Transgenic plants resistant to nematode disease caused by at least two different nematode species

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TRANSGENIC PLANTS RESISTANT TO NEMATODE DISEASE CAUSED BY AT LEAST TWO DIFFERENT NEMATODE SPECIES THE UNIVERSITY OF GEORGIA RESEARCH FOUNDATION INC. C: 61622 182245/2

BACKGROUND

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit of and priority to US provisional application number 60/618,097 filed on October 13, 2004, and US provisional application number 60/704,560 filed on August 02, 2005, copies of both applications can be found at the WIPO web site at http://www.wipo.int/pctdb/en/, and where permissible each is incorporated by reference in its entirety. 1. Technical Field

The present disclosure generally relates to compositions for controlling plant parasites and compositions for increasing root growth, more particularly to nucleic acid compositions for controlling nematode disease or increasing root growth. 2. Related Art

Nematodes are a very large group of invertebrate animals generally referred to as roundworms, threadworms, eelworms, or nema. Some nematodes are plant parasites and can feed on stems, buds, leaves, and in particular on roots. One important genus of plant parasitic nematodes is the root-knot nematode (Meloidogyne spp.). These parasitic nematodes infect a wide range of important field, vegetable, fruit and ornamental plants. In 2001 the root-knot nematode was responsible for a loss of US$200.5 million in cotton alone. WO 21106/04448() PCT/US2005/036711

Existing methods for treating or preventing root-knot nematode disease include the use of chemicals, pesticides, and fumigants. The use of pre-plant soil fumigants is highly effective in controlling root-knot and other plant-parasitic nematodes. However, the majority of the fumigant-type nematicides are no longer available and are also costly and difficult to apply properly under the prevailing conditions.

Crop rotation has also been used to control nematode disease. Rotating onion, carrot, or lettuce with a nonhost crop such as sweet corn and other grain crops, if economically possible, can be effective in controlling the northern root-knot nematode. Unfortunately, current crop rotations on organic soils are of limited value as most crops grown, including potatoes, beans, celery, lettuce, onion, and carrot are susceptible to disease.

The use of cover crops has also been attempted to control nematode disease. Cover crops grown between the main crops may provide an alternative management strategy. Ryegrain, barley, oats, sudangrass, tall fescue, annual ryegrass, and wheat have been shown to be non- or poor hosts to this nematode. Using cover crops, however, can be costly because the cover crops occupy space that could be used to grow more valuable crops.

Biological control organisms have also been used to try to control nematode disease in crops. Commercially available preparations of biological control organisms are limited in their use to regions that can support the growth of the control organism. Moreover, the outcome of using one organism to control another is unpredictable and subject to a variety of a factors such as weather and climate.

Additionally, the root-knot nematode (RKN) is a leading cause of crop loss due to plant parasitic nematodes. The most important species (M. incognita, M. javanica, M. arenaria, M. hapla, M. chitwoodi) have wide host ranges that limit nonhost rotation options. While several examples of host resistance genes in diverse crops exist, the availability of host plant resistance is substantially limited with appropriate resistance loci lacking for the majority of our.crops (Roberts, P.A. 1992. Journal of Nematology 2 WO 2006/044480 PCT/IJS2005/036711 24:213-227). In addition, the resistance is limited to only a few RKN species or populations and some resistance genes are heat-sensitive and thus unsuitable for hot production areas. Another limitation of natural resistance genes is the durability of resistance since resistance-breaking populations of RKN can develop after continuous exposure to resistant cultivars, e.g. root-knot resistant tomatoes.

Accordingly, there is need for compositions and methods for controlling, preventing, or reducing nematode disease in plants.

Still other problems affecting crops relate to poorly developed root systems. Root systems of plants are an important part of a plant, and provide many functions that are vital to plant survival. For example, root systems store nutrients for the plant, filter out toxins, help regulate plant growth, provide an absorptive network for water and nutrients, and provide mechanical structures that support the plant and strengthen the soil. Plants with larger roots have increased growth and increased stress tolerance. Increased or enhanced root growth in crop plants would be particularly advantageous because the increased root growth would increase crop yield.

In perennial crops, increased root growth would increase the regrowth rate, increase the yield potential, and increase the likelihood that plants will survive winter. In annuals, increased root size would ensure yield potential under varying environmental conditions. In root crops, enhanced root growth would mean larger yields.

Existing root stimulators typically include fertilizers or plant hormones that must be mixed or formulated in specific concentrations when applied to the plant or soil near the plant. Over application of such stimulators can have adverse effects on the plants, and under application will not achieve the desired outcome. Additionally, application of plant hormones can have undesired consequences. For example, one plant hormone used as a root initiator is auxin or indole-3-acetic acid (IAA). IAA plays important roles in a number of plant activities, including: development of the embryo, leaf formation, phototropism, gravitropism, apical dominance, fruit development, abscission as well as rootinitiation. 3 182245/2 WO 03/052110 discloses transgenic plants expressing dsRNA specific for mRNA for dual oxidases involved in crosslinking extracellular matrix proteins in the nematode cuticle. WO 03/052110 provides data from soaking nematodes with dsRNA. WO 03/052110 does not provide any data on transgenic plants that are nematode resistant due to the expression of dsRNA.

Boutla et al., Nucl. Acids Res, 30:1688-1694, 2002, discloses injection of siRNA into nematodes. Boutla does not provide any data on transgenic plants that are nematode resistant due to the expression of dsRNA. WO 01/37654 discloses nematode-resistant transgenic plants expressing dsRNA specific for a vesicular acetylcholine transporter protein, a choline acetyltransferase, ora ubiquinone oxidoreductase. US Patent Publication No. 2003/0150017 discloses nematode resistant transgenic plants expressing dsRNA specific for one of several identified genes. US 2003/0150017 does not disclose transgenic plants expressing dsRNA specific for mRNA encoding a protein encoded by SEQ ID NO: 1 or 2 ofthe present application.

Urwin, P. et al., Molecular Plant-Microbe Interactions, vof. 16, no. 5, May 2003, pages 376-381, discloses that ingestion of dsRNA by cyst nematodes leads to RNA interference.

Huang et al., Molecular Plant-Microbe Interactions, vol. 16, no. 5, May 2003, pages 376-381, discloses the sequence for 16D10 and indicates that it encodes a 13 amino acid polypeptide. Huang etal, does not disclose the function of the protein but notes that because the identified proteins had no similarity to known proteins, their function could not be predicted and they would present significant challenges to determine their roles in parasitism of plants.

Thus there is a need for new compositions and methods for stimulating or enhancing root growth or development.

SUMMARY

The invention provides a10 transgenic plant or plant cell comprising a double-stranded RNA inhibitory nucleic acid at least 21 nucleotides long and complementary to part or all of a mRNA encoding a protein encoded by SEQ ID NOs 1 or 2, wherein the double-stranded RNA inhibitory nucleic acid renders the 4 182245/1 plant or plant cell resistant to nematode disease caused by at least two different nematode species.

Notice Under Commissioner's Circular 23(P) dated April 5, 1992

Inasmuch as the invention is defined in the appended claims, it will be apparent that the portions ofthe present specification, which fall outside the scope ofthe claims, do not relate directly to the claimed invention. This Notice is not meant to disclaim any legitimate rights to which the Patentee is legally entitled, especially any rights in accordance with Section 49 of the Israel Patent Law.

ADDITIONAL ASPECTS OF THE APPLICATION

Aspects of the present disclosure generally provide nucleic acid constructs that inhibit the expression of proteins secreted by plant parasites. In some aspects, the proteins are secreted by a nematode and, optionally, modulate: gene expression ofthe plant or cell, formation of a giant cell, nematode migration through root tissue ofthe plant, cell metabolism of the plant, elicits signal transduction in the plant cell, or forms a feeding tube that enables the nematode to feed from giant-cells formed in the plant. One aspect provides inhibitory nucleic acids specific for esophageal gland cell proteins secreted by nematodes, in particular root knot nematodes. Other aspects provide transgenic cells or plants expressing or containing one or more inhibitory nucleic acids, for example inhibitory double or single stranded RNA, that inhibit or reduce the expression of nematode esophageal gland cell proteins.

Another aspect provides a transgenic plant that comprises inhibitory RNA that down regulates a target nematode parasitism gene transcript in 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18,19, 20, or more nematode species, for example RKN species. Thus, the present disclosure provides transgenic plants that are resistant to disease caused by multiple RKN species.

Representative esophageal gland cell proteins that are targeted by the disclosed inhibitory nucleic acids include one or more ofthe proteins encoded by SEQ ID NOs.1 , 2, and 5-51. In certain aspects, one or more inhibitory nucleic 4a 182245/1 acids are delivered to a parasitic nematode when the nematode enters the transgenic plant or transgenic plant cell, feeds on the transgenic plant or transgenic plant cell, or comes into physical contact with the transgenic plant or transgenic plant cell. Once the inhibitory nucleic acid is internalized by the parasitic nematode, the inhibitory nucleic acid interferes with, reduces, or inhibits the expression of a target esophageal gland cell 4b WO 2006/044480 PCT/US2005/036711 protein, for example, by directly or indirectly interfering, reducing, or inhibiting the translation of one or more mRNAs coding for one or more esophageal gland cell proteins.

Yet another aspect provides a plant cell transfected with heterologous nucleic acid encoding an inhibitory nucleic acid specific for one or more nematode esophageal gland cell proteins, wherein the heterologous nucleic acid is expressed in an amount sufficient to reduce or prevent nematode disease. In one aspect, the transgenic plant expresses the inhibitory nucleic acid, and the inhibitory nucleic acid is delivered to a nematode feeding or attempting to feed on the transgenic plant Generally, the inhibitory nucleic acid is internalized by a nematode. Exemplary methods of internalizing the inhibitory nucleic acid include ingesting the nucleic acid or absorbing the nucleic acid.

Still another aspect provides a transgenic plant comprising an inhibitory nucleic acid specific for one or more nematode parasitism polypeptides, wherein the inhibitory nucleic acid provides resistance to two or more nematode species, for example two or more root-knot nematode species.

Further aspects provide compositions for stimulating, promoting, or enhancing root growth or development in plants or trees. Certain aspects provide nucleic acid constructs encoding proteins secreted by nematode esophageal gland cells, wherein the proteins or fragments thereof stimulate or enhance root development when delivered to or in contact with a plant. Other aspects provide compositions containing one more nematode esophageal gland cell proteins or fragments thereof that stimulate root growth when in contract with a plant or plant cell. Still other aspects provide transgenic plants comprising one or more nematode esophageal gland cell proteins orfragments thereof or nucleic acids encoding one more nematode esophageal gland cell proteins orfragments thereof sufficient to stimulate, enhance, or promote root growth compared to non-transgenic or control plants. 5 WO 2006/044480 PCT/US2005/036711

Representative nematode esophageal gland cell proteins (also referred to as esophageal proteins) include one or more ofthe proteins encoded by SEQ ID NOs. 1,2, and 5-51 or combinations thereof.

Yet another aspect provides a plant cell transfected with heterologous nucleic acid encoding one or more nematode esophageal gland cell proteins, wherein the heterologous nucleic acid is expressed in an amount sufficient to stimulate, enhance, or promote root growth or development.

The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OFTHE FIGURES

Figure 1A shows A. thaliana expressing 16D10 dsRNA inoculated with M. incognita. Note that no root knot disease (galls) on roots of A. thaliana expressing 16D10 dsRNA.

Figure 1B shows control plants inoculated with M. incognita.

Figure 2 shows a photograph of a transgenic A. thaliana plant expressing 16D10 and having enhanced root growth compared to a control plant (empty vector).

Figure 3 shows a bar graph indicating enhanced root growth of four transgenic Arabidopsis T2 homozygous lines L7.L10, L11, L17 compared to control lines (L2, L3).

Figure 4 shows RT-PCR analysis of 16D10 dsRNA (RNA1 and RNA2) treated second-stage juveniles of root-knot nematode showing a significant reduction of transcripts of parasitism gene 16D10 inthe treated nematodes. Resorcinol (Res) was used to help stimulate uptake ofthe dsRNA. No reduction of transcripts with dsRNA or res alone. Mi-act -internal transcript control.

Figure 5 shows a photograph of a gel indicating that RNAi directed to 8H1 1 ΟΓ31Η06 down-regulates expression of parasitism genes 8H1 1 or 3 1H06 in nematodes. 6 WO 2006/044480 PCT/US2005/036711

Figure 6 shows DNA blot hybridization of restriction endonuclease- digested genomic DNA from four Meloidogyne species with a DIG-labeled 16D10 probe. Mi, M. incognita; Mj, M.javanica; Ma, M. arenaria; Mh, M. hapla. E, EcoRI; B, SamHI. M, 80 ng DIG-labeled molecular weight marker in kb.

Figure 7 shows a bar graph indicating reproduction (eggs per gram root) of four Meloidogyne species (Mi, M. incognita; Mj, M.javanica; Ma, M. arenaria; Mh, M. hapla) on transgenic A thaliana expressing 16D10 dsRNA was decreased compared with control plants.

DETAILED DESCRIPTION 1. Definitions

Before explaining the various embodiments ofthe disclosure, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description. Other embodiments can be practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Throughout this disclosure, various publications, patents and published patent specifications are referenced. Where permissible, the disclosures of these publications, patents and published patent specifications are hereby incorporated by reference in their entirety into the present disclosure to more fully describe the state of the art. Unless otherwise indicated, the disclosure encompasses conventional techniques of plant breeding, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition (2001); Current Protocols in Molecular Biology [(F. M.Ausubel, etal. eds., (1987)]; Plant Breeding: Principles and Prospects (Plant Breeding, Vol 1) M. D. Hayward, N. O. Bosemark, I. Romagosa; Chapman & Hall, (1993.); Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds. (1995) CURRENT 7 WO 2006/044480 PCT/US2005/036711

Protocols in Protein Science (John Wiley & Sons, Inc.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J.

Macpherson, B. D. Hames and G. R. Taylor eds. (1995)], Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture [R. t.

Freshney, ed. (1987)].

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Lewin, Genes VH, published by Oxford University Press, 2000; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Wiley-Interscience., 1999; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology, a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; Ausubel etal. (1987) Current Protocols in Molecular Biology, Green Publishing; Sambrook and Russell. (2001 ) Molecular Cloning: A Laboratory Manual 3rd. edition.·

In order to facilitate understanding ofthe disclosure, the following definitions are provided:

To "alter" the expression of a target gene in a plant cell means that the level of expression of the target gene in a plant ceil after applying a method of the present invention is different from its expression in the cell before applying the method. To alter gene expression preferably means that the expression ofthe target gene in the plant is reduced, preferably strongly reduced, more preferably the expression ofthe gene is not detectable. The alteration ofthe expression of an essential gene may result in a knockout mutant phenotype in plant cells or plants derived therefrom. Alternatively, altered expression can included upregulating expression of plant genes. "Antisense RNA" is an RNA strand having a sequence complementary to a target gene mRNA, and thought to induce RNAi by binding to the target gene mRNA. "Sense RNA" has a sequence complementary to the antisense RNA, and annealed to its complementary antisense RNA to form siRNA. These antisense and sense RNAs have been conventionally synthesized with an RNA synthesizer. In the present invention, these RNAs are intracellularA expressed from DNAs coding for 8 WO 2006/044480

PCT/US200S/03671I antisense and sense RNAs (antisense and sense code DNAs) respectively using the siRNA expression system.

The term "biological sample" refers to a body sample from any animal, such as a mammal, for example, a human. The biological sample can be obtained from vascular, diabetic, or cancer patients, for example. A biological sample can be biological fluids such as serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, and tissue culture medium, as well as tissue extracts such as homogenized tissue, cellular extracts, or whole cells or tissue. The biological sample can be, for example, serum, plasma, or urine.

As used herein, "buffer" refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components.

When referring to expression, "control sequences" means DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and the like. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

The term "cell" refers to a membrane-bound biological unit capable of replication or division.

The term "construct" refers to a recombinant genetic molecule comprising one or more isolated polynucleotide sequences ofthe invention.

Genetic constructs used for transgene expression, in a host organism comprise in the 5'-3' direction, a promoter sequence; a sequence encoding an inhibitory nucleic acid disclosed herein; and a termination sequence. The open reading frame may be orientated in either a sense or anti-sense direction. The construct may also comprise selectable marker gene(s) and other regulatory elements for expression.

As used herein, the term "control element" or "regulatory element" are used interchangably herein to mean sequences positioned within or adjacent to a promoter sequence so as to influence promoter activity. Control 9 WO 2006/044480 PCT/US2005/036711 elements may be positive or negative control elements. Positive control elements require binding of a regulatory element for initiation of transcription. Many such positive and negative control elements are known. Where heterologous control elements are added to promoters to alter promoter activity as described herein, they are positioned within or adjacent the promoter sequence so as to aid the promoter's regulated activity in expressing an operationally linked polynucleotide sequence.

