNA Constructs Causing Significant Mortality Levels in Southern Corn Rootworm (SCR) Larvae Feeding Bioassays

<td>Vector</td><td>Sequence expressed as dsRNA</td>

<td>RNAi-plC17546:003</td><td>LIB5462-091-A1-M1-G3 Dv.1 CG6217 KNICKKOPF UNK</td>

<td>RNAi-plC17504:055</td><td>Diabrotica V-ATPase subunit 2 sequence virgifera, complete EST sequence serving as positive control</td>

<td>RNAi-pMON96155:001</td><td>Dv.10_CG6141; probable L9 ribosomal protein: ortholog of CG6141</td>

<td>RNAi-pMON96158:001</td><td>Dv.11_CG2746; probable L19 ribosomal protein: ortholog of CG2746</td>

<td>RNAi-pMON96154:003</td><td>Dv.14 CG 17927; probable myosin heavy chain: ortholog</td>

<td></td><td>CG17927; cells grown in complete S medium</td>

<td>RNAi-pMON78438:001</td><td>Dv.17 CG10149; probable p44.5 proteasome subunit, rpn6: from ortholog CG10149</td>

<td>RNAi-pMON78449:001</td><td>Dv.19_CG18174; probable proteasome regulatory particle, lid subcomplex, rpn11:CG18174 ortholog</td>

<td>RNAi-pMON78440:001</td><td>Dv.20_CG3180; probable RNA-directed RNA polymerase II: ortholog of CG3180</td>

<td>RNAi-pMON96156:001</td><td>Dv.20_CG3180; probable 150 kD RNA polymerase II subunit: ortholog of CG3180</td>

<td>RNAi-pMON78420:001</td><td>Dv.21 CG3320; likely Rabbi: ortholog of CG3320</td>

<td>RNAi-pMON78427:006</td><td>Dv.35 CG3762; probable Vha68-2: ortholog of CG3762</td>

<td>RNAi-pMON78428:001</td><td>Dv.49_CG8055_1; probable binding, vehicle activity: deCG8055 ortholog 1</td>

<td>RNAi-pMON96177:001</td><td>Dv.61 CG3725 1; probable calcium ATPase: CG3725 ortholog</td>

<td>RNAi-pMON96166:001</td><td>Dv.70 CG5771; probable Rab 11 protein: ortholog of CG5771</td>

<td>RNAi-pMON96174:001</td><td>Dv.86 CG5055; probable bazooka: CG5055 ortholog</td>

<td>RNAi-pMON97111:001</td><td>Dv.105_CG1250 1; GTPase activator, involved in intracellular protein transport: CG1250 ortholog</td>

<td>RNAi-pMON97107:001</td><td>Dv.107_CG14813; COPI vesicle coating; participates in the transport of proteins from the Golgi to the ER: ortholog of CG14813</td>

Example 4

Transformation of transgenic plants and bioassays

Briefly, the sequence encoding a dsRNA construct as described above is ligated at its 5' end to a sequence consisting of an e35S promoter, operably linked to a maize hsp70 intron, and ligated at its 3' end. to a NOS3' transcription terminator and a polyadenylation sequence. This expression cassette is then placed towards the 3' end of a glyphosate selection cassette. These linked cassettes are then placed into a functional Agrobacterium tumefaciens plant transformation vector, used to transform maize tissue to obtain glyphosate tolerance, select for events, and transfer to soil. Western corn rootworm (WCR, Diabrotica virgifera) larvae feeding on plant roots Reither. Transgenic maize roots are grown in Petri dishes with MSOD medium containing the antibiotics and glyphosate for in vitro selection. Two WCR larvae per root are placed on each plate using a fine-tipped brush. The plates are sealed with plastic sheets to prevent the escape of the larvae. Assays are placed in a Percival incubator at 27gC and 60% RH in complete darkness. Contamination and the quality of the larvae are monitored. After six days of feeding on root tissue, the larvae are transferred to a WCR diet in a 96-well dish. Feeding of the larvae on the diet is allowed for eight days, thus extending the entire trial to a total of fourteen days. For analysis, the mass and survival of the larvae are recorded. A one-way ANOVA is performed on the larval mass data, and a Dunnett's test is performed to look for statistical significance, compared to an untransformed negative control. The fade of WCR larvae is measured after feeding two events, and compared to the growth of larvae fed negative control plants.