The term ’'heterologous" refers to elements occurring where they are not normally found. For example, a promoter may be linked to a heterologous nucleic acid sequence, e.g., a sequence that is not normally found operably linked to the promoter. When used herein to describe a promoter element, heterologous means a promoter element that differs from that normally found in the native promoter, either in sequence, species, or number. For example, a heterologous control element in a promoter sequence may be a control/regulatory element of a different, promoter added to enhance promoter control, or an additional control element ofthe same promoter.

As used herein, the term "homologues" is generic to "orthologues" and "paralogues".

The term "host plant" refers to a plant subject to nematode disease.

As used herein, the phrase "induce expression" means to increase the amount or rate of transcription and/or translation from specific genes by exposure of the cells containing such genes to an effector or inducer reagent or condition.

An "inducer" is a chemical or physical agent which, when applied to a population of cells, will increase the amount of transcription from specific genes. These are usually small molecules whose effects are specific to particular operons or groups of genes, and can include sugars, phosphate, alcohol, metal ions, hormones, heat, cold, and the like. For example, isopropyl (beta)-D-thiogalactopyranoside (IPTG) and lactose are inducers of the tad I promoter, and L-arabinose is a suitable inducer ofthe arabinose promoter. 10 WO 20(16/044480 PCT/US2005/036711

The term "isolated," when used to describe the various compositions disclosed herein, means a substance that has been identified and separated and/or recovered from a component of its natural environment. For example an isolated polypeptide or polynucleotide is free of association with at least one component with which it is naturally associated. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide or polynucleotide and may include enzymes, and other proteinaceous or nonproteinaceous solutes. An isolated substance includes the substance in situ within recombinant cells. Ordinarily, however, an isolated substance will be prepared by at least one purification step.

An "isolated" nucleic acid molecule or polynucleotide is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source. The isolated nucleic can be, for example, free of association with all components with which it is naturally associated. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. "IPTG" is the compound "isopropyl (beta)-D- thiogalactopyranoside", and is used herein as an inducer of lac operon. IPTG binds to a lac repressor effecting a conformational change in the iac repressor that results in dissociation ofthe lac repressor from the lac operator. With the lac repressor unbound, an operably linked promoter is activated and downstream genes are transcribed.

The term 7ac operator" refers to a nucleic acid sequence that can be bound by a lac repressor, lad, as described, for example, in Jacob etal., 1961 , J. Mol. Biol., 3: 318-356. A promoter is not activated when the lac repressor is bound to the lac operator. When the lac repressor is induced to dissociate from the operator, the promoter is activated.

The term "leader sequence" refers to a nucleic acid sequence positioned upstream of a coding sequence of interest. Leader sequences described herein contain specific sequences known to bind efficiently to 11 WO 20(16/04448(1 PCT/US2005/036711 ribosomes, thus delivering a greater efficiency of translation initiation of some polynucleotides.

As used herein, the term "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. The mammal can be, for example, human.

The term "nematode esophageal glands or nematode esophageal gland cell" refers to three large, transcriptionally active gland cells, one dorsal and two subventral, located in the esophagus of a nematode and that are the principal sources of secretions (parasitism proteins) involved in infection and parasitism of plants by plant-parasitic nematodes in the orders Tylenchida and Aphelenchida. A nucleic acid sequence or polynucleotide is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader Is operably linked to DNA for a polypeptide rf it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably (inked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

The term "orthologues" refers to separate occurrences of the same gene in multiple species. The separate occurrences have similar, albeit nonidentical, amino acid sequences, the degree of sequence similarity depending, in part, upon the evolutionary distance of the species from a common ancestor having the same gene.

As used herein, the term "paralogues" indicates separate occurrences of a gene in one species. The separate occurrences have similar, albeit 12 WO 20(16/044480 PCT/US2OO5/O36713 nonidentical, amino acid sequences, the degree of sequence similarity depending, in part, upon the evolutionary distance from the gene duplication event giving rise to the separate occurrences.

The term "parasitism proteins, parasitism polypeptides, esophageal polypeptides, or nematode esophageal gland cell secretory polypeptide" refers to the principal molecules involved in nematode parasitism of plants; products of parasitism genes expressed in plant-parasitic nematode esophageal gland cells and injected through their stylet into host tissues to mediate parasitism of plants. "Percent (%) nucleic acid sequence identity" is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W/Z, where W is the number of nucleotides scored as identical matches by the sequence alignment program in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will he appreciated that where the length of nucleic acid sequence C is not equal to the length of 13 WO 2006/044480 PCT/US2005/036711 nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.

The term "plant" is used in it broadest sense. It includes, but is not limited to, any species of woody, ornamental or decorative, crop or cereal, fruit or vegetable plant, and photosynthetic green algae (e.g., Chlamydomonas reinhardtii). It also refers to a plurality of plant cells that are largely differentiated into a structure that is present at any stage of a plant's development Such structures include, but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc. The term "plant tissue" includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture. The term "plant part" as used herein refers to a plant structure, a plant organ, or a plant tissue. A non-naturally occurring plant refers to a plant that does not occur in nature without human intervention. Non-naturally occurring plants include transgenic plants and plants produced by non-transgenic means such as plant breeding.

The term "plant cell" refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.

The term "plant cell culture" refers to cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.

The term "plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant. A "plant organ" refers to a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo. 14 WO 2006/044480 PCT/US2005/036713 "Plant tissue" refers to a group of plant cells organized into a structural and functional unit. Any tissue of a plant whether in a plant or in culture is included. This term ^includes, but is not limited to, whole plants, plant organs, plant seeds,, tissue culture and any groups of. plant cells organized into structural and/or functional units. The use ofthis term in conjunction with, or in the absence of, any specific type of.planbtissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue. "Plasmids" are designated by a lower case "p" preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be-apparent to the ordinarily skilled artisan.

As used herein,· "polypeptide" refers generally to peptides and proteins.having more than about ten amino acids. The polypeptides can be "exogenous," meaning that they are "heterologous," i.e., foreign to the host cell being utilized^such as* human polypeptide produced by a bacterial cell. "Primate" is. construed to mean any of an order of* mammals,,; comprising humans, apes, monkeys, and related forms, such as lemurs and tarsiers. ., .

The term "promoter" refers to a regulatory nucleic acid sequence, typically located upstream (51) of a gene or protein coding sequence that, in conjunction with various elements, is responsible for regulating the expression of the gene or protein coding sequence. The promoters suitable for use in the constructs of this disclosure are functional in plants and in host organisms used for expressing the inventive polynucleotides. Many plant promoters are publicly known. These include constitutive promoters, inducible promoters, tissue- and cell-specific promoters and developmentaliy-regulated promoters. Exemplary promoters and fusion promoters are described, e.g., in U.S. Pat. No. 6,717,034, which is herein incorporated by reference in its entirety. 15 WO 2((06/044480 PCT/US2005/03671.1 "Purifying" means increasing the degree of purity of a substance in a composition by removing (completely or partially) at least one contaminant from the composition. A "purification step" may be part of an overall purification process resulting in an "essentially pure" composition. An essentially pure composition contains at least about 90% by weight of the substance of interest, based on total weight of the composition, and can contain at least about 95% by weight.

The term "regulatory element" or "control element" refers to DNA sequences controlling initiation of transcription. Examples of control or regulatory elements include, but are not limited to, a TATA box, operators, enhancers, and the like. Regulatory or control elements include negative control elements and positive control elements. A negative control element is one that is removed for activation. Many such negative control elements are known, for example operator/repressor systems. For example, binding of IPTG to the lac repressor dissociates from the lac operator to activate and permit transcription. Other negative elements include the E. colt trp and lambda systems. A positive control element is one that is added for activation. Many such positive control elements are known.

Promoters naturally containing both positive and negative regulatory elements are rare. The metE promoter is one example. See, for example, Neidhardt, Ed., 1996, Escherishia coli and Salmonella, Second Ed., pages 1300-1309. Descriptions of known positive and negative control elements can be found, for example, in this reference. Positioning of a positive or negative control element within or adjacent to the promoter to achieve added regulation ofthe promoter is known, and is described, for example, in Escherishia coii and Salmonella (Supra) at pages 1232-1245.

Small RNA molecules are single stranded or double stranded RNA molecules generally less than 200 nucleotides in length. Such molecules are generally less than 100 nucleotides and usually vary from 10 to 100 nucleotides in length. In a preferred format, small RNA molecules have 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. Small RNAs include microRNAs (miRNA) and small interfering 16 WO 2006/044480

PCT/US2005/03671I RNAs (siRNAs). MiRNAs are produced by the cleavage of short stem-loop precursors by Dicer-like enzymes; whereas, siRNAs are produced by the cleavage of long double-stranded RNA molecules. MiRNAs are single-stranded, whereas siRNAs are double-stranded.

The term "siRNA" means a small interfering RNA that is a short-length double-stranded RNA that is not toxic. Generally, there is no particular limitation in the length of siRNA as long as it does not show toxicity. "siRNAs" can be, for example, 15 to 49 bp, preferably 15 to 35 bp, and more preferably 21 to 30 bp long. Alternatively, the double-stranded RNA portion of a final transcription product of siRNA to be expressed can be, for example, 15 to 49 bp, preferably 15 to 35 bp, and more preferably 21 to 30 bp long. The double-stranded RNA portions of siRNAs in which two RNA strands pair up are not limited to the completely paired ones, and may contain nonpairing portions due to mismatch (the corresponding nucleotides are not complementary), bulge (lacking in the corresponding complementary nucleotide on one strand), and the like. Nonpairing portions can be contained to the extent that they do not interfere with siRNA formation. The "bulge" used herein preferably comprise 1 to 2 nonpairing nucleotides, and the double-stranded RNA region of siRNAs in which two RNA strands pair up contains preferably 1 to 7, more preferably 1 to 5 bulges. In addition, the "mismatch" used herein is contained in the double-stranded RNA region of siRNAs in which two RNA strands pair up, preferably 1 to 7, more preferably 1 to 5, in number. In a preferable mismatch, one ofthe nucleotides is guanine, and the other is uracil. Such a mismatch is due to a mutation from C to T, G to A, or mixtures thereof in DNA coding for sense RNA, but not particularly limited to them. Furthermore, in the present invention, the double-stranded RNA region of siRNAs in which two RNA strands pair up may contain both bulge and mismatched, which sum up to, preferably 1 to 7, more preferably 1 to 5 in number.

The terminal structure of siRNA may be either blunt or cohesive (overhanging) as long as siRNA can silence, reduce, or inhibit the target gene expression due to its RNAi effect. The cohesive (overhanging) end 17 WO 2006/044480 PCT/U S2005/036711 structure is not limited only to the 3' overhang, and the 51 overhanging structure may be included as long as it is capable of inducing the RNAi effect. In addition, the number of overhanging nucleotide is not limited to the already reported 2 or 3, but can be any numbers as long as the overhang is capable of inducing the RNAi effect. For example, the overhang consists of 1 to 8, preferably 2 to 4 nucleotides. Herein, the total length of siRNA having cohesive end structure is expressed as the sum of the length of the paired double-stranded portion and that of a pair comprising overhanging singlestrands at both ends. For example, in the case of 19 bp double-stranded RNA portion with 4 nucleotide overhangs at both ends, the total length is expressed as 23 bp. Furthermore, since this overhanging sequence has low specificity to a target gene, it is not necessarily complementary (antisense) or identical (sense) to the target gene sequence. Furthermore, as long as siRNA is able to maintain its gene silencing effect on the target gene, siRNA may contain a low molecular weight RNA (which may be a natural RNA molecule such as tRNA, rRNA or viral RNA, or an artificial RNA molecule), for example, in the overhanging portion at its one end.

In addition, the terminal structure of the "siRNA" is not necessarily the cut off structure at both ends as described above, and may have a stem-loop structure in which ends of one side of double-stranded RNA are connected by a linker RNA. The length of the double-stranded RNA region (stem-loop portion) can be, for example, 15 to 49 bp, preferably 15 to 35 bp, and more preferably 21 to 30 bp long. Alternatively, the length ofthe double-stranded RNA region that is a final transcription product of siRNAs to be expressed is, for example, 15 to 49 bp, preferably 15 to 35 bp, and more preferably 21 to 30 bp long. Furthermore, there is no particular limitation in the length ofthe linker as long as it has a length so as not to hinder the pairing of the stem portion. For example, for stable pairing of the stem portion and suppression of the recombination between DNAs coding for the portion, the linker portion may have a clover-leaf tRNA structure. Even though the linker has a length that hinders pairing of the stem portion, it is possible, for example, to construct the linker portion to include introns so that the introns are excised 18 WO 2006/044480 PCT/US2005/036711 during processing of precursor RNA into mature RNA, thereby allowing pairing ofthe stem portion. In the case of a stem-loop siRNA, either end {head or tail) of RNA with no loop structure may have a low molecular weight RNA. As described above, this low molecular weight RNA may be a natural RNA molecule such as tRNA, rRNA or viral RNA, or an artificial RNA molecule. "Signal peptide" refers to a short (15-60 amino acids long) peptide chain that directs the post translational transport of a protein; usually directs the peptide to the secretory pathway of the cell. "Transformed," "transgenic," "transfected" and "recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome ofthe host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A "non-transformed," "non-transgenic," or "non-recombinant" host refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule. A "transformed cell" refers to a cell into which has been introduced a nucleic acid molecule, for example by molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, plant or animal cell, including transfection with viral vectors, transformation by

Agrobacterium, with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration and includes transient as well as stable transformants.

The term "transgenic plant" refers to a plant or tree that contains recombinant genetic material not normally found in plants or trees of this type and which has been introduced into the plant in question (or Into progenitors of the plant) by human manipulation. Thus, a plant that is grown 19 WO 2006/044480 PCT/US2005/036711 from a plant cel! into which recombinant DNA is introduced by transformation is a transgenic plant, as are all offspring of that plant that contain the introduced transgene (whether produced sexually or asexually). It is understood that the term transgenic plant encompasses the entire plant or tree and parts ofthe plant or tree, for instance grains, seeds, flowers, leaves, roots, fruit, pollen, stems etc.

The term "translation initiation enhancer sequence", as used herein, refers to a nucleic acid sequence that can determining a site and efficiency of initiation of translation of a gene (See, for example, McCarthy et al., 1990, Trends in Genetics, 6:78-85). A translation initiation enhancer sequence can extend to include sequences 5' and 31tothe ribosome binding site. The ribosome binding site is defined to include, minimally, the Shine-Dalgarno region and the start codon, in addition to any bases in between. In addition, the translation initiation enhancer sequence can include an untranslated leader or the end of an upstream cistron, and thus a translational stop codon. See, for example, US Patent No. 5,840,523.

The term "vector" refers to a nucleic acid molecule which is used to introduce a polynucleotide sequence into a host cell, thereby producing a transformed host cell. A "vector" may comprise genetic material in addition to the above-described genetic construct, e.g., one or more nucleic acid sequences that permit it to replicate in one or more host cells, such as origin(s) of replication, selectable marker genes and other genetic elements known in the art (e.g., sequences for integrating the genetic material into the genome of the host cell, and so on). 2. Exemplary Embodiments Nematode Resistant Transgenic Plants

It has been discovered that interrupting the feeding cycle of nematodes by down-regulating one or more nematode parasitism genes is an effective method for reducing, preventing, or treating nematode disease in plants. Nematode parasitism genes refers to genes expressed in the esophageal gland cells encoding for secretory proteins exported from the 20 WO 2006/044480 PCT/US2005/036711 gland cell to be released through the nematode's stylet into host tissue. In particular, it has been discovered that interfering with the expression of proteins secreted by nematodes related to the formation of specialized feeding cells in host plants is an effective method for reducing, treating, or preventing nematode disease in plants. Representative parasitism genes encoding secreted proteins that can be targeted, for example with inhibitory RNA include, include but are not limited to those genes listed in Table 2, or a fragment thereof.