Once they reach an appropriate size, transgenic maize plants (Reither) generated are planted in 10-inch pots containing Metromix soil. When plants reach the V4 growth stage, approximately 1000 Western Corn Rootworm (WCR, Diabrotica vitiera) eggs are laid in the root zone. Non-transgenic maize of the same genotype, at a similar growth stage, is infested to serve as a negative control. Eggs are pre-incubated so that hatching occurs within 24 hours of infestation. Feeding of the larvae with the root system is allowed for 3 weeks. The plants are removed from the soil and washed, in order to be able to evaluate the feeding of the larvae on the roots. Root damage is scored using a Node Injury Scale (ELN) to rate the level of damage, where 0 indicates no damage, 1 indicates pruning of a root nodule to 1.5 inches, 2 Indicates 2-nodule pruning, while 3 indicates 3-nodule pruning. Because the plants used for evaluation are taken directly from tissue culture after transformation, and because transformation events are unique, only one plant per event is evaluated on this occasion, and no statistics are available. Plants in the test show signs or symptoms of larval feeding, indicating a successful infestation has been achieved. Negative control roots received moderate to severe damage, averaging about 0.2 on the Nodule Injury Scale.

Example 5

Implementation of Gene Suppression in Insect Pests Using a ta-s¡RNA-Mediated Silencing Method

An alternative method for silencing genes in a plant pest is the recently discovered class of trans-acting small interfering RNAs (ta-siRNAs) (Dalmay et al., 2000; Mourrain et al., 2000; Peragine et al., 2004; Vázquez et al. , 2004). ta-s¡RNAs are derived from single-stranded RNA transcripts that are targeted by naturally occurring miRNAs within the cell. Methods for using microRNAs to trigger ta-s¡RNAs to achieve gene silencing in plants are described in US Provisional Patent Application No.yes 60/643,136 (Carrington et al. 2004), and are incorporated herein by reference in their entirety. At least one pest-specific miRNA expressed in the gut epithelial cells of corn rootworm larvae was identified. This pest-specific miRNA is then used to identify at least one transcribed RNA target sequence, complementary to the miRNA being expressed in the cell. The corresponding target sequence is a short sequence of no more than 21 contiguous nucleotides that, when part of an RNA transcript and contacted by its corresponding miRNA in a cell type with a functional iRNA pathway, leads to miRNA-mediated cleavage. a machine to cut said transcript. Once the miRNA target sequences have been identified, at least one miRNA target sequence is fused to a second sequence that corresponds to part of a pest gene to be silenced using this method. For example, the miRNA target sequence(s) are fused to any of SEQ ID NO:1 through SEQ ID NO:906, or a fragment thereof, such as a sequence of the corn caterpillar vacuolar ATPase (V-ATPase) gene. The miRNA target sequence can be placed at the 5' end, 3' end, or in the middle of the target sequence. It is preferable to use several miRNA target sequences corresponding to multiple miRNA genes, or to use the same miRNA target sequence several times in the chimera of miRNA target sequence and target gene sequence. The target gene sequence can be of any length, with a minimum of 21 bp.

The chimera of miRNA target sequences with the target gene sequence is expressed in plant cells using any of several appropriate promoters and other transcriptional regulatory elements, as long as transcription occurs in the cell types subjected to being supplied in the diet. of the pest, for example corn roots for the control of the corn caterpillar.

This method may have the additional advantage of delivering longer RNA molecules to the target pest. Typically, plant-produced double-stranded RNAs are rapidly processed by Dicer into short RNAs that may not be effective when fed exogenously to some pests. In this method, a single-stranded transcript is produced in the plant cell, taken up by the pest, and converted to a double-stranded RNA in the pest cell where it is then processed into a ta-s¡RNA capable of post-transcriptionally silencing one or more genes in one or more target pests.

EXAMPLE 6

Method for Providing a DNA Sequence for Double-stranded RNA-Mediated Gene Silencing

This example illustrates a method for providing a DNA sequence for double-stranded RNA-mediated gene silencing. More specifically, this method describes the selection of an improved DNA useful for double-stranded RNA-mediated gene silencing by (a) selecting, from a target gene, an initial DNA sequence that includes more than 21 contiguous nucleotides; (b) identification of at least one shorter DNA sequence derived from regions of the initial DNA sequence consisting of predicted regions that do not generate undesirable polypeptides and do not exhibit identity to known sequences such as homologues/orthologs, and (c ) selection of a DNA sequence for double-stranded RNA-mediated gene silencing that includes the at least one shorter DNA sequence. Undesirable polypeptides include, but are not limited to, polypeptides homologous to allergenic polypeptides and polypeptides homologous to known polypeptide toxins.

The WCR V-ATPase has been shown to function in corn rootworm feeding assays to assess double-stranded RNA-mediated gene silencing as a means of controlling larval growth. A cDNA sequence is chosen from a target gene, such as the Western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte) vacuolar ATPase (V-ATPase) gene, for use as a DNA sequence. initial. This initial DNA sequence can be investigated to identify regions within which each contiguous fragment includes at least 21 fewer nucleotide matches than the 21 of the 21 contiguous nucleotides of known vertebrate sequences. Sequence segments that are greater than about 100 contiguous nucleotides that are free of such 21/21 matches are identified. Thus, criteria including segment length, GC content, sequence, predicted function based on sequence or function of a corresponding gene in a model organism can be used, and any predicted secondary structure can be used (eg Elbashir, et al. al., 2001b) to choose and design sequence(s) for use. Different combinations of these sequence segments are combined to construct chimeric DNA sequences for expression as dsRNA and use in insect feeding bioassays as described above.

Example 7

Additional results from insect feeding bioassays with selected sequences in the EST database

Additional sequences established to be effective in causing larval fainting and/or mortality when ingested by rootworm larvae are illustrated in this example as double-stranded RNA sequences. Methods for rearing corn rootworm larvae, applying dsRNA, and performing insect bioassays are as described in Examples 1-3. The results of the studies confirmed significant (p<0.05) levels of larval fading and/or mortality when using dsRNAs containing portions of sequences homologous to a variety of different gene classes. Sequences and vectors that provide significant fading and/or mortality, and SEQ ID NQ Corresponding values for the sequences, expressed as dsRNA, are provided in Table 6 below. pMON98503, an example of a binary vector used in the transformation of maize, contains the following elements between the left and right DNAT borders, prior to transfer into a plant cell: e35S - HSP70 - DV49 (antisense targeting) - universal spacer - DV49 (reading frame orientation) - hsp17; ACT (promoter and intron) - transit sequence CTP2 - CP4 - NOS.: pMON98504, another example of a binary vector used in the transformation of maize contains the following elements between the left and right borders: e35S - HSP70 - C1 (antisense orientation) - universal separator - C1 (reading frame orientation) - hsp17; ACT (promoter and intron) - transit sequence CTP2 - CP4 - NOS.

Table 6: Additional dsRNA Constructs Exhibiting a Significant Fading and/or Mortality Effect in Insect Feeding Bioassays with Southern Corn Rootworm or Western Corn Rootworm Larvae__________________________________________

<td>Vector</td><td>Sequence expressed as dsRNA</td><td>I KNOW THAT ID No.2</td>

<td>pMON98356</td><td>Dv164; unknown function, WCR single sequence</td><td> 726</td>

<td>pMON98354</td><td>Dv172; unknown function, WCR single sequence</td><td> 727</td>

<td>pMON97191</td><td>Dv189; unknown function, WCR single sequence</td><td> 728</td>

<td>pMON98359</td><td>dv200; unknown function, WCR single sequence</td><td> 729</td>

<td>pMON38880</td><td>Dv207 F39H11.5; probable pbs-7, endopeptidase: ortholog</td><td> 730</td>

<td>Vector</td><td>Sequence expressed as dsRNA</td><td>I KNOW THAT ID No.and</td>

<td>pMON101054</td><td>Dv208_F58F121; probable mitochondrial F1F0-ATP synthase, delta subunit/ATP16: ortholog of F58F12 1</td><td> 731</td>

<td>pMON98437</td><td>Dv210_K11H122; probable rpl-15, constituent ribosome structure: K11H12 ortholog 2</td><td> 732</td>

<td>pMON98435</td><td>Dv211_R12E2_3; probable rpn-8, translation initiation factor, 26S proteasome regulatory complex, RPN8 subunit: ortholog of R12E2 3</td><td> 733</td>

<td>pMON98447</td><td>Dv212_C17H12_14; probable hav-8, ATPase exporter hydrogen: ortholog of C17H12 14</td><td> 734</td>

<td>pMON98448</td><td>Dv213_B0464_1; probable drs-1, tRNA ligase: ortholog of B0464 1</td><td> 735</td>

<td>pMON101059</td><td>Dv214 F53G12 10; probable rpl-7, constituent ribosome structure: ortholog of F53G12 10</td><td> 736</td>

<td>pMON98442</td><td>Dv216_C52E4_4; probable rpt-1, ATPase subunit of the 19S proteasome regulatory complex: ortholog of C52E4 4</td><td> 737</td>

<td>pMON98441</td><td>Dv218_K01G5_4; probable ran-1, monomeric GTPase small: ortholog of K01G5 4</td><td> 738</td>

<td>pMON98440</td><td>Dv219_C15H117; probable pas-1, endopeptidase: ortholog of C15H11 7</td><td> 739</td>

<td>pMON101081</td><td>Dv223_R10E11.1; probable cbp-1, homologue of the transcription cofactors CBP and p300: ortholog of R10E11.1</td><td> 740</td>

<td>pMON101050</td><td>Dv224_F11 C3_3; probable unc-54, ATP binding; activity motor: ortholog of F11C3 3</td><td> 741</td>

<td>pMON101051</td><td>Dv225_C37H5_8; probable hsp-6, heat attack protein 6: C37H5 ortholog 8</td><td> 742</td>

<td>Vector</td><td>Sequence expressed as dsRNA</td><td>I KNOW THAT ID No.and</td>

<td>pMON38888</td><td>Dv226_C47E12.5; probable uba-1, ubiquitin-activating enzyme: C47E12.5 ortholog</td><td> 743</td>