Nematode disease results in substantial losses of valuable crops. Root-knot nematodes, Meloidogyne species, are among nature's most successful parasites. They parasitize more than 2,000 plant species from diverse plant families and represent a tremendous threat to crop production world-wide. These biotrophic pathogens have evolved highly specialized and complex feeding relationships with their hosts. A successful nematode-host interaction requires molecular signals from the parasite to modify, directly or indirectly, plant root cells into elaborate feeding cells, called giant-cells, which are the sole source of nutrients needed for nematode development and reproduction. Plant-parasitic nematodes release proteinaceous secretions through a hollow protrusibfe stylet into plant cells when feeding. These secretions, collectively called the parasitome are encoded by parasitism genes expressed in large and transcriptionally active esophageal gland cells (Davis, E. L., R. Allen, and R.S. Hussey. 1994. Developmental expression of esophageal gland antigens and their detection in stylet secretions of Meloidogyne incognita. Fundam. Appl. Nematol. 17:255-262.; Hussey, R. S., E. L. Davis, and T. J. Baum. 2002. Secrets in secretions: genes that control nematode parasitism of plants. Braz. J, Plant Physiol. 14:183-194.). The profound cellular modifications induced by Meloidogyne species to form the giant-cells are the result of an alteration in host root cell gene expression and phenotype that is driven by the molecular signals secreted through the nematode's stylet.

One embodiment provides a plant or cell comprising one or more inhibitory RNAs specific for one or more mRNAs of one or more nematode 21 WO 2((((6/04448(( PCT/US2005/036711 parasitism genes. For example, the present disclosure provides transgenic plants that express one or more inhibitory RNAs that down regulate nematode parasitism gene expression when the one or more inhibitory RNAs are absorbed or ingested by a nematode. The transgenic plant can be designed to express inhibitory RNA that down-regulates the target parasitism gene transcript in at least two different nematode species, for example two different RKN species. Another embodiment provides a transgenic plant that comprises inhibitory RNA that down regulates the target parasitism gene transcript in 3,4,5, 6, 7,8,9, 10, 11,12, 13, 14, 15,16, 17, 18, 19, 20, or more nematode species, for example RKN species. Thus, the present disclosure provides transgenic plants that are resistant to disease caused by multiple RKN species.

Another embodiment, provides a transgenic plant comprising inhibitory RNA specific for one or more nematode parasitism genes in an amount effective to provide the plant with resistance to all RKN species, for example those RKN species referenced in Jepson, S. B. 1987. Identification of root-knot nematodes (Meloidogyne species). C. A. B. International,

Oxford, United Kingdom. 1-265 pages, which, where permissible, is incorporated by reference in its entirety.

Another embodiment provides a transgenic plant or transgenic cell containing or expressing one or more inhibitory nucleic acids specific for at least a portion of a nucleic acid encoding one or more secretory polypeptides of a parasitic nematode. The inhibitory nucleic acid is typically a small inhibitory RNA or microRNA that is specific for mRNA encoding a nematode esophageal gland cell protein or polypeptide. It will be appreciated by one of skill in the art that the inhibitory nucleic acid can be RNA, DNA, or a combination thereof. Additionally, the inhibitory nucleic acid may be single or multi-stranded and may be anti-sense or enzymatic. In one embodiment, the inhibitory nucleic acid interferes, inhibits, or reduces the translation of a target mRNA. For example, the inhibitory nucleic acid can bind to a target mRNA and induce or promote the degradation of the target mRNA or physically prevent the cellular translational machinery from translating the 22 WO 2006/044480 PCT/US2005/036711 target mRNA into a functional protein. Inhibition of the secretory polypeptide can be compared to controls, for example plants or cells that do not contain or express the inhibitory nucleic acid. A "control" refers to a sample of material which is known to be identical to a sample containing the disclosed inhibitory nucleic acid in every regard, except that the control sample does not contain or express the inhibitory nucleic acid.

The term "esophageal gland cell protein or polypeptide" refers to a secretory polypeptide encoded by a nematode parasitism gene. In one embodiment, the esophageal gland cell protein or polypeptide to be down-regulated generally is a secreted protein that modulates expression of at least one host plant gene. Exemplary nematode polypeptides that are down-regulated in the disclosed compositions and methods include, but are not limited to polypeptides orfragments thereof encoded by SEQ ID NOs 1, 2, or 5-51 .orfragments thereof. The secretory polypeptide can increase or decrease expression of host plant genes either directly or indirectly. For example, direct modulation can occur when the esophageal gland cell protein or polypeptide binds to a host plant nucleic acid, including genomic DNA, RNA, and mRNA. Indirect modulation can occur for example when the polypeptide binds with one or more other proteins or factors to form a complex. The complex can then bind to a host plant nucleic acid to either promote or suppress transcription or translation. Down-regulation ofthe secretory protein alleviates or reduces at least one symptom associated with nematode disease. Exemplary symptoms of nematode disease include, but are not limited to the formation of galls, giant cells, lesions, stunting, nutrient and water deficiencies, .dieback, and numbers of nematodes infecting a plant. Levels of reduction or inhibition of nematode disease in transgenic plants or cells can be compared to levels of nematode disease in control plants or cells. In one embodiment, the inhibitory nucleic acid reduces, inhibits, alleviates, treats or prevents nematode disease.

In another embodiment, the esophageal gland cell protein or polypeptide to be down-regulated is encoded by a parasitism gene involved in the formation of a giant cell. In still other embodiments, the targeted 23 WO 2006/044480 PCT/VS2005/036711 parasitism gene encodes a polypeptide or nucleic acid involved in nematode migration through root tissue, alters cell metabolism, elicits signal transduction in the recipient cell, or forms a feeding tube that enables the nematode to feed from the giant-cells. Additionally, the esophageal gland cell protein or polypeptide can cause cell wall modifications and potentially interact with signal transduction receptors in the extracellular space, influence cellular metabolism, cell cycle, selective protein degradation, localized defense response, and regulatory activity within the plant cell nucleus.

Exemplary plant genes that are modulated by the esophageal gland cell protein or polypeptide include, but are not limited to genes involved in the formation of specialized nematode feeding cells also known as giant cells. For example, nematode parasitism gene 16D10 encodes a protein that binds to a scarecrowlike transcription regulator. Representative plant genes that can be modulated by nematode esophageal gland cell polypeptides include, but are notlimited to WUN1, POX, CAT, GST, Mia-1, Mia-2, Mia-3, Mia-4, CHS1-CHS3, LOX, Chitinase, Trypsin inhibitor, Miraculin, HMGR, TSW12, LEAD, LEMM19, 06-19, C27-45, TASU, UBC DB#103, RPE, ISDGh, IPPP, LPPL, mUCp, endomembrane protein, 20s proteasome, DAP decarboxylase, GRP, ENOD40, A TAO 1 or combinations thereof (Gheysen, G. and Fenoll, C. 2002. Annual Review of Phytopathology 40:191 .which, where permissible, is incorporated by reference in its entirety). Generally, the plant gene is directly or indirectly involved in root cell growth, root cell division or the production of specific nutrients ingested by the parasitic nematode. The gene can be one expressed in a root celt or any other cell of the plant.

In one embodiment, expression of a targeted nematode secretory protein is reduced, inhibited, or blocked, as compared to a control, when the inhibitory nucleic acid is delivered to the nematode'. Delivery of the inhibitory nucleic acid can be achieved, for example, when the nematode comes into contact with the inhibitory nucleic acid as the nematode feeds on the transgenic plant or cell. The nematode can ingest the inhibitory nucleic acid 24 WO 2006/044480 PCT/US2005/036711 during feeding, or the nucleic acid can be transported across a cellular membrane of the nematode by active transport or passive diffusion. It will be appreciated that the inhibitory nucleic acid can be delivered to the nematode in combination or alternation with an agent that induces or promotes the uptake ofthe inhibitory nucleic acid by the nematode. An exemplary inducing agent includes, but is not limited to resorcinol (3-hydroxyphenol).

In one embodiment, the transgenic plant or transgenic cell expresses the inhibitory nucleic in an amount effective to modulate the expression of a nematode esophageal gland cell polypeptide or protein in a nematode when the inhibitory nucleic acid is delivered to the nematode. Levels of expression of the inhibitory nucleic acid in a transgenic plant or cell can be controlled using methods known in the art, for example using vectors with strong promoters or constitutively active promoters, high copy number vectors, etc. The plant or cell can be stably or transiently transfected.

An exemplary parasitic nematode includes, but is not limited to members of Meloidogyne spp. also referred to as root-knot nematodes. Representative species include, but are not limited to M. arenaria, M. incognita, M. javanica, M. hapla, M. chitwoodi and M. naasi.

Representative phylogenetic families of host plants include Acanthaceae, Aceraceae, Actinidiaceae, Agavaceae, Aizoaceae, Amaranthaceae, Annonaceae, Apiaceae, Apocynaceae, Araceae, Arafiaceae, Arecaceae, Aristolochiaceae, Balsaminaceae, Barringtoniaceae, Baseiiaceae, Berberidaceae, Betuiaceae, Bignoniaceae, Bixaceae, Bombacaceae, Boraginaceae, Buxaceae, Byttneriaceae, Cactaceae, Caesa/piniaceae, Cannaceae, Capparaceae, Caprifo/iaceae, Caricaceae, Caryophyitaceae, Casuarinaceae, Casuarinaceae, Ceiastraceae, Chenopodiaceae, Chenopodiaceae, Chloranthaceae, Commelinaceae, Convolvulaceae, Cornaceae, Corylaceae, Crassulaceae, Cucurbitaceae, Cupressaceae, Cyatheaceae, Cyperaceae, Datiscaceae, Dilleniaceae, Dioscoreaceae, Dipsacaceae, Ebenaceae, Ericaceae, Euphorbiaceae, Fabaceae, Flacourtiaceae, Fumariaceae, Gentianaceae, Geraniaceae, Gesneriaceae, Ginkgoaceae, Goodeniaceae, Guttiferae, Haemodoraceae, 25 WO 2006/(144480 PCT/US2005/036711

Hamamelidaceae, Heliconiaceae, Hydrophyltaceae, Hypericaceae,

Iridaceae, Juglandaceae, Juncaceae, Labiatae, Lamiaceae, Lauraceae, Lifiaceae, Linaceae, Lobeliaceae, Loganiaceae, Lythraceae, Magnoliaceae, Malpighiaceae, Malvaceae, Marantaceae, Melastomataceae, Meliaceae, Menispermaceae, Mimosaceae, Moraceae, Musaceae, Myoporaceae, Myricaceae, Myristicaceae, Myrtaceae, Nyctaginaceae, Oieaceae, Onagraceae, Orchidaceae, Othnaceae, Oxalidaceae, Paeoniaceae, Pandanaceae, Papaveraceae, Pedaliaceae, Phytoiaccaceae, Pinaceae, Piperaceae, Pittosporaceae, Plantaginaceae, Platanaceae,

Plumbaginaceae, Poaceae, Podostemaceae, Polemoniaceae,

Polygaiaceae, Portulacaceae, Primuiaceae, Proteaceae, Punicaceae, Ranunculaceae, Resedaceae, Rhamnaceae, Rosaceae, Rubiaceae, Rutaceae, Saiicaceae, Santalaceae, Sapindaceae, Sarraceniaceae, Saxifragaceae, Scrophuiariaceae, Smilacaceae, Solanaceae, Sterculiaceae, Styracaceae, Tamaricaceae, Taxodiaceae, Tetragoniaceae, Theaceae, Theophrastaceae, Thymelaeaceae, Tiliaceae, Tropaeolaceae, Turneraceae, Typhaceae, Ulmaceae, Urticaceae, Valerianaceae, Verbenaceae, Violaceae, Vitaceae, Zamiaceae, Zingiberaceae, or Zygophy/laceae.

Common names of host plants that can be transfected with an inhibitory nucleic acid according the present disclosure include, but are not limited to tomato, eggplant, potato, melon, cucumber, carrot, lettuce, artichoke, celery, cucurbits {melon, watermelon, etc.), barley, corn, peanut, soybean, sugar beet, cotton, cowpea, beans, alfalfa, tobacco, citrus, clover, pepper, grape, coffee, olive, or tea.

It will be appreciated by one of skill in the art that the present disclosure encompasses any ofthe fifty or more known root-knot nematode species.

Another embodiment provides a composition having an inhibitory nucleic acid specific for an mRNA or fragment thereof encoding a polypeptide encoded by one or more of SEQ ID NOs. 1,2 or 5-51 or a fragment or homologues thereof, in an amount sufficient to inhibit expression of the polypeptide encoded by one or more of SEQ ID NOs 1,2 or 5-51 or 26 WO 2006/044480 PCT/US2005/036711 homologues thereof when delivered to a nematode, for example when the nematode is feeding on a plant or cell expressing or containing the inhibitory nucleic acid. The composition can contain one or more nematicides, pesticides, fungicides, or combinations thereof. Representative nematicides include, but are not limited to chloropicrin, methyl bromide, 1,3-dichloropropene, sodium methyl dithiocarbamate, sodium tetrathiocarbonate; and carbamates such as 2-methyl-2-(methylthio)propionaldehyde O-methylcarbamoyloxime (aldicarb), 2,3-Dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate (carbofuran), methyl 2-(dimethylamino)-N- [[(methylamino)carbonyl]oxy]-2-oxoethanimidothioate (oxamyl), 2-methyl-2-(methylsulfonyl)propanal O-[(methylamino)carbonyl]oxime (aldoxycarb), 0,0-diethyl O-[4-(methylsulfinyl)phenyl] phosphorothioate (fensulfothion), O-Ethyl S.S-dipropylphosphorodithioate (ethoprop), and Ethyl-3-methyl-4-(methylthio)phenyl(1-methylethyi)phosphoramidate (phenamiphos).

Another embodiment provides a cell containing a nucleic acid encoding an inhibitory nucleic acid specific for an mRNA or fragment thereof, wherein the mRNA encodes a esophageal gland cell protein or polypeptide that directly or indirectly modulates: root cell gene expression, nematode migration through root tissue, cell metabolism, signal transduction, or is involved in the formation of a feeding tube that enables the nematode to feed from the giant-cells of at least one plant gene involved in the formation of a giant cell. Additionally, the esophageal gland cell protein or polypeptide or esophageal polypeptide can cause cell wall modifications and potentially interact with signal transduction receptors in the extracellular space, influence cellular metabolism, cell cycle, selective protein degradation, localized defense response, and regulatory activity within the plant cell nucleus. The cell can be prokaryotic or eukaryotic, and generally is a plant cell, particularly a root cell.

Still another embodiment provides a method for providing nematode resistance to a plant by contacting the plant with one or more inhibitory nucleic acids specific for one or more nematode esophageal gland cell proteins in an amount sufficient to reduce nematode disease, wherein the 27 WO 2(106/044480 PCT/US2005/036711 one or more nematode esophageal gland cell proteins modulate: gene expression ofthe plant or cell, formation of a giant cell, nematode migration through root tissue ofthe plant, cell metabolism ofthe plant, elicits signal transduction in the plant cell, or forms a feeding tube that enables the nematode to feed from giant-cells formed in the plant. One aspect provides inhibitory nucleic acids specific for esophageal gland cell proteins secreted by nematodes, in particular root knot nematodes. The inhibitory nucleic acid can be sprayed onto the plant or otherwise delivered to the plant so that the inhibitory nucleic acid comes into contact with a parasitic nematode.

Yet another embodiment provides transgenic plants or plant cells containing an inhibitory nucleic acid, for example siRNA or microRNA, that down regulates root-knot nematode esophageal gland cell proteins when delivered to a nematode feeding on the plant or plant cell. RNA interference is known in the art. See for example, Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al, International PCT Publication No. WO 01/36646; Fire, international PCT Publication No.WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; Li et al., International PCT Publication No. WO 00/44914; and Trick et al., US20040098761.

In one embodiment, the nematode is not a soybean cyst nematode.

In another embodiment, the inhibitory nucleic acid is not directly lethal to embryonic or adult nematodes or is not involved in nematode fertility, but instead inhibits the ability of the nematode to feed on or obtain nutrients from the transgenic plant or. plant cell.

In some embodiments, inhibitory double stranded RNA (dsRNA) is derived from an "exogenous template". Such a template may be all or part of a plant or nematode nucleotide sequence; it may be a DNA gene sequence ora cDNA produced from an mRNA isolated from a parasitic nematode, for example by. reverse transcriptase. When the template is all or a part of a DNA gene sequence, it is preferred if it is from one or more or all 28 WO 2006/044480 PCT/US2005/036711 exons ofthe gene. While the dsRNA is derived from an endogenous or exogenous template, there is no limitation on the manner in which it could be synthesized. For example, the siRNA can be chemically synthesized, produced by in vitro transcription; produced by digestion of long dsRNA by an RNase ill family enzyme (e.g., Dicer, RNase’ III); expressed in cells from an siRNA expression plasmid or viral vector; or expressed in cells from a PCR-derived siRNA expression cassette

SiRNA prepared in vitro is then introduced directly into cells by transfection, electroporation, or by another method. Alternatively, transfection of DNA-based vectors and cassettes that express siRNAs within the cells can be used. RNAi may be synthesized in vitro or in vivo, using manual and/or automated procedures. In vitro synthesis may be chemical or enzymatic, for example using cloned RNA polymerase (e.g., T3, T7, SP6) for transcription ofthe endogenous DNA (or cDNA) template, ora mixture of both.