<td>pMON38887</td><td>Dv227 F54A3.3; probable chaperonin complex, TCP-1 gamma subunit: F54A3.3 ortholog</td><td> 744</td>

<td>pMON101110</td><td>Dv229_D1081.8; probable Myb-like DNA binding: ortholog from D1081.8</td><td> 745</td>

<td>pMON101052</td><td>Dv230_F55A11_2; probable syn-3, transporter of proteins; syntaxin: ortholog of F55A11 2</td><td> 746</td>

<td>pMON101107</td><td>Dv231_C30C11.1; probable mitochondrial rlbosomal protein L32: ortholog of C30C11.1</td><td> 747</td>

<td>pMON101055</td><td>Dv232_B0250_1; probable rpl-2, structural constituent ribosome: ortholog of B0250 1</td><td> 748</td>

<td>pMON98446</td><td>Dv233 F54C9 5; probable rpl-5, 5S rRNA binding, ribosome structural constituent: ortholog F54C9 5</td><td> 749</td>

<td>pMON101138</td><td>Dv235_C04F12.4; probable rpl-14, ribosomal subunit major L14 protein: C04F12.4 ortholog</td><td> 750</td>

<td>pMON98449</td><td>Dv236_C01G8_5; probable erm-1, Ezñn/Radixin/Moesin (ERM), cytoskeletal linker family: ortholog of C01G8 5</td><td> 751</td>

<td>pMON98439</td><td>Dv237_F57B9_10; probable rpn-6, regulatory particle of proteasome, not ATPase-like: F57B9 ortholog 10</td><td> 752</td>

<td>pMON98436</td><td>Dv240_F53A3_3; probable rps-22, structural constituent ribosome: ortholog of F53A3 3</td><td> 753</td>

<td>pMON101078</td><td>Dv241_F32H2.5; probable alcohol dehydrogenase, zinc-dependent: ortholog of F32H2.5</td><td> 754</td>

<td>pMON101058</td><td>Dv242 B0336 2; probable arf-1, monomeric GTPase small: ortholog of B0336 2</td><td> 755</td>

<td>pMON101057</td><td>Dv244_C14B9_7; probable rpl-21, structural constituent ribosome: ortholog of C14B9 7</td><td> 756</td>

<td>Vector</td><td>Sequence expressed as dsRNA</td><td>I KNOW THAT ID No.and</td>

<td>pMON98444</td><td>Dv245_C26F1_4; probable rps-30, ribosomal protein, small subunit: ortholog of C26F1 4</td><td> 757</td>

<td>pMON98434</td><td>Dv247 C13B9 3; probable delta subunit of the complex catomer (COPI): ortholog of C13B9 3</td><td> 758</td>

<td>pMON101053</td><td>Dv248_F38E11_5; probable vesicle coating complex COPI complex, beta' subunit: F38E11 ortholog 5</td><td> 759</td>

<td>pMON98445</td><td>Dv249_F37C12_9; probable rps-14, constituent ribosome structure: ortholog of F37C12 9</td><td> 760</td>

<td>pMON101056</td><td>Dv250_CD4_6; probable pas-6, endopeptidase: ortholog of CD4 6</td><td> 761</td>

<td>pMON101104</td><td>Dv251_D1007.12; probable rpl-24.1, structural constituent of ribosome: ortholog of D1007.12</td><td> 762</td>

<td>pMON101088</td><td>Dv252_C49H3.11; probable rps-2, structural constituent from ribosome: C49H3.11 ortholog</td><td> 763</td>

<td>pMON101079</td><td>Dv253_C26D10.2; probable hel-1, ATP-dependent RNA helicase: C26D10.2 ortholog</td><td> 764</td>

<td>pMON101085</td><td>Dv254_B0336.10; probable rpl-23, structural constituent ribosome: ortholog of B0336.10</td><td> 765</td>

<td>pMON38879</td><td>Dv255_C36A4.2; Probable member of the cytochrome P450 family: C36A4.2 ortholog</td><td> 766</td>

<td>pMON101087</td><td>Dv256_K05C4.1; probable pbs-5, beta subunit of proteasome: ortholog of K05C4.1</td><td> 767</td>

<td>pMON101082</td><td>Dv257_F29G9.5; probable rpt-2, 26S proteasome regulatory complex: ortholog of F29G9.5</td><td> 768</td>

<td>pMON101084</td><td>Dv258 F40F8.10; probable rps-9, structural constituent of ribosome: ortholog of F4 0F8.10</td><td> 769</td>

<td>pMON101083</td><td>Dv259_K07D4.3; probable rpn-11, 26S proteasome regulatory complex, RPN11 subunit: ortholog of K07D4.3</td><td> 770</td>

<td>Vector</td><td>Sequence expressed as dsRNA</td><td>I KNOW THAT ID No.and</td>