In vivo, the dsRNA may be synthesised using recombinant techniques well known in the art (see e.g., Sambrook, et al., Molecular Cloning; A Laboratory Manual, Third Edition (2001), For example, bacterial cells can be transformed with an expression vector which comprises the DNA template from which the dsRNA is to be derived. Alternatively, the cells, of a plant for example, in which inhibition of gene expression is required may be transformed with an expression vector or by other means. Bidirectional transcription of one or more copies of the template may be by endogenous RNA polymerase of the transformed cell or by a cloned RNA polymerase (e.g., T3, T7, SP6) coded for by the expression vector ora different expression vector. The use and production of an expression, construct are known inthe art (see WO98/32016; U.S. Pat Nos. 5,593,874, 5,698,425, 5712,135, 5,789,214, and 5,804,693). Inhibition of gene expression may be targeted by specific transcription in an organ, tissue, or cell type; an environmental condition (e.g. temperature, chemical); and/or engineering transcription at a developmental stage or age, especially when the dsRNA Is synthesized in vivo in the plant cell for example. dsRNA may also be 29 WO 2006/044480 PCT/US2005/036711 defivered to specific tissues or cell types using known gene delivery systems. Components of these systems include the seed-specific lectin promoter and the flower specific promoter from APETALA3. These vectors are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art.

If synthesized outside the cell, the RNA may be purified prior to introduction into the cell. Purification may be by extraction with a solvent (such as phenol/chloroform) or resin, precipitation (for example in ethanol), electrophoresis, chromatography, or a combination thereof. However, purification may result in loss of dsRNA and may therefore be minimal or not carried out at all. The RNA may be dried for storage or dissolved in an aqueous solution, which may contain buffers or salts to promote annealing, and/or stabilization of the RNA strands.

Suitable dsRNA can also contain one or more modified bases, or have a modified a backbone to increase stability or for other reasons. For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. Moreover, dsRNA comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, can be used. It will be appreciated that a great variety of modifications have been made to RNA that serve many useful purposes known to those of skill in the art. The term dsRNA as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of dsRNA, provided that it is derived from an endogenous template.

The double-stranded structure may be formed by a single self-complementary RNA strand or two separate complementary RNA strands. RNA duplex formation may be initiated either inside or outside the plant cell.

The sequence of at least one strand of the dsRNA contains a region

complementary to at least a part of the target mRNA sufficient for the dsRNA

to specifically hybridize to the target mRNA. In one embodiment, the siRNA is substantially identical to.at least a portion ofthe target mRNA. "Identity", as known in the art, is the relationship between two or more polynucleotide 30 WO 2006/044480 PCT/US2005/03671] (or polypeptide) sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polynucleotide sequences, as determined by the match between strings of such sequences. Identity can be readily calculated (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed.,

Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M„ and Griffin, H. G., eds., Humana Press, N.J., 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Grtbskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide sequences, the term is well known to skilled artisans (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods commonly employed to determine identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, J., etal., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215:403 (1990)). Another software package well known in the art for carrying out this procedure is the CLUSTAL program. It compares the sequences of two polynucleotides and finds the optimal alignment by inserting spaces in either sequence as appropriate. The identity for an optimat alignment can also be calculated using a software package such as BLASTx. This program aligns the largest stretch of similar sequence and assigns a value to the fit. For any one pattern comparison several regions of similarity may be found, each having a different score. 31 WO 2006/044480 PCT/US2005/036711

One skilled in the art will appreciate that two polynucleotides of different lengths may be compared over the entire length of the longer fragment. Alternatively small regions may be compared. Normally sequences of the same length are compared for a useful comparison to be made.

In one embodiment, the inhibitory nucleic acid has 100% sequence identity with at (east a part of the target mRNA. However, inhibitory nucleic acids having 70%, 80% or greater than 90% or 95% sequence identity may be used. Thus sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence can be tolerated.

The duplex region of the RNA may have a nucleotide sequence that is capable of hybridizing with a portion ofthe target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C or 70° C hybridization for 12-16 hours; followed by washing).

While the optimum length of the dsRNA may vary according to the target gene and experimental conditions, the duplex region of the RNA may be at least 19, 20, 21-23, 25, 50, 100, 200, 300, 400 or more bases long.

Target genes, are nematode genes encoding secreted proteins, in particular secreted proteins that modulate: gene expression ofthe plant or cell, formation of a giant cell, nematode migration through root tissue of the plant, cell metabolism of the plant, elicits signal transduction in the plant cell, or forms a feeding tube that enables the nematode to feed from giant-cells formed in the plant. One aspect provides inhibitory nucleic acids specific for esophageal gland cell proteins secreted by nematodes, in particular root knot nematodes. Typically, the dsRNA or inhibitory nucleic acid is substantially identical to the whole of the target gene, i.e. the coding portion of the gene. However, the dsRNA or inhibitory nucleic acid can be substantially identical to a part of the target gene. The size of this part depends on the particular target gene and can be determined by those skilled in the art by varying the size of the dsRNA and observing whether expression of the gene has been inhibited. 32 WO 2006/044480 PCT/US200S/036713

Plants with Enhanced Root Growth

One embodiment provides a transgenic plant or transgenic cell containing or expressing one or more nucleic acids encoding one or more nematode esophageal gland cell polypeptides orfragments thereof of a parasitic nematode. Expression ofthe one or more nematode esophageal gland cell polypeptides orfragments thereof in a plant or plant cell promotes, stimulates, or enhances root growth of the transgenic plant compared to non-transgenic plants or control plants. A root includes a seminal root, adventitious root, first orderlateral root, second order laterals, etc., feeder roots primary roots, secondary roots, and coarse roots.

The nematode esophageal gland celt polypeptides orfragments used with the disclosed embodiments can increase the size of roots, the number of roots, the surface area of roots, and the overall quality of a root system. Root crops can be produced with the disclosed compositions and methods that are larger than root crops produced in the absence of the disclosed compositions and methods. Other crops, produced using the disclosed compositions and methods can be resistant to drought, erosion, or increased environmental stress. Environmental stress includes changes in climate such as rainfall, temperature, and humidity.

Exemplary nematode esophageal gland cell polypeptides or fragments thereof include, but are not limited to polypeptides encoded by SEQ ID NOs 1,2, or 5-51 .fragments thereof, or combinations thereof. The nematode esophageal gland cell·polypeptides orfragments can increase, stimulate, orenhance root growth directly or indirectly. For example, direct modulation can occur when the nematode secretory polypeptide binds to a host plant nucleic acid, including genomic DNA, RNA, and mRNA. Indirect modulation can occur for example when the polypeptide binds with one or more other proteins or factors toform a complex. The complex can then bind to a host plant nucleic acid to either promote or suppress transcription or translation.

In one embodiment, the transgenic plant or transgenic cell expresses the nematode esophageal gland cell polypeptide orfragment thereof in an 33 WO 2006/044480 PCT/US2005/036711 amount effective to stimulate, enhance or promote root growth or development. Alternatively, the nematode esophageal gland cell polypeptide or fragment thereof can be delivered directly to the plant. Levels of nematode esophageal gland cell polypeptide or fragment thereof expression in a transgenic plant or cell can be controlled using methods known in the art, for example using vectors with strong promoters or constitutively active promoters, high copy number vectors, etc. The plant or cell can be stably or transiently transfected.

An exemplary nematode includes, but is not limited to members of Meloidogyne spp. also referred to as root-knot nematodes. Representative species include, but are not limited to M. arenaria, M. incognita, M.javanica, M. hapla, and M. naasi.

Representative phylogenetic families of host plants include Acanthaceae, Aceraceae, Actinidiaceae; Agavaceae, Aizoaceae, Amaranthaceae, Annonaceae, Apiaceae, Apocynaceae, Araceae, Araiiaceae, Arecaceae, Aristolochiaceae, Baisaminaceae, Barringtoniaceae, Baseftaceae, Berberidaceae, Betulaceae, Bignoniaceae, Bixaceae, Bombacaceae, Boraginaceae, Buxaceae, Byttneriaceae, Cactaceae, Caesa/piniaceae, Cannaceae, Capparaceae, Caprifoiiaceae, Caricaceae, Caryophyllaceae, Casuarinaceae, Casuarinaceae, Celastraceae, Chenopodiaceae, Chenopodiaceae; Chloranthaceae, Commelinaceae, Convolvulaceae, Cornaceae, Corylaceae, Crassulaceae, Cucurbitaceae, Cupressaceae, Cyatheaceae, Cyperaceae, Datiscaceae, DHIeniaceae, Dioscoreaceae, Dipsacaceae, Ebenaceae, Ericaceae, Euphorbiaceae, Fabaceae, Flacourtiaceae, Fumariaceae, Gentianaceae, Geraniaceae, Gesneriaceae, Ginkgoaceae, Goodeniaceae, Guttiferae, Haemodoraceae, Hamameiidaceae, Heliconiaceae, Hydrophyllaceae, Hypericaceae,

Iridaceae, Juglandaceae, Juncaceae, Labiatae, Lamiaceae, Lauraceae, Liiiaceae, Linaceae, Lobeliaceae, Loganiaceae, Lythraceae, Magnoliaceae, Malpighiaceae, Malvaceae, Marantaceae, Meiastomataceae, Meliaceae, Menispermaceae, Mimosaceae, Moraceae, Musaceae, Myoporaceae, Myricaceae, Myristlcaceae, Myrtaceae, Nyctaginaceae, Oleaceae, 34 WO 2006/044480 PCT/US2005/036711

Onagraceae, Orchidaceae, Othnaceae, Oxalidaceae, Paeoniaceae,

Pandanaceae, Papaveraceae, Pedaliaceae, Phytoiaccaceae, Pinaceae,

Piperaceae, Pittosporaceae, Plantaginaceae, Platanaceae,

Plumbaginaceae, Poaceae, Podostemaceae, Polemoniaceae,

Polygaiaceae, Portulacaceae, Primuiaceae, Proteaceae, Punicaceae, Ranunculaceae, Resedaceae, Rhamnaceae, Rosaceae, Rubiaceae, Rutaceae, Saiicaceae, Santalaceae, Sapindaceae, Sarraceniaceae, Saxifragaceae, Scrophuiariaceae, Smilacaceae, Solanaceae, Sterculiaceae, Styracaceae, Tamaricaceae, Taxodiaceae, Tetragoniaceae, Theaceae, Theophrastaceae, Thymelaeaceae, Tiliaceae, Tropaeolaceae, Turneraceae, Typhaceae, Ulmaceae, Urticaceae, Valerianaceae, Verbenaceae, Violaceae, Vitaceae, Zamiaceae, Zingiberaceae, or Zygophy/laceae.

Common names of host plants that can be transfected with nucleic acid encoding a RKN esophageal gland cell secretory polypeptide according the present disclosure include, but are not limited to tomato, eggplant, potato, melon, cucumber, carrot, lettuce, artichoke, celery, cucurbits (melon, watermelon, etc.), barley, corn, peanut, soybean, sugar beet, cotton, cowpea, beans, alfalfa, tobacco, citrus, clover, pepper, grape, coffee, olive, or tea.

It will be appreciated by one of skill in the art that the present disclosure encompasses any nematode that secretes a protein that alters the expression of a host gene. For example, one embodiment provides a transgenic plant or cell containing a nucleic acid encoding a protein secreted by a member ot Meloidogyne spp., wherein the secreted protein stimulates, enhances, or promotes root growth or development.

Another embodiment provides a composition comprising a nucleic acid having a sequence of SEQ ID NOs. 1,2 or 5-51 ora fragment or homologues thereof. The composition stimulates, promotes, or enhances, root growth or development when delivered to a plant or plant cell.

Still another embodiment provides a composition comprising a one or more polypeptides or fragments thereof encoded by SEQ ID NOs 1,2 or 5-51 or homologues thereof when delivered to a plant or plant cell. 35 WO 2006/044480 PCT/US2005/036711

Root stimulating compositions disclosed herein can optionally contain a growth enhancer, fertilizer, one or more nemiticides, pesticides, fungicides, or combinations thereof. Representative nematicides include, but are not limited to chloropicrin, methyl bromide, 1,3-dichloropropene, sodium methyl dithiocarbamate, sodium tetrathiocarbonate; and carbamates such as 2-methyl-2-(methylthio)propionaldehyde O-methylcarbamoyloxime (aldicarb), 2,3-Dihydro-2,2-dimethyl-7:benzofuranol methylcarbamate (carbofuran), methyl 2-(dimethylamino)-N-[[(methylamino)carbonyl]oxy]-2- oxoethanimidothioate (oxamyl), 2-methyl-2-(methyisulfonyl)propanal 0-[(methylamino)carbonyljoxime (aldoxycarb), 0,0-diethyl O-[4-(methylsulfinyi)phenyl] phosphorothioate (fensulfothion), Ο-Ethyl S,S-dipropylphosphorodithioate (ethoprop), and Ethyl-3-methyl-4-(methylthio)phenyl(1-methylethyl)phosphoramidate (phenamiphos).

Another embodiment provides a cell, for example a plant cell, containing one or more nucleic acids encoding a nematode secretory polypeptide orfragment thereof wherein the nematode secretory polypeptides directly or indirectly stimulate, enhance or promote root growth or development. The cell can be prokaryotic or eukaryotic, and generally is a plant cell, particularly a root cell.

Still another embodiment provides a method for providing drought resistance to a plant by contacting the plant with one or'more nematode esophageal proteins or nucleic acids encoding nematode esophageal proteins in an amount sufficient to stimulate, promote, or enhance root development The composition can be sprayed onto the plant, applied to the soil surrounding the plant or otherwise delivered to the plant so that the composition contacts the plant.

Plant Transformation Technology DNA molecules and RNA molecules of the present disclosure are

incorporated in plant or bacterial cells using conventional recombinant DNA technology. Generally, a DNA or an RNA molecule ofthe present disclosure is comprised in a transformation vector. A large number of such vector 36 WO 20(16/044480 PCT/US2005/036711 systems known in the art may be used, such as plasmids. The components of the expression system are also modified, e.g., to increase expression of the introduced RNA fragments. For example, truncated sequences, nucleotide substitutions or other modifications may be employed. Expression systems known in the art may be used to transform virtually any plant cell under suitable conditions. A transgene comprising a DNA molecule of the present invention is preferably stably transformed and integrated into the genome ofthe host cells. Transformed cells are preferably regenerated into whole plants. Detailed description of transformation techniques are within the knowledge of those skilled in the art.

Reporter genes or selectable marker genes may be included in the expression cassette. Examples of suitable reporter genes known in the art can be found in, for example, Jefferson et al. (1991) in Plant Molecular Biology Manual, ed. Gelvin etal. (Kluwer Academic Publishers), pp. 1-33; DeWet et al. ¢1987) Mol. Cell. Biol. 7:725-737; Goff et al. (1990) EMBO J. . 9:2517-2522; Kain etal. (1995) Bio Techniques 19:650-655; and Chiu etal. (1996) Current Biology 6:325-330.

Selectable marker genes for selection of transformed cells or tissues can include genes that confer antibiotic resistance or resistance to herbicides. Examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella etal. (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella et al. (1983) Nature 303:209-21 3; Meijer et al. (1991) Plant Mol. Biol. 16:807-820); hygromycin (Waldron etal. (1985) Plant Mol. Biol. 5:103-108; Zhijian et al. (1995) Plant Science 108:219-227); streptomycin (Jones etal. (1987)

Mol: Gen. Genet 210:86-91): spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res. 5:131-137); bleomycin, (Hille et al. (1990) Plant Mol. Biol 7:171-176); sulfonamide (Guerineau etal. 1990) Plant Mol. Biol. 15:127- . 136); bromoxynil (Stalker, et al. (1988) Science 242:41 9423); glyphosate (Shaw et al. (1986) Science 233:478481 ); phosphinothricin (DeBlock et al. (1987) EMBO J. 6:2513-2518). 37 WO 2006/044480 PCT/US2005/036711

Other genes that could serve utility in the recovery of transgenic events but might not be required in the final product would include, but are not limited to, examples such as GUS (b-glucoronidase; Jefferson (1987) Plant Mol. Biol. Rep. 5:387), GFP (green florescence protein; Chalfie et ai. (1994) Science 263:802), luciferase (Riggs etal. (1987) Nucleic Acids Res. 15(19):81 15 and Luehrsen etal. (1992) Methods Enzymol. 216:397-414) and the maize genes encoding for anthocyanin production (Ludwig et al. (1990) Science 247:449).

The expression cassette comprising a promoter sequence operably linked to a heterologous nucleotide sequence of interest can be used to transform any plant. In this manner, genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained.

Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway etal. (1986) Biotechniques 4:320-334), electroporation (Riggs etal. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (Townsend etal., U.S. Pat. No. 5,563,055; Zhao etal. WO US98/01268), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford etal., U.S. Pat. No. 4,945,050; Tomes etal. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell,

Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe etal. (1988) Biotechnology 6:923-. 926). Also see Weissinger etal. (1988) Ann. Rev. Genet. 22:421-477;

Sanford et al. (1987) Particulate Science and technology 5:27-37 (onion); Christou etal. (1988) Plant Physiol. 87:671-674 (soybean); McCabe etal. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) in Vitro Cell Dev. Biol. 27P: 175-1 82 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Dafta et al. (1990) Biotechnology 8:736-740 38 WO 2006/044480 PCT/US2005/036711 (rice); Klein etal. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising etal., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) "Direct DNATransfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture; Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; Bowen etal., U.S. Pat. No. 5,736,369 (cereals); Bytebier etal. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liiiaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and.Kaeppler etal. (1992) Theor. Appl. Genet 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li etal. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference in their entirety.

The cells that have been transformed may be grown into plants in accordance with conventional techniques. See, for example; McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either'pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two. or more generations may be grown to ensure that constitutive expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure constitutive expression , of the desired phenotypic characteristic has been achieved.

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application ofthe chemical induces 39 WO 2006/(144480 PCT/US2005/036711 gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize 1n2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid. Other chemicai-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena etal. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,1 56), herein incorporated by reference in their entirety.

Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CAMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy etal. (1990) Plant Cell 2:163-1 71); ubiquitin (Christensen etal. (1989) Plant.Mol. Biol. 12:619-632 and Christensen etal. ¢1992) Plant Mol. Biol. 18:675-689); pEMU (Last et ai. (1991 )Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142.

Where low level expression is desired, weak promoters may be used. Generally, by "weak promoter" is intended a promoter that drives expression of a coding sequence at a low level. By low level is intended at levels of about 1/1000 transcripts to about 1/100,000. transcripts to about 1/500,000 transcripts. Alternatively, it is recognized that weak promoters also encompasses promoters that are expressed in only a few cells and not in others to give a total low level of expression. Where a promoter is expressed 40 WO 2006/044480 PCT/US2005/036711 at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels.

Such weak constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), the core 35S CaMV promoter, and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. "Tissue-preferred" promoters can be used to target a gene expression within a particular tissue. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2)255-265; Kawamata etal. (1997) Plant Cell Physiol. 38(7):792-803: Hansen et al (1997) Mol. Gen. Genet. 254(3):337-343:

Russell etal. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1 331 -1341 ; Van Camp et al (1996) Plant Physiol. 112(2):525-535: Canevascini et al. (1996) Plant Physiol. 112(2):51 3-524; Yamamoto etal. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994)

Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1 129-1 138; Matsuoka etal. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia etal. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression, "Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seed-germinating" promoters (those promoters active during seed germination). See Thompson etal. (1989) BioEssays 10:108, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 , (maize 19 kDa zein); milps (myo-inositol-1 -phosphate synthase); and celA (cellulose synthase). Gama-zein is a preferred endosperm-specific promoter. Glob-1 is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean.beta.-phaseolin, napin,.beta.-coriglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1,etc. 41 WO 2006/044480 PCT/US2005/036711

Leaf-specific promoters are known in the art. See, for example,

Yamamoto etal. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant

Physiol. 105:357-67; Yamamoto etal. (1994) Plant Cell Physiol. 35(5):773- 778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol.

Biol. 23(6): 1129-1 138; and Matsuoka etal. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred promoters are known and may be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2): 207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(1 0):1 051 -1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol, Biol. 14(3):433-443 (root-specific promoter ofthe mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1 ):l Γ-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401 ,836; 5,1 10,732; and 5,023,179.

Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5):3335-3342): 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer etal. (1990) J. Bioenerg. Biomemb. 22(6):789-810): tryptophan synthase (Zhao etal. (1995) J. Biol. Chem. 270(1 1):6081 -6087); plastocyanin (Lawrence etal. (1997) J. Biol. Chem. 272(33):20357-20363): chorismate synthase (Schmidt et al. (1993) J. Biol. Chem. 268(36):27447-27457): and the light harvesting chlorophyll a/b binding protein (LHBP). (Lamppa etal. (1988) J. Biol. Chem. 263:14996-14999). See also Von Heijne et al. (1991) Plant Mol. BioL Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. 42 WO 2(1(16/044480 PCT/US2005/036711

Res. Commun. 196:1414-1421 ; and Shah etal. (1986) Science 233:478- 481 . .

Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting ofthe DNA to the plastid genome through homologous recombination^ Additionally, plastid transformation may be accomplished by transactivation of a silent plastid-bome transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride etal. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

The nucleic acids of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Pat. Nb. 5,380,831 , herein incorporated by reference.

Plants transformed in accordance with the present disclosure may be monocots or dicots and include, but are not limited to, any nematode host plant.

Requirements for Construction of Plant Expression Cassettes

Nucleic acid sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable promoter expressible in plants. The expression cassettes may also comprise any further sequences required or selected for the. expression of the transgene. Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting ofthe gene product to specific organelles and cell compartments. These expression cassettes can then be easily transferred to the plant transformation vectors described infra. 43 WO 2006/044480 PCT/US2005/036711

The following is a description of various components of typical expression cassettes.

Promoters

The selection of the promoter used in expression cassettes determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection reflects the desired location of accumulation of the gene product. Alternatively, the selected promoter drives expression of the gene under various inducing conditions.

Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters known in the art may be used. For example, for constitutive expression, the CaMV 35S promoter, the rice actin promoter, or the ubiquitin promoter may be used. For. example, for regulatable expression, the chemically inducible PR-1 promoter from tobacco or Arabidopsis may be used (see, e.g., U.S. Pat No. 5,689,044). ' A suitable category of promoters is that which is wound inducible. Numerous promoters have been described which are expressed at wound sites. Preferred promoters ofthis kind include those described by Stanford et al. Mol. Gen. Genet. 2 15: 200-208 (1989), Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell. 1: 151-1 58 (1989), Rohrmeier & Lehle, Plant Moiec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), and Warner et al. Plant J. 3: 191-201 (1993).

Suitable tissue specific expression patterns include green tissue specific, root specific, stem specific, and flower specific. Promoters suitable . for expression in green.tissue include'many which regulate genes involved in photosynthesis, and many of these have been cloned from both monocotyledons and dicotyledons. A suitable promoter is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula, Plant 44 WO 2006/044480

PCT/US2005/0367U

Molec.Biol. 12: 579-589 (1989)). A suitable promoter for root specific

expression is that described by de Framond (FEBS 290: 103-106 (1991 ); EP 0 452 269 and a root-specific promoter is that from the T-1 gene. A suitable stem specific promoter is that described in U.S. Pat. No. 5,625,136 and which drives expression of the maize trpA gene.

Transcriptional Terminators A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S.terminator, the tm1 terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These are used in both monocotyledonous and dicotyledonous plants.

Sequences for the Enhancement or Regulation of Expression

Numerous sequences have been found to enhance gene expression from within the transcriptional. unit and these sequences can be used in conjunction with the genes to increase their expression in transgenic plants. For example, various intron sequences such as introns ofthe maize Adh 1 gene have been shown to enhance expression, particularly in monocotyledonous cells. In addition, a number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells.

Coding Sequence Optimization

The coding sequence of the selected gene may be genetically engineered by altering the coding sequence for optimal expression in the crop species of interest Methods for modifying coding sequences to achieve optimal expression in a particular crop species are well known (see, e.g. Perlak etal., Proc. Natl. Acad. Sci. USA 88: 3324 (1991); and Koziel et al, Bio/technol. 11:194 (1993)). 45 WO 2006/044480 PCT/US2005/036711

Another embodiment provides an RNA molecule directly transformed into the plastid genome. Plastid transformation technology is extensively described in U.S. Pat. Nos. 5,451 ,513,5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91,7301-7305. The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87,8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4,39-45). The presence of cloning sites between these markers allowed creation of a plastid targeting vector for introduction of foreign DNA molecules (Staub, J. M.f and Maliga, P. (1993) EMBO J. 12,601-606). Substantial increases in transformation frequency are obtained by replacement ofthe recessive rRNA orr-protein antibiotic , resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3 t adenyltransferase (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90,913-917). Previously, this marker had been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19: 4083-4089). Other selectable markers useful for plastid transformation are known in the art and are encompassed within the scope of the invention.. 46 WO 2006/044480 PCT/US2005/036711

Construction of Plant Transformation Vectors

Numerous transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this disclosure can be used in conjunction with any such vectors. The selection of vector depends upon the selected transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers are preferred. Selection markers used routinely in transformation include the npt1 1 gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra. Gene 19: 259-268 (1982); Bevan etal., Nature 304: 184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White etal., Nucl. Acids Res 18: 1062 (1990), Spencer etal. Theor. Appl. Genet 79: 625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931). the manA gene, which allows for positive selection in the presence of mannose (Miles and Guest (1984) Gene, 32: 41-48; U.S. Patent No. 5,767,378), and the dhfr gene, which confers resistance to methotrexate (Bourouis etal., EMBO J. 2 (7): 1099-1 104 (1983)), and the EPSPS gene, which confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642).

Many vectors are available for transformation using Agrobacteriiim tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984). Typical vectors suitable for Agrobacterium transformation include the binary vectors pCIB200 and pCIB2001 , as well as the binary vector pCIB 10 and hygromycin selection derivatives thereof. (See, for example, U.S. Pat. No. 5,639'949).

Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences are utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on 47 WO 2006/044480 PCT/US2005/036711

Agrobacterium include transformation via particle bombardment, protoplast uptake {e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Typical vectors suitable for non-Agrobacterium transformation include pCIB3064, pSOG 19, and pSOG35. (See, for example, U.S. Pat. No. 5,639,949).

Transformation Techniques

Once the DNA sequence of interest is cloned into an expression system, it is transformed into a plant cell. Methods for transformation and regeneration of plants are well known in the art. For example, Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles. In addition, bacteria from the genus Agrobacterium can be utilized to transform plant cells.

Transformation techniques for dicotyledons are well known inthe art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This is accomplished by REG or electroporation mediated, uptake, particle bombardment-mediated delivery, or microinjection. in each case the transformed cells may be regenerated to whole plants using standard techniques known in the art.

Transformation of most monocotyledon species has now become somewhat routine. Preferred techniques include direct gene transfer into protoplasts using PEG, or electroporation techniques, particle bombardment into callus, tissue or organized structures, as well as Agrobacterium-mediated transformation.

Plants from transformation events are grown, propagated and bred to yield progeny with the desired trait, and seeds are obtained with the desired trait, using processes well known in the art. The methods can result in plant cells comprising the RNA fragments ofthe present invention, wherein the 48 WO 2006/044480 PCT/US2005/036711 expression of said target gene in said plant cell is altered by said RNA fragments, a plant and the progeny thereof derived from the plant cell, and seeds derived from the plant.

The disclosed inhibitory nucleic acids or RKN esophageal gland cell secretory polypeptides may be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to subjects. Suitable components are the dsRNA and a vehicle that promotes introduction ofthe dsRNA. Such a kit may also include instructions to allow a user of the kit to practice the invention.

Another embodiment provides a method for providing resistance to nematode disease by introducing into a nematode host plant cell an RNA comprising a double stranded structure having a nucleotide sequence which is complementary to at least a part of the target mRNA; and optionally verifying inhibition of expression of the target mRNA. ' One embodiment provides a method for treating or preventing nematode disease in a plant by contacting a parasitic nematode in or on the plant with a with dsRNA having a sequence which is, complementary to at least a part of a mRNA encoding a nematode secretory protein, for example an esophageal gland cell protein; wherein the secretory protein modulates gene expression of plant.

Still another embodiment provides a plant cell, for example, containing an expression construct, the-construct coding for an RNA which forms a double stranded structure having a nucleotide sequence which is complementary to at least a part of a target mRNA that encodes a nematode secretory protein, for example an esophageal gland cell protein, as well as a transgenic plant containing such a cell.

In another embodiment, the RNA fragments are comprised in two different RNA molecules. In this case, the RNA fragments are mixed before being introduced into said cell, e.g. ,under conditions allowing them to form a double-stranded RNA molecule. In another embodiment, the RNA fragments are introduced into said cell sequentially. Preferably, the time interval 49 WO 2006/044480 PCT/US2005/036711 between the introduction of each of the RNA molecules is short, preferably less than one hour.

In still another embodiment, the RNA fragments are comprised in one RNA molecule. By using one single RNA molecule, the two complementary RNA fragments are in close proximity such that pairing and double strand formation is favored. In such case, the RNA molecule is preferably capable of folding such that said RNA fragments comprised therein form a double-stranded region. In this case, the complementary parts ofthe RNA fragments recognize one another, pair with each other and form the double-stranded RNA molecule. In another embodiment, the RNA fragments are incubated under conditions allowing them to form a double-stranded RNA molecule prior to introduction into the cell. In yet another embodiment, the RNA molecule comprises a linker between the sense RNA fragment and the antisense RNA fragment. The linker preferably comprises a RNA sequence encoded by an expression cassette comprising a functional gene, e.g. a selectable marker gene. In another embodiment, the linker comprises a RNA sequence encoded by regulatory sequences, which e.g. comprise intron processing signals.

Another embodiment provides a dsRNA construct having a promoter operably linked to said dsRNA and might further comprise said dsRNA molecule. The promoter can be a heterologous promoter, for example a tissue specific promoter, a developmentally regulated promoter, a constitutive promoter, divergent or an inducible promoter. Termination signal are also Optionally included in the DNA molecules.

The single RNA molecule or the two distinct RNA molecules are preferably capable of forming a double-stranded region, in which the complementary parts ofthe RNA fragments recognize one another, pair with each other and form the double-stranded RNA molecule.

Another embodiment provides the disclosed transgenic plant material in the form of feedstock, pellets,-granules, flakes and the like. The inhibitory nucleic acids disclosed here can be in seeds and seed products derived from the transgenic, plants described above. Another embodiment provides 50 WO 2006/044480 PCT/US2005/036711 a composition comprising the disclosed inhibitory nucleic acids that can be coated on seeds. The coating can be formulated so that the inhibitory nucleic acids remain able to inhibit nematode secretory proteins as the seed matures and develops roots. A further embodiment provides provides chimeric or fusion proteins containing the disclosed nematode esophageal gland cell proteins or fragments thereof. As used herein, a "chimeric protein" or "fusion protein" includes a nematode esophageal gland cell protein orfragment thereof linked to a foreign polypeptide. A "foreign polypeptide" is polypeptide that is not substantially homologous to a nematode esophageal gland cell protein orfragment thereof. The foreign polypeptide can be fused to the N-terminus or C-terminus ofthe nematode esophageal gland cell protein orfragment thereof.

The fusion protein can include a moiety which has a high affinity for a ligand. For example, the fusion protein can be a GST fusion protein in which a nematode esophageal gland cell protein orfragment thereof is fused to the C-terminus of GST. Such'fusion proteins.can facilitate the purification ofthe polypeptide. Alternatively, the fusion protein can contain a heterologous signal sequence at its N-terminus. In certain host cells, expression, secretion or transport of a protein can be increased through use of a heterologous signal sequence. For example, in a plant cell, a polypeptide ofthe invention may be fused with a chloroplast transit peptide; The chloroplast transit peptide allows the polypeptide to be transported from the cytoplasm of the plant cell into the chloroplast, thereby increase, root growth. Expression vectors are commercially available that already encode afusion moiety (e.g., a GST polypeptide). A nucleic acid encoding a nematode esophageal gland cell protein orfragment thereof can be cloned into such an expression vector so that the fusion moiety is linked in-frame to the polypeptide.

The following are only exemplary examples. It should be understood that the invention is not limited to these examples. Other important applications of disclosure would be readily recognized by those of ordinary 51 WO 2006/044480 PCT/US2005/036711 skills in the art. Other uses which are potentially recognizable by those of ordinary skills in the art are also part of the disclosure.

The references mentioned herein are incorporated in their entirety to the fullest extent permitted by applicable law.

EXAMPLES

Example 1: Nematodes and Plants

Meloidogyne species were propagated on roots of greenhouse-grown tomato (Lycopersicon esculentum cv. Marion or Better-Boy). Meloidogyne eggs were collected as described (Hussey and Barker, 1973). Pre-parasitic second-stage juveniles (pre-J2) were collected via hatching eggs on25-pm-pore sieves in deionized water in plastic bowls. The different-parasitic stages of M. incognita were collected by root blending and sieving (Ding et al. 1998). Mixed parasitic stages (MS) of M. incognita for in situ hybridizations were collected 13-15 days after inoculation of eggs as described in De Boer et al. (1998). Similarly, pre:J2 and MS of Heterogera glycines were collected from infected soybean (Glycine max) roots. Caenorhabditis elegans was cultured on OP50 of E. co//(Brenner, 1974). One-month-old host plant leaves were collected from growth-chamber, grown Nicotiana tabacum cv. Petite Havana SR1 , and Arabidopsis thaliana ecotype Col-O.

Example 2: Nucleic Acid Manipulation . Pre-J2 of packed nematodes were frozen in 1.5-ml microcentrifuge tubes with liquid nitrogen and ground with a smooth-end metal bar. The frozen nematode fragments were mixed with 0.5ml extraction solution (100 . mM NaCl, 100 mM Tris-HCl [ρΗβ.Ο], 50 mM EDTA,. 1% sodium dodecyl sulfate, 4 mg/ml proteinase K and 10 pg/ml RNase) and incubated at 37°C for 1 hr. DNA was extracted with phenol/chloroform and then precipitated with isopropanol (Sambrook et al., J989). The DNA was re-suspended'in H2O. Tobacco and Arabidopsis genomic DNA was extracted using standard techniques (Dellaporta 1993). 52 WO 2006/044480 PCT/US2005/036711 mRNAs were extracted and purified from ground plant tissues using Dynabeads mRNA DIRECT kit (Dynal, Lake Success, NY), eluted with 10μΙ diethylpyrocarbonate (DEPC)-treated water, and converted into first-strand . cDNA by reverse transcription (RT)-PCR SMART PCR cDNA Synthesis kit (BD Biosciences, Palo Alto, CA), following the manufacturer's instructions. RT-PCR reactions contained the following components: 4.0 μΙ of· 5χ first-strand buffer, 2.0μΙ of2OmM DTT, 2.0μΙοί 10mM 50χ dNTP, 1μΙ of 10μΜ 3'-CDS primer, ΊΟμΙ of isolated mRNA and 1μΙ of Superscript ll reverse transcriptase (200 units/μι, Gibco BRL, Rockville, MD). The reaction was incubated at42°Cfor 1 hr.

Example 3: Isolation of 16D10 cDNA Clone

Clone 16D10 encoding a secretory signaling peptide was identified during random sequencing of a gland-cell specific cDNA library of M. incognita (Huang etal., 2003) and designated as 16D10. The full-length double-strand cDNA sequences of 16D10 \n pGEM-T Easy vector were, obtained by using T7 and SP6 primers in sequencing reactions. The longest open reading frame ofthe 16D10 cDNA (364 bp) encoded a deduced' protein of 43 aa including a 30 aa N-terminal hydrophobic signal peptide as . predicted by.Signal P (Nielsen etai, 1997). While the mature. 16D10 peptide of-13 aa (G'KKRSGPNpGGNN, Mx 1,223 Da)(SEQ ID NO:52) provided., no significant BLASTX similarity, it did contain 8 aa (K- PSGPNP-N) (SEQ ID NO:53) ofthe conserved C-terminal 13 aa motif (KRLVPSGPNPLHN)(SEQ ID NO:54) ofthe functional domain ot Arabidopsis CLV3-like proteins (Cock and McCormick, 2001 ) as well as a cAMP/cGMP-dependent protein kinase, phosphorylation site [KKpS] as predicted by PROSITE (Hofmann et.al, 1999).

Example 4: Genomic Clones in Meloidogyne Species

One pair of the gene-specific primers. 16D10GF (5’-GAGAAAATAAAATATAAATTATTCCTC-S') (SEQ ID NO:55) and 16D10GR (5'-CAGATATAATTTTATTCAG-S ’) (SEQ ID NO:56) designed from the most 53 WO 2006/044480 PCT/US2005/036711 extreme 5’- and 3'-ends of the cDNA sequence of M. incognita 16D10, were used to amplify, the corresponding genomic sequences (or the highest homologues) from 200ng of M. incognita, M. javanica, M. arenaria and M. hapla genomic DNA. The PCR products were cut from a 1.2% agarose gel, and purified with a QIAquick gel extraction kit (Qiagen, Valencia, CA). The purified products were cloned into pGEM-Τ Easy vector (Promega, Madison, WI) for sequencing. The 16D10 homologues from the Meloidogyne species shared over 95% identity at the nucleotide level and the deduced proteins encoded by putative cDNAs were identical to that of M. incognita 16D10.

Example 5: Southern Blot Analysis

For each sample, 10 pg of genomic DNA was completely digested with 50 units of EcoRI or BamHI (New England Biolabs, Beverly, MA), separated ona 0.7% (w/v) agarose gel, transferred onto a Hybond-N Nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ) and blotting using a standard protocol (Sambrook et al., 1989). 16D10 probe was generated by amplification of the corresponding full-length cDNA from insert in pGEM-Τ Easy vector with T7 and SP6 primers. Gel-purified PCR products were labeled by PCR with a PCR-DIG probe synthesis system (Roche. Applied Science, Indianapolis, IN). About 15ng of DIG-labeled probe per ml was used for each-hybridization. Hybridizations were performed in DIG Easy Hyb solution (Roche Applied Science, Indianapolis, IN) at4CPCfor16 h followed by two 5-min washes in 2%SSC/0.1% SDS solution at RT. The membranes were then washed twice at 68°C with 0.5χ SSC/0.1 % SDS solution for 30 min. After incubating the membrane in 1% blocking reagent for 1 hr, .the membranes were incubated with a 1 .-10,000 dilution of sheep anti-DIG alkaline phosphatase (AP) conjugate for 30 min. Unbound antibody was removed by two 15-min washes with maleic acid washing buffer (100 mM maleic acid, 150 mM NaCI,'pH7.5, and 0.3% Tween 20). The membrane was incubated in AP detection buffer (100 mM, Tris-HCl, pH9.5, 100 m.M NaCl, and 50 mM MgCI2)for 10 min followed bya 1:50 dilution ofthe chemiluminescent substrate disodium 3-(4-methoxyspiro{1 ,2-dioxetane-3,2- 54 WO 2006/044480 PCT/US2O05/036713 (5'-chloro)tricyclo[3.3.1 ,i3'?]decan}-4-yl)pheryl phosphate (CSPD) (Roche Applied Science) before sealing the membrane in two sheets of transparency film and exposing it to X-ray film for 1.5 hr. A blot containing genomic DNA from M. incognita, M.javanica, M. arenaria and M. hapla hybridized with a 16D10 cDNA probe showed that 16D10 was present in each of the four agriculturally important Meloidogyne species with 3-4 copies or homologues (Figure 6). No hybridization was detected with genomic DNAs from the soybean cyst nematode H. glycines, the non-parasitic free-living nematode Caenorhabditis elegans, and plants (tobacco and Arabidopsis).

Example 6: Sequence Analyses

Sequence similarity searches were carried out using the BLAST programs PSI-BLASTP and BLASTX at the National Center for Biotechnology Information (NCBI) (Altschul etal., 1998). Multiple sequence alignments of Meloidogyne 16D10 genomic DNA sequences were generated using ClustalWI .8 (Jeanmougin et al., 1994). Prediction of a signal peptide for secretion and the cleavage site was performed via the SignalP program (Nielsen et al., 1997).

Example 7: in situ Hybridization

Specific forward and reverse primers for 16D10 cDNA clone were used to synthesize digoxigenin (DIG)-labeled sense and antisense cDNA probes (Roche Applied Science, Indianapolis, IN) by asymmetric PCR (Huang et al., 2003). In situ hybridization was performed using formalin-fixed, permeabilized pre-parasitic juveniles and mixed parasitic stages of M. incognita (De Boer et al., 1998; Hiiang et a!., 2003). cDNA probes that hybridized within the nematode were detected with alkaline phosphatase-conjugated anti-DIG antibody and substrate, and specimens were observed with a compound light microscope (De Boer et al., 1998). In situ mRNA hybridization revealed that 16D10 was strongly expressed in the two 55 WO 2006/04448« PCT/US2005/036711 subventral esophageal gland cells of M. incognita at the early parasitic stages.

Example 8: Immunofluorescence Assay

The purified 16D10 polyclonal antiserum was used to localize 16D10 expression in sections of pre-parasitic J2, mixed parasitic stages of M. incognita with indirect immunofluorescence as described previously by Goverse etal. (1994). Following fixation in freshly prepared 2% paraformaldehyde in PBS buffer (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, pH7.4) for 5 days at4°C,the nematodes were washed three times in PBS buffer and once in deionized water. The fixed nematodes were cut into sections and incubated in 0.6mg of proteinase K (Roche Applied Science, Indianapolis, IN) per ml in phosphate buffered saline (PBS) buffer at 37°C for 1 hr. After washed once with PBS, the partially digested nematodes were placed in a -80 °C freezer for 20 min, incubated in dry-ice cold methanol for 3min, and then incubated in dry-ice cold acetone for 15min. The nematodes were washed once with blocking solution (10% goat serum, 0.02% NaN3,1mM phenylmethylsulfonyi fluoride, 1χ PBS) amended with protease inhibitors as previously described (Goverse et al., 1994), incubated at 4°C for 3 days and then used immediately for immunofluorescence. The blocked nematodes were aliquoted to wells of a 96-well Multiscreen plate (Millipore, Bedford, MA), and agitated in a -1:250 dilution ofthe 16D10 purified polyclonal antibody in ELISA diluent (0.05% Tween, 0.02% NaN3, 1% BSA, 1χ PBS) in a moisture chamber overnight at RT. Nematode sections were washed three times for 5 min each with PBST (1* PBS, 0.5% Triton X-100) and agitated in a 1:1000 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Sigma-Aldrich,. St. Louis, MO) in Tris-Saline-BSA (0.15M NaCl, 0.01 MTris, pH7.2, 0.2% Triton X-100, 3% BSA) in the dark for 3h at RT. Sections were washed twice in PBST and once with distilled water. . Treated sections were transferred in a 15-μ1 drop of water to individual wells on Multitest slides (ICN-Flow,

Horsham, PA) that previously coated with 5plof 0.1 % poly-L-lysine (Sigma 56 WO 2006/044480 PCT/US2005/036711

Chemical). Sections were airy dried on slides, covered with a 3μΙ drop of antiquenching agent {0.02mg/ml phenylenediamine in SOOmM carbonate buffer, pH8.6, mixed with nonfluorescent glycerol), and a coverslip was applied. Specimens were observed on an Olympus fluorescence microscope. Negative control consisted of pre-immune rabbit serum. The purified 16D10 antiserum bound to secretory granules within the subventral gland cells of pre-parasitic and parasitic J2 and their cytoplasmic extensions and expanded ampullae, which are Ideated posterior to the pump chamber at the metacarpus. No specific labeling with the rabbit preimmune serum was observed in any nematode specimens.

Example 9: Protein Extraction

Nematode proteins were extracted by grinding pre-parasitic J2 and mixed parasitic stages of M. incognita and H. glycines in 200 μΙ of extraction buffer [10OmM Tris-HCl, pH7.0, 150mM NaCl and 1χ complete protease inhibitors (Roche Applied Science, Indianapolis, IN)] in microcentrifuge tubes in liquid nitrogen. Plant proteins (0.5 g) were extracted by grinding transgenic seedlings or root tissues in 200 μΐ of extraction buffer [50 mM Tris-HCl, pH7.0, 150 mM NaCl, 1χ complete protease inhibitors (Roche Applied Science)] in microcentrifuge tubes in liquid nitrogen. Supernatant was recovered from homogenates after centhfugation at 13,000 rpm for 10 min. All protein concentrations were estimated (with a Bio-Rad Protein Assay Kit II) with BSA as a standard. As the positive control, the 16D10 peptide (GKKPSGPNPGGNN; >95% purity)(SEQ ID NO:52) was synthesized from Sigma-Genosys, TX for immunodetection assays (see examples 12-13).

Example 10: Collection of Stylet Secretions

Stylet secretions from M. incognita J2 were produced and collected in vitro as described by Davis et al. (1994). Pre-parasitic J2 were incubated in 0.4% resorcinol (Sigma-Aldrich, St. Louis, MO) for 6 hr at room temperature in a humid chamber. Stylet secretions were solubilized via adding an equal volume of;0.1 MTris-NaOH, pH1 1.0. Solubilized stylet secretions were 57 WO 2006/044480 PCT/US2005/036711 concentrated with StrataClean (Stratagene, La Jolla, CA). Briefly, soluble secretory proteins were trapped via suspending 1.5ml of beads in the supernatant of induction mixture (460ml). and incubating it for 1 hr under constant mixing. The beads were centrifuged, re-suspended in 2χ SDS-PAGE sample buffer, and boiled for 3 min to release the absorbed proteins. The concentrated stylet secretions were used in enzyme-linked immunosorbent assay (ELISA) and immunoblotting analyses using the purified 16D10 antiserum (see Examples 11-13). Both assays identified 16D10 peptide in the stylet secretions as well as total extracts of J2 and mixed parasitic stages of M. incognita.

Example 11: Production of Antisera

Polyclonal antiserum to 16D10 was produced by immunizing two rabbits with a synthetic mature (i.e., without the N-terminal signal peptide) 16D10 peptide (GKKPSGPNPGGNN)(SEQ ID NO:52) from Eurogentec, Inc. (Herstal, Belgium). The antiserum was affinity-purified from 15 ml of last crude sera with the peptide antigen. Peptide affinity-purified 16D10 . polyclonal antiserum was used to localize 16D10 expression in specimens of M. incognita using immunofluorescence microscopy (Goverse etai, 1994), and for immunodetection of 16D10 in stylet secretions and transgenic plant-expressed or in vitro translated 16D10.

Example 12: Western Dot-blot Analysis

Protein samples (2μΙ) were spotted onto Hybond ECL nitrocellulose. The nitrocellulose membrane was allowed to air dry for 20 min. The membrane was incubated in a blocking solution (2% nonfat dry milk, 1%Tris-buffered-saline-Tween , [TBS-T: 20 mM Tris-HCl, pH7.4, 0.8% NaCl, 0.1 % Tween 20] overnight at 37°C and then treated with the purified 16D10 polyclonal antiserum ¢1:2,000), followed by anti-rabbit IgG (whole molecule) alkaline phosphatase conjugate ¢1:30,000) (Sigma). The membrane was washed three times in 1χ TBS-T buffer at room temperature, and incubated in the substrate solution (45μΙ of nitroblue tetrazolium [NBT] solution and 58 WO 2006/04448(1 PCT/US2005/036711 35μΙ of 0-bromoA-chloro-S-indolyl-phosphate toluidinium [BCIP] solution in 10 ml of AP buffer [100 mM Tris-HCl, pH9.5, 100 mM NaCl, 5 mM MgCI]) at room temperature until color develops.

Example 13: ELISA Assay ELISA was modified from Pratt et al. (1986). Dynatech Immulon plate wells were coated overnight at 4°C with proteins diluted in borate saline (0.2M sodium borate, 75mM NaCl, pH8.5) from the following sources: 2μΙ of 1000χ concentrated stylet secretions of M. incognita J2, 10μρ of total extracted proteins of pre-J2 of M. incognita, MS of M. incognita, pre-J2 and MS of H. glycines, or 1Ομο of BSA (Sigma Chemical) as a negative control. As a positive control, wells were coated with 100ng of synthetic 16D10 peptide (>95% purity, Sigma-Genosys, TX). Wells were rinsed three times with wash buffers (10mM Tris.HCI, ρΗδ.ο, 0.5M NaCl) and blocked with 1% BSA in PBS (32.9mM Na2HPO4, 1.77mM NaH2PO4, 0.14M NaCl, pH7.4) for 30;min at room temperature. After being rinsed once with wash buffer, each coated welLwas incubated with 16D10 purified polyclonal·antisera diluted. 1:1,000 with 0.5% .BSA .in PBS;for 1 hr-at room temperature. Negative controls included omitting, incubation with'the primary polyclonal antibody, and incubation with the' rabbit, pre-immurie serum. The wells .were washed three times, incubated with' alkaline phosphatase-conjugated goat anti-rabbit antibody (Sigma Chemical) at 1:5,000 dilution for 1 hr at room temperature, washed three times before phosphate colorimetric substrate was added. The substrate, p-nitrophenyl phosphate was prepared according to manufacturer's directions in alkaline phosphatase buffer (1M diethanolamine, 0.5mM MgCI2, pH 9.8) and incubated in the treated wells 30 min at room temperature before the reaction was stopped with 3 N NaOH. Absorbance was measured at 405 nm and 490 nm on an ELISA reader.