<td>pMON101080</td><td>Dv260_F49C12.8; probable rpn-7, regulatory particle of proteasome, non-ATPase-like: F49C12.8 ortholog</td><td> 771</td>

<td>pMON101115</td><td>Dv261_D1054.2; probable pas-2, endopeptidase: ortholog from D1054.2</td><td> 772</td>

<td>pMON101141</td><td>Dv263_F55A3.3; probable metalloexopeptidase: ortholog from F55A3.3</td><td> 773</td>

<td>pMON101126</td><td>Dv264_F56F3.5; probable rps-1, structural constituent of ribosome: ortholog of F56F3.5</td><td> 774</td>

<td>pMON101133</td><td>Dv266_C09D4.5; probable rpl-19, structural constituent of ribosome: ortholog of C09D4.5</td><td> 775</td>

<td>pMON38881</td><td>Dv268_R06A4.9; probable polyadenylation factor I complex, PFS2 subunit: ortholog of R06A4.9</td><td> 776</td>

<td>pMON101135</td><td>Dv271 F37C12.4; probable rpl-36, structural constituent of ribosome: ortholog of F37C12.4</td><td> 777</td>

<td>pMON101132</td><td>Dv273_F54E7.2; probable rps-12, structural constituent ribosome: ortholog of F54E7.2</td><td> 778</td>

<td>pMON101139</td><td>Dv274_C23G10.4; probable rpn-2, regulatory particle of proteasome, non-ATPase-like: C23G10.4 ortholog</td><td> 779</td>

<td>pMON101130</td><td>Dv275_C03D6.8; probable rpl-24.2, structural constituent of ribosome: ortholog of C03D6.8</td><td> 780</td>

<td>pMON101119</td><td>Dv276_C26E6.4; probable DNA-directed RNA polymerase: C26E6.4 ortholog</td><td> 781</td>

<td>pMON101134</td><td>Dv277 R13A5.8; probable rpl-9, structural constituent of ribosome: ortholog of R13A5.8</td><td> 782</td>

<td>pMON101127</td><td>Dv279_F42C5.8; probable rps-8, structural constituent of ribosome: ortholog of F42C5.8</td><td> 783</td>

<td>pMON101122</td><td>Dv280_F13B10.2; probable rpl-3, major ribosomal subunit L3: ortholog of F13B10.2</td><td> 784</td>

<td>pMON101116</td><td>Dv281 T05C12.7; probable cct-1, component of</td><td> 785</td>

<td>Vector</td><td>Sequence expressed as dsRNA</td><td>I KNOW THAT ID No.and</td>

<td></td><td>chaperonin complex, TCP-1 alpha subunit: T05C12.7 ortholog</td><td></td>

<td>pMON101125</td><td>Dv282 F07D10.1; probable rpl-11.2, structural constituent of ribosome: ortholog of F07D10.1</td><td> 786</td>

<td>pMON38883</td><td>Dv283_T05H4.6; probable peptide chain-releasing factor 1 (eRF1): ortholog of Dv283 T05H4.6</td><td> 787</td>

<td>pMON101124</td><td>Dv284_C47E8.5; probable daf-21, heat attack protein 90, chaperone activity: C47E8.5 ortholog</td><td> 788</td>

<td>pMON101120</td><td>Dv285_M03F4.2; probable act-4, actin: ortholog of M03F4.2</td><td> 789</td>

<td>pMON101137</td><td>Dv286_F25H5.4; probable translation elongation factor eft-2: ortholog of F25H5.4</td><td> 790</td>

<td>pMON101140</td><td>Dv287 F26D10.3; probable hsp-1, heat attack protein: ortholog of F26D10.3</td><td> 791</td>

<td>pMON101117</td><td>Dv288_F28D1.7; probable rps-23, structural constituent of ribosome: ortholog of F28D1.7</td><td> 792</td>

<td>pMON38886</td><td>Dv290_CG 11979; probable H-exporting ATPase: ortholog of CG11979</td><td> 793</td>

<td>pMON38885</td><td>Dv291_CG 13628; probable H-exporting ATPase: ortholog of CG13628</td><td> 794</td>

<td>pMON101103</td><td>Dv293_CG31237; probable RNA polymerase II targeting DNA: CG31237 ortholog</td><td> 795</td>

<td>pMON101096</td><td>Dv294 CG8669; probable cryptocephalus; transcription factor, involved in molting cycle, pupation and metamorphosis: ortholog of CG8669</td><td> 796</td>

<td>pMON101095</td><td>Dv295 CG8048; probable 44 kD Vacuolar H+ ATPase, subunit C: ortholog of CG8048</td><td> 797</td>

<td>pMON101100</td><td>Dv298_CG9032; probable H-ATPase exporter: CG9032 ortholog</td><td> 798</td>

<td>Vector</td><td>Sequence expressed as dsRNA</td><td>I KNOW THAT ID No.and</td>