Example 14: Plasmid Construction

The coding regions of 16D10 with or without a signal peptide sequence were amplified from the full-length cDNA clone with primers 59 WO 2006/044480 PCT/US2005/036711

16D10SF (5"-CGGGGIACCLAGATGTTTACTAATTCAATTΑΑ-S') (SEQ ID NO:57) or16D10F (5-CGGGGTACC rAGATGGGCAAAAAGCCTAGTG-3') (SEQ ID NO:58) and 16D10R (S'-GC TCTAGA TCAATTATTTCCTCCAGG-3') (SEQ ID NO:59) that introduced Kpn\ orXba\ restriction sites (underlined) and the stop/start codons (in italics), cloned into the Kpn\ and Xba\ sites of binary vector pBIX under the control of CaMV 35S promoter to generate pBIX(16D10S) and pBIX(16D10), respectively, and confirmed by sequencing. pBIX was derived from pBI101 (BD Biosciences, Palo Alto, CA) and contains a nos promoter-nptll-nos terminator cassette, a 35S promoter-gusA-nos terminator, and a second 35S promoter with a polylinker having Kpn\ and Xba\ sites. The hybrid expressed sequence of civ3 and 16D10 was generated by PCR amplifications from Arabidopsis genomic DNA using primers C3K (5’-GG GGTACC ATGGATTCTAAAAGCTTTG-S'), (SEQ ID NO:60) that introduced Kpn\ restriction site (underlined) and C3R (5’-CCACTAGGCTTTTTGCCAAGGAACAAGAAGCAG-S ’) (SEQ ID NO:61 ) for signal sequence, and from 16D10 cDNA using primers C3F (5'.-CTTCTGCTTCTTGTTCCTTGGCAAAAAGCCTAGTGG-S’) (SEQ ID NO:62) and 16D10X (S'-GCTCTAGATCAATTATTTCCTCCAGG-S') (SEQ ID NO:63) that introduced Xba\ restriction site (underlined) for mature peptide coding sequence using Vent polymerase (New England Biolabs, Beverly, MA). The two products were then used to prime each other in a fusion PCR reaction. The resulting fragment was cloned into pBIX to generate pBIX(C3S-16D10) and verified by sequencing.

Example 15: Tobacco Hairy-root Transformation

The plasmids pBIX(16D10), pBIX(16D10S) and the empty vector pBIX as a control were transferred into Agrobacterium rhizogenes ATCC 15834 by electroporation (Shen and Forde, 1989) and transformed into tobacco (Nicotiana tabacum cv Petite Havana SR1) using the A. rhyzogenes-medlated cotyledon transformation (Christey, 1997). Transformed hairy, roots were generated from inoculated tobacco cotyledons on Gamborg's B-5 plates containing 0.8% Noble agar with 100 mg/L 60 WO 2006/044480 FCT/US2005/036711

kanamycin and timentins (230.8 mg/L ticarcilfin disodium plus 7.69 mg/L clavulanate potassium). Individual hairy root tips (about 0.5 cm) were cultured for 3 weeks at 24°C in the dark, and 2 to 3 roots from individual hairy root system were subjected to GUS-staining (Jefferson etai, 1987).

The kanamycin-resistant and GUS-positive root lines with no bacterial contamination, confirmed by PCR analyses, were used to establish hairy root lines. The root-tips were sub-cultured for root growth assay on Gamborg's B-5 plates without hormones every 2 weeks and the cut roots were kept in culture on the old plates at 24°C in the dark for assays. For root-growth assays, plates were cultured horizontally in the dark and 5 hairy roots-from each transgenic line in each ofthe three repeats were investigated. Relative RT-PCR and immunoblotting analyses of transgenic hairy roots or calli with a single transgenic copy identified as described (Does etai, 1991 )were carried out using the same procedures as in those of transgenic Arabidopsis. Expression of 16D10 in the cytoplasm of hairy root cells increased root growth at the rate of approximately 65% [mean root length after 2 weeks of 5.20 ± 0.61 cm(n = 90) in 16D10 transgenic lines, compared to 3.15 ± 0.34 cm (n = 90) in control lines], generated extensive lateral roots and led to the formation' of calli where roots were cut for subculturing at 5 weeks. RT-PCR analysis of 16D10 expression showed that the-steady-state mRNA levels in calli were higher than in the. hairy roots. Immunoblotting analysis with the purified 16D10 antiserum revealed that 16D10 was produced in both hairy roots and calli. No expression of 16D10 was detected in the control vector-transformed hairy roots.

Example IG: Arabidopsis Floral-dip Transformation

The plasmids pBIX(16D10), pBIX(C3S-16D10) and the empty vector pBIX as a control were introduced into Agrobacterium tumefaciens C58C1 by electroporation (Shen and Forde, 1989) and transformed into A. thaliana wild-type Col-0 plants by the floral dip method (Clough and Bent, 1998). Segregation of kanamycin resistance, GUS-straining (Jefferson etal, 1987), and 16D10 expression coupled to PCR analyses confirmed generation ofthe 61 WO 2006/044480 PCT/US2005/036711 transgenic homogenous T2 lines. Inverse PCR (Does etal, 1991 ) identified the homogenous lines with a single transgenic copy in the genome for molecular and root growth assays. Thirty plants from each transgenic line in each ofthe three repeats were in vitro cultured on MS plates with 3% sucrose with 16 h light (24°C)/8 h dark (2O°C) cycles and the plates were kept vertically for root growth assay.

Four transgenic Arabidopsis T2 homozygous lines containing a singlecopy of 16D10 without a signal peptide under the control ofthe 35S promoter were generated. Two transgenic lines originating from the blank transformation vector were also generated as controls. RT-PCR and immunoblotting analyses confirmed that 16D10 was expressed in all ofthe 16D10 transgenic lines, but not in the control lines. Compared to controls, expression of 16D10 in the cytoplasm ot Arabidopsis cells increased the length of primary roots 85% [mean 54.01 ± 8.75 mm in four 16D10 transgenic lines (n = 90/line), and 29.20 ±4.50 mm in 2 control lines (n = 90/line)] and the number of lateral branches and adventitious roots increased 1.4-fold and 2.08-fold, respectively (Figure 2 and Figure 3). Increased primary root growth was closely correlated with increased lateral root number and increased adventitious root number. Measurements of the root tip growth rate over 3 days revealed an increase (20%) in length only in the meristematic zone of 16D10 roots, indicating increase in cell number and not cell size contributed to the enhanced root growth.

Example 17: Complementation Tests

Since the mature 16D10 peptide of 13 aa (GKKPSGPNPGGNN) (SEQ ID NO:52) contained 8 aa (K- PSGPNP-N) (SEQ ID NO:53) ofthe conserved C-terminal 13 aa motif (KRLVPSGPNPLHN) (SEQ ID NO:54) of the functional domain ot Arabidopsis CLV3-like proteins (Cock and McCormick, 2001), the plasmid pBIX(Clv3S-16D10) encoding M. incognita 16D10 with A. thaliana CLAVATA3 signal peptide was .transferred into the A. thaliana civ3 mutants clv3-1 (intermediate), civ3-2 and clv3-6 via A. tumefaciens C58C1 -mediated floral-dip transformation (Clough and Bent, 62 WO 2006/044480 PCT/US2005/036711 1998) for functional complementation tests. As controls, the plasmids pBIX(16D10) and pBIX were also introduced into the clv3 mutants. Three transgenic T2 homozygous lines for each construct were also generated. The phenotypes (flower and shoot apical meristem) of 16D10-transformed clv3 lines were investigated and compared with those of vector-transformed lines, A. thaliana wild-type ecotype Col-0 and the clv3 mutant progeny as described in Fletcher etal. (1999). While 16D10 contained the functional domain of Arabidopsis· CLV3-like proteins, expression of 16D10 inthe apoplast or cytoplasm of Arabidopsis cfv3 mutants did not restore wild type phenotype, indicating 16D10 does not function as CLV3-like proteins.

Example 18: Histological Analysis

Primary root tissues of A. thaliana were fixed and dehydrated (Dolan etai, 1993), and embedded in Spurrs resin using Low Viscosity Embedding kit (Electron Microscopy Sciences, Hatfield, PA) according to the manufacturer's instructions. Thin sections (0.4 pM)were made on.a Reichert-Jung Ultracut E and stained with. 1%,toluidine blue. Transverse root sections in and above, the root meristem and longitudinal sections ’at the root-tip revealed that the average,.cell-size and. number of ceil types and cell-layer did not differ in the transgenic lines, compared, to wild .type.. Root morphology was also not altered in our transgenic plants, and increased, growth was accompanied: by accelerated development of the root system. Thus ectopic 16D10 expression enhanced root growth rate and induced lateral root initiation, possibly by stimuliation of cell division in meristems, increasing the rate of cell production without altering meristem organization.

Example 19: Relative RT-PCR

Reverse transcription (RT)-PCR was conducted on mRNA extracted from equivalent amounts of plant tissue. The 16D10 gene-specific primers 16D10F and 16D10R as described above, were used in subsequent PCR amplifications. In controls, the. primers UBQ1 (5- GATCTTTGCCGGAAAACAATTGGAGGATGGT-S’) (SEQ ID NO:64) and 63 WO 2006/044480 PCT/lJS2005/<)36711

UBQ2 (5’-CGACTTGTCATTAGAAAGAAAGAGATAACAGG-S') (SEQ ID NO:65) designed from the uniformly expressed UBQ10 gene (GenBank accession no. NM_202787) of A. thaliana wild-type ecotype Col-O, were used to amplify a 483bp unique sequence of UBQ10 from transgenic Arabidopsis lines. The primers ActF (δ'-CCGGTCGTGGTCTTACTGAT-S ’) (SEQ ID NO:66) and ActR (5-GCACCGATTGTGATGACTTG-S ’) (SEQ ID NO:67) designed from the uniformly expressed actin gene (GenBank accession no. U60494) of N. tabacum cv Petite Havana SR1 were used to amplify a 271 bp unique sequence of the tobacco actin (Tob104) gene from transgenic tobacco hairy roots. PCRs containing the following components: 5μ! of 10χ BD Advantage 2 PCR buffer, 1.0μΙ of 10mM dNTP mix, 1.5μΙ of 5' primer, 1.5μΙ of 3’ primer, 2μΙ of cDNA, 38μΙ of water, and Ι.ομΙ of 50χ BD Advantage 2 Polymerase Mix (BD Biosciences, Palo Alto, CA). PCR cycles consisted of an initial denaturation step at 94°C for 2 min, followed by 35 cycles of 94°C for 1 min, 55°C for 30 seconds, 72°C for 40 seconds, and a final 10-min elongation step at 72°C. Ten-microliter aliquots of each RT-PCR reaction were electrophoresed on a 2% agarose gel, transferred to nylon membranes, and hybridized with corresponding DIG-tabeled DNA probes. RT-PCR analysis revealed that 16D10 transcripts were steadily present in the16D10 transgenic tobacco hairy roots and Arabidopsis lines, but absent in the vector-transformed control lines.

Example 20: Yeast Two-hybrid Screens

The MATCHMAKER yeast two-hybrid system ll (BD Biosciences,

Palo Alto, CA) was used in the yeast two-hybrid screening. The cDNA encoding the mature peptide of 16D10 was cloned in frame into the GAL4-binding domain (BD) of pGBKT7 to generate pGBKT7(16D10) and expressed as bait to screen a tomato root cDNA library constructed from mRNA from tomato root tissues in the GAL4 activation domain (AD) of pGADT7. Twelve full-length SCL-encoding cDNAs (AtSCLl ,AtSCL3, AtSCL5, AtSCL6, AtSCLQ, AtSCL13, AtSCL14, AtSCL2\, AtSCR, AtSHR, AtRGA, AtGA/J were amplified from a root cDNA pool made from mRNA 64 WO 2006/044480 PCT/US2005/036711 from A. thaliana root tissues with specific primers of each gene based on the corresponding sequences in GenBank databases (Bolle, 2004), and cloned in frame into pGADT7. Each of the constructs was introduced with pGBKT7(16D10) into the yeast strain AH109. cDNAs encoding the specific regions of AtSCL6 and AtSCL21 were cloned into pGADT7, and then cotransformed with pGBKT7(16D10) into the strain AH109. Ail procedures including cDNA library screening, selection of positive clones and the assay of β-galactosidase activity, were performed by following the protocol of MATCHMAKER yeast two-hybrid system 11 (BD Biosciences, Palo Alto, CA). Two Arabidopsis SCL proteins, AtSCL.6 and AtSCL.21 , interacted with 16D10 in yeast. Domain analysis revealed the specific interaction of 16D10 with the SAW domain of AtSCL.6 and AtSCL21 , and no interaction of 16D10 with the rest of the domains of the SCL proteins, and indicated that the SCL transcription factor(s) was a putative target ofthe secreted 16D10 during RKN parasitism of plants.

Example 21: RNAi by Soaking

Forty-two bp and 271 bp sequences of 16D10 were respectively amplified from the full-length cDNA clone using the primers 16D10T7F1 (5'-TAATACGACTCACTATAGGG CCTCAAAAATACCATAAAG-3')(SEQ ID NO:68) and 16D10T7R1 (5'- TAATACGACTCACTATAGGG GAAATTAACAAAGGAAACC-S'), (SEQ ID NO:69), and 16D10T7F2 (5’- TAATACGACTCACTATAGGG GGCAAAAAGCCTAGTGGGC-S) (SEQ ID NO:70) and 16D10T7R2 (5'-

TAATACGACTCACTATAGGG TCAATTATTTCCTCCAGG-S') (SEQ ID NO:71) each of that incorporates the RNA primer site T7 (underlined). The gel-purified PCR products were used as templates for synthesis of sense and antisense 16D10 RNAs in a single reaction in vitro using the MEGAscript RNAi kit (Ambion, Austin, TX) according to manufacturer's instructions, except that the reactions were incubated for 16 hr to increase RNA yield. The amount and quality of generated double-strand (ds) RNA 65 WO 2006/044480 PCT/US2005/036711 were estimated and quantitated by standard procedures (Sambrook et al., 1989). The dsRNA products were ethanol precipitated and re-suspended in nuclease-free water to 10-1 5μρ/μι.

Approximately 10,000 freshly hatched J2s of M. incognita were soaked in M9 buffer (10.9mM Na2HPO4, 5mM KH2PO4, 4JmM NH4CI, and 2.2mM NaCl) containing 1 mg/ml of 16D10 dsRNA, 1% resorcinol, 0.13mg/ml FITC isomer I, 0.05% gelatin and 3mM spermidine, and incubated for 4 hr in the dark at RT ona rotator. Resorcinol (Res) was used to help stimulate uptake of the dsRNA. Control samples were incubated in the same solution but without resorcinol or dsRNA. After soaking, nematodes were thoroughly washed five times with nuclease-free water by centrifugation and about 100% of treated nematodes were observed with an Olympus fluorescence microscope to take up FITC, a marker for uptake of dsRNA. The FITC-labeled transgenic J2 were assayed to determine silencing of the 16D10 transcripts by relative RT-PCR analysis, using first-strand cDNAs synthesized from mRNA of equivalent number of treated J2 as templates and a 284bp amplified fragment of the M. incognita constitutively expressed actin gene (GenBank accession no. BE225475) as a control. The ingestion of short or full-length 16D10 dsRNA by second-stage juveniles of root-knot nematode caused a significant reduction of 16D10 transcripts in ( the treated nematodes (Figure 4), providing direct evidence for in vivo targeting of 16D10 in root-knot nematodes by RNAi. J2s of M. incognita were also soaked as described above with 1 mg/ml of dsRNA specific for 8H1 1 (SEQ ID NO:17) or 31H06 (SEQ ID NO:33). Relative RT-PCR analysis revealed that ingestion of 8H1 1 and 31H06 dsRNA by second-stage juveniles of root-knot nematode caused a significant reduction of transcripts of these two additional parasitism genes in the treated nematodes (Figure 5).