<td>pMON101111</td><td>Dv299_CG 17369; probable H-exporting ATPase: ortholog of CG17369</td><td> 799</td>

<td>pMON101129</td><td>Dv303_CG4152; probable RNA helicase-dependent ATP: CG4152 ortholog</td><td> 800</td>

<td>pMON101136</td><td>Dv305_CG4916; probable RNA helicase-dependent ATP: CG4919 ortholog</td><td> 801</td>

<td>pMON101131</td><td>Dv315_CG9160; probable NADH dehydrogenase: ortholog by CG9160</td><td> 802</td>

<td>pMON101123</td><td>Dv316_CG8764; probable ubiquinol-cytochrome-c reductase: ortholog of CG8764</td><td> 803</td>

<td>pMON98364</td><td>C4_Dv49_CG8055 concatemer; probable binding, vehicle activity: CG8055 ortholog</td><td> 804</td>

<td>pMON98365</td><td>C5 Dv49 CG8055 concatemer; probable binding, vehicle activity: CG8055 ortholog</td><td> 805</td>

<td>pMON98368</td><td>C6 concatemer of highly effective WCR targets, -50% GC criteria, consists of segments in 5'-3' order of Dv26, Dv49, Dv23, Dv20, Dv13, Dv22, Dv18</td><td> 806</td>

<td>pMON98369</td><td>C7 insect-specific target concatemer, -50% GC criteria, consists of segments in 5'-3' order of Dv6, Dv1, Dv88, Dv93, Dv4, Dv113, Dv127, Dv99</td><td> 807</td>

<td>pMON98372</td><td>C8 concatemer; probable sodium/potassium exchanger ATPase: ortholog of CG9261</td><td> 808</td>

<td>pMON98373</td><td>C9 concatemer; probable sodium/potassium exchanger ATPase: ortholog of CG9261</td><td> 809</td>

<td>pMON98366</td><td>C10 gene concatemer with likely same/different mode of action, -50% GC criteria</td><td> 810</td>

<td>pMON98367</td><td>C12 concatemer of highly effective WCR targets, -50% GC criteria, consists of segments in 5'-3' order of Dv23-Dv13-Dv26-Dv18-Dv49-Dv22-Dv20.</td><td> 811</td>

<td>Vector</td><td>Sequence expressed as dsRNA</td><td>I KNOW THAT ID No.and</td>

<td>pMON98371</td><td>C14 target gene concatemer active in several different organisms, -50% GC criteria</td><td> 812</td>

<td>pMON98503</td><td>Comprising DV49 probable ESCRT-lll (endosomal sorting complex required for transport lll), subunit complex from Diabrotica virgifera</td><td> 820</td>

<td>pMON98504</td><td>Comprising probable Vha68-2: ortholog of CG3762; 250 bp C1 concatamer;</td><td> 821</td>

<td>pMON102862</td><td>Dv319_CG14750; probable ESCRTII, Vps25: ortholog of CG14750</td><td> 835</td>

<td>pMON102863</td><td>Dv320_CG9712; probable ESCRTI, Vps23: ortholog of CG9712</td><td> 836</td>

<td>pMON102861</td><td>Dv321_CG12770; probable ESCRTI, Vps28: ortholog of CG12770</td><td> 837</td>

<td>pMON102865</td><td>Dv322_CG14542; probable ESCRT lll, Vps2: ortholog of GC 14542</td><td> 838</td>

<td>pMON102866</td><td>Dv323_CG4071; probable ESCRT lll, Vsp20: ortholog of CG4071</td><td> 839</td>

<td>pMON102871</td><td>Dv326_CG3564; probable protein carrier, component of vesicle coating COPI: ortholog of CG3564</td><td> 840</td>

<td>pMON102873</td><td>Dv327_CG6223; probable coating element, vesicle coating component COPI: CG6223 ortholog</td><td> 841</td>

<td>pMON102877</td><td>Dv328_CG6948; probable clathrin light chain, cavity lining: ortholog of CG6948</td><td> 842</td>

<td>pMON102872</td><td>Dv330_CG9543; probable COPI vesicle coating: CG9543 ortholog</td><td> 843</td>

<td>pMON102879</td><td>Dv331_CG5183; probable KDEL binding sequence: CG5183 ortholog</td><td> 844</td>

<td>Vector</td><td>Sequence expressed as dsRNA</td><td>I KNOW THAT ID No.and</td>

<td>pMON102867</td><td>Dv335_F11C1.6; probable nhr-25, DNA binding: ortholog from F11C1.6</td><td> 845</td>

<td>pMON102870</td><td>Dv337 CG18734; probable furin 2, serine-like endopeptidase: ortholog of CG18734</td><td> 846</td>

<td>pMON102875</td><td>Dv329_CG7961; probable coating element, vesicle coating component COPI: CG7961 ortholog</td><td> 874</td>

Efficacy tests were performed as follows, using the progeny of maize plants transformed with control insect constructs:

1. Seven days after sowing: 10,000 WCR eggs per event (10 plants per event) are incubated at 25SC, at 60% RH in complete darkness for seven days.

2. Fourteen days after infestation: plants are transplanted from 4” pots with soil to 8” pots; v4 root tips can be sampled for gene expression studies.

3. Fourteen days after sowing: WCR eggs are washed out of the soil. Eggs and soil are placed on a 60 mesh screen, and placed on a 30 mesh screen over the 60 mesh screen to protect the eggs from water current. Wash thoroughly with lukewarm water, using a spray nozzle, until all soil is removed.

4. The eggs are suspended in a 2% (w/v) solution of Difeo agar, 25 ml of solution per ml of eggs. Eggs are placed in the soil in about 3-4 aliquots, using, for example, an Eppendorf repeating pipet, at about 1000 eggs per plant. Holes are made in the ground using a trowel before infestation, and covered after infestation.

5. Twenty-eight days after planting, v8 root tips can be sampled for gene expression studies.

6. The trial is evaluated thirty-five days after sowing. Plants are cut using pruning knives, leaving approximately 6” of stem. Pieces of the plants containing event information are pierced and tied to the stem. As much soil is removed from the root system as possible. The rest of the dirt is washed away using a spray hose.

7. Roots are examined and assigned an Olsen scale damage rating (0-3)

NIS, for damage caused by WCR larvae.

In FIG. 1 and FIG. 2 illustrates the results of control insects, obtained after attack of F1 maize plants (derived from plants transformed with pMON98503 or pMON98504) with WCR. In the culture chamber, with tests performed essentially as described above, NIS scores equal to or less than the economic injury threshold were observed in the progeny of events obtained by transformation with pMON98503 or pMON98504.

Full length EST DNA sequences were assembled for the selected genes described in Examples 3 and 7, and these sequences exhibited significant activity against western corn rootworm. These EST sequences are indicated in Table 7:

Table 7: Armed EST sequences for white WCR

<td>armed sequence</td><td>SEQ ID No.3</td>

<td>Full Dv9 (aka apple); probable ortholog of CG2331</td><td> 813</td>

<td>Complete Dv10 (Rp19); probable ortholog of CG6141</td><td> 814</td>

<td>Full Dv11 (Rpl 19); probable ortholog of CG2746</td><td> 815</td>

<td>Full Dv13 (Rps4); probable ortholog of CG11276</td><td> 816</td>

<td>v-ATPase A complete Dv35; ortholog of CG3762</td><td> 817</td>

<td>Complete Dv49, ortholog of CG8055</td><td> 818</td>

<td>Complete Dv248, probable ortholog of CG6699</td><td> 819</td>

Example 8

Creation and efficacy results for Dv49 and Dv248 sequences

Portions of the EST and adjacent sequences of Dv49 (SEQ ID N9 818) and Dv248 (SEQ ID No.3 819) for further bioactivity assays, based on criteria including predicted function, phenotype of deletion mutants of corresponding coding regions in other organisms, segment length, GC content, sequence similarity known, and the predicted secondary structure (eg, Elbashir, et al., 2001b). The respective Dv49 and Dv248 sequences were synthesized in vitro on the basis of their predicted activity against WCR, individually and in groups, as illustrated in FIGS. 3 and 4, and in Table 8, and applied to WCR larvae.

Table 8: Fragments of Dv49 and Dv248 with which the efficacy against WCR was evaluated

<td>Fragment</td><td>SEQ ID No.9</td>

Fragments F1-F3 correspond to portions of the complete Dv49 transcript. F4-F6 fragments correspond to portions of the transcript or surrounding region of Dv248. The F7F13 fragments are concatemers of two or more of the F1-F6 fragments, as illustrated in FIG. 4. The F13 fragment (SEQ ID Nyes 834) represents the C38 concatemer (Dv49-Dv248).

The F1-F13 dose response data are illustrated in FIGS. 5-7. As illustrated in FIG. 5, the activity (% larval mortality) of the F4-F6 fragments, comprising sequences derived from Dv248 (FIG. 4), was significantly better than the control, when fed to WCR larvae at 0 .1ppm. The F6 fragment also showed significant activity when used at 0.02 ppm. The activity of the F5 fragment at the lower dose is probably an artifact, since the surviving larvae did not show fading.

As illustrated in FIG. 6, each of the F7-F10 fragments exhibits statistically significant activity when used to feed WCR larvae at 0.1 ppm. Fragment F9 and F10 also had statistically significant activity when fed WCR larvae at 0.02 ppm, and F10 fragment also had statistically significant activity when used to feed WCR larvae at rate of 0.01 ppm. The F8 activity at the 0.01 ppm dose may be an artifact.

As illustrated in FIG. 7, F11-F13 fragments show statistically significant activity when used to feed WCR larvae at 0.02 ppm or higher. Additionally, the largest fragments (F12 and F13) show activity at doses of 0.005 ppm and higher.

Example 9

Sequences of additional active concatemers derived from the C6 concatemer

As illustrated in Table 9, portions of the active C6 concatemer (SEQ ID Ng806), derived from pMON98368, in diet analysis bioassays (performed as described, for example, in Example 2), and said concatemer inhibited corn rootworm (WCR) growth and/or survival. . The complete C6 concatemer contains 7 target subfragments of 70 bp each, as indicated in Table 6 and Table 9.

Table 9: Efficacy of complete C6 concatemer and selected subportions in a rootworm diet bioassay (SEQ ID Nyes 806, 847-873).______________________________________________

<td>concatemer</td><td>segments</td><td>% mortality at a rate of 1 ppm</td><td>SEQ ID No.2</td>

<td>C6_complete</td><td>Dv26-Dv49-Dv23-Dv20-Dv13-Dv22- dv18</td><td> 100</td><td> 806</td>

<td>concatemer</td><td>segments</td><td>% mortality at a rate of 1 ppm</td><td>SEQ ID No.2</td>

The complete C7 concatemer contains 8 target subfragments of 70 bp each, as indicated in Table 6. Similarly, the complete C12 concatemer contains 7 target subfragments of Dv23Dv13-Dv26-Dv18-Dv49-Dv22-Dv20, with a length which varies between 53 and 80 bp, as indicated in Table 6 and Table 10.

Table 10: Composition of the complete C12 concatemer and selected subportions (SEQ ID N9 811, 887-892).__________________________________________________________________________________

<td rowspan="2">concatemer</td><td rowspan="2">segments</td><td colspan="2">Included sequence (SEQ ID</td>

<td>No.2) in orientation 5'-3'</td><td>the</td>

<td>C12 complete</td><td>Dv23-Dv13-Dv26-Dv18-Dv49-Dv22-Dv20</td><td colspan="2"> 811</td>

<td rowspan="2">concatemer</td><td rowspan="2">segments</td><td colspan="2">Included sequence (SEQ ID</td>

<td>No.2) in orientation 5'-3'</td><td>the</td>

Additional sequences for use in targeting for other coleopteran pests, including Diabrotica sp., are listed in Table 11. and other Coccinellidae and Chrysomelidae.

Table 11: Target sequences for additional Coleoptera

<td>sequence and source</td><td>SEQ ID No.2</td>

<td>Dbar248 CG6699 (O. barberi)</td><td> 875</td>

<td>Dbal248 CG6699 (D. balteata)</td><td> 876</td>

<td>Du248 CG6699 (D. undecimpunctata howardi)</td><td> 877</td>

<td>Dz248 CG6699 (D. virgifera zea)</td><td> 878</td>

<td>Dv248 CG6699 (O. virgifera virgifera)</td><td> 879</td>

<td>Ev248 CG6699 (Epilachna varivestis)</td><td> 880</td>

<td>Ld248 CG6699 (Leptinotarsa decemlineata)</td><td> 881</td>

<td>Dbal49 CG8055 2 (D. balteata}</td><td> 882</td>

<td>Db49 CG8055 2 (D. barben}</td><td> 883</td>

<td>Du49 CG8055 2 (D. undecimpurictata howardi)</td><td> 884</td>

<td>Dz49 CG8055 2 (D. virgifera zea}</td><td> 885</td>

<td>Dv49 CG8055 2 (D. virgifera virgifera}</td><td> 886</td>

★ * *

All of the compositions and methods disclosed and claimed herein can be made and performed without undue experimentation in view of the present invention. While the compositions and methods of this invention have been described in terms of the aforementioned illustrative embodiments, it will be apparent to those skilled in the art that variations, changes, modifications, and alterations may be applied to the composition, methods, and in the steps or in the sequence of steps of the methods described in the present documentation, without departing from the true concept, spirit, and scope of the invention. More specifically, it will be apparent that it is possible to substitute certain agents that are both chemically and physiologically related for the agents described herein with similar or the same results. All such substitutions and similar modifications obvious to those skilled in the art are considered to be within the spirit, scope and concept of the invention defined in the appended claims.

References

The following references, to the extent that they provide exemplary procedures or other details supplementary to those described herein, are specifically incorporated herein by reference.

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Methods for genetic control of insect infestations in plants and compositions thereof

Abstract

Term

SIGNATURE OF THE APPLICANT

SIGNATURE OF ATTORNEY

CASE 5853

TECHNICAL MEMORY

INCORPORATION AS A REFERENCE OF THE LIST OF SEQUENCES PRESENTED IN

A COMPACT DISC

INVOKED PRIORITY

BACKGROUND OF THE INVENTION

SUMMARY OF THE INVENTION

BRIEF DESCRIPTION OF THE FIGURES

EXAMPLES

EXAMPLE 1

EXAMPLE 6

Priority claims1

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