Example 22: In planta Delivery of RNAi

The sense and anti-sense cDNAs (42bp or 271 bp) of 16D10 were amplified from the full-length cDNA clone with the gene-specific primers 66 WO 2((06/((44480 PCT/US2005/036711 16D10Xho1 (δ'-CCG CTCGAG GGCAAAAAGCCTAGTGGGC-S ’) (SEQ ID N0.72) and 16D10Kpn1 (S'-CGGGGTACCTCAATTATTTCCTCCAGG-S') (SEQ ID NO:73), 16D1 OCIal (S'-CCATCGAT TCAATTATTTCCTCCAGG-S') . (SEQ ID NO:74) and 16D10Xba1 (5*-

GCTCTAGA GGCAAAAAGCCTAGTGGGC-3') . (SEQ ID NO:75), 16D10Xho3 (5-CCG CTCGAG_CGTCAAAAATACCATAAAG-3’(SEQ ID NO:76) and 16D10Kpn2 fS'-CGG GGTACC GAAATTAACAAAGGAAACC-S *) (SEQ ID NO:77), 16D10Cla2 (5'-CCAICGAIGAAATTAACAAAGGAAACC-S') (SEQ ID NO:78) and 16D10Xba3 (51GCICIAGACCTCAAAAATACCATAAAG-S') (SEQ ID NO:79) that introduced Xho\, Kpn\, Cia\ or Xbal restriction sites (underlined), respectively. The PCR products were gel-purified, and digested with the restriction enzymes Xho\ and Kpnl, or C/al and Xba\, respectively. The digested-PCR products were cloned into the Xho-Kpn\ sites, and the Cla\-Xba\ sites of pHANNIBAL to generate pHANNIBAL(16D10#1 ) and pHANNIBAL(16D10#2), respectively. The sense and antisense 16D10 cDNAs of pHANNIBAL-derived plasmids were subcloned as Nof\ fragments into the binary vector pART27 (Gleave, 1992) to produce highly effective intron-containing "hairpin" RNA (ihpRNA) silencing constructs (Wesley et al., 2001 ).The pART27-derived constructs were electroporation transformed into A. tumefaciens C58C1 .The transformants were selected on LB media containing rifampicin (50mg/L), gentamycin (25mg/L) and spectinomycin (100 mg/L), and then introduced into A. thaliana ecotype Col-0 via floral-dip transformation as described above, transgenic homologous T2 lines constitutively transcribing the specific ihpRNA of 16D10 under the CaMV35S promoter were generated for resistance assays to the root-knot nematodes, Meloidogyne species.

Example 23: Resistance Assays

Seeds from the A. thaliana transgenic lines generated from transformation of pART27-derived constructs were surface sterilized in 70% (v/v) ethanol for 1 min and 3% (v/v) sodium hypochloride for 5 min, and then rinsed 5 times in sterile distilled water. The sterilized seeds were geminated and grown on 67 WO 2006/044480 PCT/US2005/036711

Gamborg’s B-5 medium for 3 weeks. M. incognita eggs were sterilized and then inoculated about 500 eggs for each plant near to the roots as described (Sijmons et al., 1991). The number and size of galls on the infected roots were analyzed after inoculation of 3 weeks, and the infected roots were stained red with acid fuschin as described (Hwang et al., 2000) and assayed by the number of eggs per gram of roots after inoculation of 8 weeks. Transgenic Arabidopsis lines expressing 16D10 dsRNA were resistant to the four major Meloidogyne species - M. incognita, M.javanica, M. arenaria, and M. hapla. Root galling assay showed a 63-90% reduction in the number (and size) of galls on the 16D10 dsRNA transgenic Arabidopsis lines, compared to galls on the vector-transformed line (Figures 1A, 1B and Table 1). Nematode reproduction assay revealed a 70-97% reduction in the number of RKN eggs per gram root in the 16D10 dsRNA transgenic lines when compared to the control plants (Figure 7).

Table 1

Gall production on transgenic A. thaliana expressing 16D10 dsRNA and inoculated with four Meloidogyne species (M. incognita, M.javanica, M. arenaria, M. hapla) compared with control plants.

Galling No. (Mean value) CK 16D10i-l 16D10i-2 T L M S T L M S T L M S M. incognita 13.50 4.83 7.67 1.00 1.50 0 0.25 1.25 3.40 0 0.80 2.60 M. javanica 14.38 7.41 5.47 1,50 3.29 0.14 1.00 2.15 2.50 0.13 1.25 1.12 M. araneria 11.75 6.75 3.25 1.75 3.00 0.17 1.50 1.33 3.50 0.25 0.75 2.50 M, hapla 10.21 3.46 6.25 0.50 3.63 0.25 2.33 1.05 3.78 0.50 1.67 1.61 T: total L: large (>2 mm) M: medium (1-2 mm) S: small (<1 mm) n: 8-16

Example 24: 16d10 Sequence Data 16D10 Genomic DNA sequence (840bp)

GAGAAAATAAAATATAAATTATTCCTCAAAAATACCATAAAGGTTAGCCA

ATATTAATTCTTTTGAAATTTTCTTTGCTTCCATAAATTAAAAAAAATTG 68 WO 20(16/044480 PCT/US2005/036711

TTTTTAAGTGAGGGAATGTGGATTAAGCATCTTTCTTATTTTTAAAATTT

TTGATAGAGTGTAGCGACAGTCAATCAAAATATTTTGATTTTTTTAAAG

TTAAAAATTAAGGATGATAAAGAAGTTTAAAATGTAGGTGGAAATATA

AGTATACCGAAAAACATCTTTTATTTTTAAGTTTAAACAAGCAGTAAAA

CTTTGTCTGGTTTTATCACCGGGCAACTGTAAGGGAAGCTTTAATAAA

AATTTTGTAAGATACGAAAATCATTGTCCCCAGTAGCTTGAGTGATCG

AAGCGCCTGGTTGCCATTAAGTTTTTTGCTTGAGACTTATATAACAAGT

ATATATCAAACCGGATTATAAAGTTAAAGAACAGAAAAAATTTCACGG

AATAAATATTGGCTAACCACTCAATTTATTTAATTATTCTTCAATCAAAA

AATGTTTACTAATTCAATTAAAAATTTAATTATTTATTTAATGCCTTTAATG

GTTACTTTAATGCTTTTGTCTGTCTCATTTGTGGATGCAGGCAAAAAGCC

TAGTGGGCCAAATCCTGGAGGAAATAATTGAAGAAAAATGATTGAAGAA

AAACGTTTAAATTAAACGATAAATGGGAAATAATGGAATTTAAATTAAGC

TAATTTTGATGGTTTCCTTTGTTAATTTCAACATAAAATTAATTGAATTTA CTGAATAAAATTATATCTGAAAAAAA(SEQ ID NO:1) (One 476-bp intron sequence is bolded). 16D10 cDNA sequence (364bp)

GAGAAAATAAAATATAAATTATTCCTCAAAAATACCATAAAGTTAATTATT

CTTCAATCAAAAAAATGTTTACTAATTCAATTAAAAATTTAATTATTTATTT

AATGCCTTTAATGGTTACTTTAATGCTTTTGTCTGTCTCATTTGTGGATG

CAGGCAAAAAGCCTAGTGGGCCAAATCCTGGAGGAAATAATTGAAGAA

AAATGATTGAAGAAAAACGTTTAAATTAAACGATAAATGGGAAATAATGG

AATTTAAATTAAGCTAATTTTGATGGTTTCCTTTGTTAATTTCAACATAAA

ATTAATTGAATTTACTGAATAAAATTATATCTGAAAAAAAAAAAAAAAAAA AAAAAAAAAAA (SEQ ID NO:2) 16D10 cDNA sequence region used for making 16D10 RNAi constructs

GAGAAAATAAAATATAAATTATBCTCAAAAATACCATAAAGTTAATTATT

CTTCAATCAAAAAAArGTTTACTAATTCMTTAAAAATTTAATTATTTATTT 69 WO 2006/044480 PCT/US2005/036711

AATGCCTTTAATGGTTACTTTAATGCTTTTGTCTGTCTCATTTGTGGATG

CAGGCAAAAAGCCTAGTGGGCCAAATCCTGGAGGAAATAAT7G AAGA

AAAATGATTGAAGAAAAACGTTTAAATTAAACGATAAATGGGAAATAATG GAATTTAAATTAAGCTAATTTTGATGGTTTCCTTTGTTAATTTC AACATAA AATTAATTGAATTTACTGAATAAAATTATATCTGAAAAAAAAAAAAAAAAA AAAAAAAAAAAA (SEQ ID NO:2) [The bold 42-bp sequence was used for constructing pHANNIBAL(16D10#1 ), and the underlined 271 -bp sequence was used for constructing pHANNIBAL(16D10#2)] pHANNIBAL(16D10#1 ): (a) Construct: (Xho\+42bp 16D10 sense-strand-sequence + Kpn\ - 54bp) + Pdk intron + (C/al +42bp 16D10 antisense-strand-sequence + Xba\ ~ 54bp)

Xhol CICGAGGGCAAAAAGCCT AGTGGGCCAAATCCTGGAGGAAAT AATTGAGG1ACC ------ ------ Kpn\ C/al

ATCGATTCAATTATTTCCTCCAGGATTTGGCCCACTAGGCTTTTTGCCICIAGA

Xbal SEQ ID NO:3) (b) PCR detection: primers H1F1 & H1R1 (234bp PCR product) Primers H1F2 & H1R2 (273bp PCR product) pHANNIBAL(16D10#2) (1). Construct #2: (Xho\ + 271 bp 16D10 sense-strand-sequence + Kpn\ = 283bp) + Pdk intron + (C/al + 271 bp 16D10 antisense-strand-sequence + Xba\ ~ 283bp) 70 WO 2006/044480 PCT/US2005/036711

Xho\

CTCGAG CCTCAAAAATACCATAAAGTTAATTATTCTTCAATCAAAAAA ATGTTT ACTAATTC AATT AAAAATTT AATT ATTT ATTT AATGCCTTT AAT GGTTACTTTAATGCTTTTGTCTGTCTCATTTGTGGATGCAGGCAAAAAG CCTAGTGGGCCAAATCCTGGAGGAAATAATTGAAGAAAAATGATTGA AGAAAAACGTTTAAATTAAACGATAAATGGGAAATAATGGAATTTAAA TTAAGCTAATTTTGATGGTTTCCTTTGTTAATTTC GGTACC

Kpn\----------------------Pdk intron--------- C/al ATCGATGAAATTAACAAAGGAAACCATCAAAATTAGCTTAATTTAAATT CC ATT ATTTCCC ATTT ATC GTTTAATTTAAACGTTTTTCTTCAATCATTTT TCTTCAATTATTTCCTCCAGGATTTGGCCCACTAGGCTTTTTGCCTGCA TCCACAAATGAGACAGACAAAAGCATTAAAGTAACCATTAAAGGCAT TAAATAAATAATTAAATTTTTAATTGAATTAGTAAACATTTTTTTGATTG AAGAATAATTAACTTTATGGTATTTTTGAGG TCTAGA (SEQ ID NO:4)

Xal 71 WO 2006/044480 PCT/US2005/036711

Table 2: >2E07>msp 1 »bankit4B2031»>AF531160

GATCAAACAATCTCCTCAACAACTAAAAAAACTCAAAAAACACCCCAAAA

CCAAACTAAAAAATCAAAAATGTCCATCTTCCTCACTTCTGCTCTTCTAA

TCATTTCATTAATCGCTATGACCGAGGGAGCAGGCGATCGAAGCGCTTCA

ACCTCTACTGGTTGTACAACCTATrTTGGAATGCTAGATCATGCGGATAC

CAAGGAAAATAACAAAAGAAAAACTTTCAAACCCAACGATAAAACCATAT

C C AAC ACTTTGC AAGTG ATTG GTG G GACAAAGTTC AGC AATACCTC GGTG

GCGTTGGTTGTCGGTGATGAGGTGTTATGTATGGCTAAGACAGGAGGTTC

AGGCGATTGCGGAATGCGCTACGATGCGTTGACTGGATCAATGAAATTTA TTCACCGACAACAAGTGTGTCATCCAGCTTGTAGGCTAC GATATT AAAAC T AAT AT AACTCTTCTC AAAATTAATGATGTCGACTTCAAAATTGTC CCT A CTGATAAGAAAATTTCCCCGAAGGCTTGTACTATGAAAATGTGAGGGAAA AAAGTAAAGAAAATGTGTAAATATGGAAGGATAAAAACTAAACAAAAAAG AATGTGAAGTAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID N0:5) >2G02>msp2»bankit478474>»AF531 161

GGATTTAAAAAATTAATTT AAAAAAAGTGAAAAATTCAATTAAAATT AAA AAATAI "rrrrCAATGAATTTATTTTCTAt ’ΓΓΓΓΓΙΑΙ “ΓΓΓί'ΑΤΤΠΧΑΑΤ

CGGGTTTATTTGGGCTGAATGTAGCGGAGATTGTTCTATAGAGAACCAAT

ATAATTATAAATGTGAGGATAGAAGTGAATTTTGTGAAGAATGGGGAAAA

TACTGCGAAAATGTCTTTCTTCACAAATGTGTAAGAAAGGCTTGTCCAAA

GAAATGTAAAGTTTGTCATAGTTCTGGTGAAGAACCTAAACCAAATCCTA

CAACTATAACAACGGCATCAACAATAACAACACCATTAGCAACAACACCT

CAAAACTCAGCAGTTACTTCGGCAACCTCAAAAAGTGCTACTCCATCAAA

AACTTATTCAACCGAGACAACCGAATGTGCTAACACAACTACTGAGGAAT

ATGAAGCAACTATTGAGGAATATCAAACAACTACAGAAGAATATGAAGAG

GTAACAACCCCTATAATTACAACCACCAATCCAACAACTTATTTATTAAT

GACTACAATAGTTGAAGAAATTAGTGACGACGAATTCAAAGACGCAAAGA

AGATGAAATGTAAATCATGTAATGCAAAAAGGAAGAAATTGGCTGAAATT

TATGACAAATATTATCCGAAAGTTAAGATTCATGCTAAATTGTAAATTAT

GATGGAAAATGTTnTGAATTGTGAAAAT AAAAATTT AATT AACCCAAAA AAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID N0:6) 72 WO 2006/044480 PCT/US2005/036711 >2G1 Q>msp27»bankit482965>»AY1 35363___ GATAGCACAGATCTAl "ΓΓΓΙΑΘΑΙ "ΓΓΓΓΓΙΑΘΟ'ΓΓΓΓί'ΑΘΑΑΑΑΤΤΤΤΑΑ

TTTAAAAATTATGTTTATTCTTCCAAAACCTTTTTATCTTTTAATTTTAC

TAATTATTTCAACAATTTTCCTTTTATTTTTAATTCTTCGTTTGCCTTCA

ATTTCTTACCAAACAAATCAATGTCAACATTTATGGGACTCTTCCGAGTG

CAAAAATTTAAATAATTCTTTAAATTGGCATCCAATAACTTGTTTTATTG

ACGGAAAGCAAAAAAGAGTTCCCTGTCTTCAACAAAATGATFTAAAAGAA

GTTTATCTTCCATTCAATTCAl "rrrfGAAAAAACAATTTGATTTGTATGG

AGAGACTGACAAAAGCAI "ΓΓΓΓΓΓΑΑΤΤΤΤΑΑΑΑΤΤΤΑ TTITIAACAAAA

TTTGCGGACAGATA AAATTTG TCCGCAAAATATTTGCGG ACAAATAATTT TTTTTGTTGTTAGGCI "ΠΤΠΑΘΘΘΤΑΙ “TlTITTCTACAATI ΤΓΓΓΘΘΑΘ

TrrrrrrCTACAAl "ΓΓΓΓΓΘΘΑΘΤΤΑΑΤΤΟΤΑΑΑΤΤΑΤΑΑΤΤΤΤΑΤΤΑΤΑ

TATTTTATAATTTTAAAAT ΤΓΓΓΓΓ CTrrrrAAGAAAACAATTCTTTTGA TTATTTTACTTCAAACATTCCTCCACGTC ΤΓΓΓίΑΑΑΑΑΤΑΑΑΑΑΤΑΑΑΑ

TGGTGGCTGCAAATCCAATTGAACAATTTAGCAATGTGGCTATTCGTCAA

AGAATAAAATGTTTAAAACCTGAAAATGGATTACCAATGAGCGTTCAATG

GAGTCCAATTCCCTACTTCTATCCTGTTCAAATACTCCAATTTGGCTTTG

ATTATTTT ATG AG AAATCGAACAG AACAG AGG AAATTAATTG AAAG AAGG

TTATCAAACAAAGATGATTTCTTGGTACTAAAAAGTGGAGAGAAAGTTAG

CGAATTTTCAACTrrrrrCTGATTTGCCATTTTACCTTTTCTGCAAAATT GAATCAATGGATGCTTCCTTGTAATAI "rrrrGAGAAGATTGGGGGAATT (SEQ ID N0:7) >4D01 >msp3»bankit478504>»AF531 162

GACAATAAACGATCCAATTTCCTAAAATTTTTTAAAAATTTTTAAAATTT ATTTTATGCCCC ΤΓΓΓΓΘΤΤΤΑΤΤΤΑΑΑΟΑΑΑΤΤΤΘΟΤΤΘΑΤΤΑΤΤΑΑΤΘ

C C AAAATT AATTTT ATTATTTT ATTTAATTATTTATGGAATTTT ATT GTT

AATAAGTTTAAGTGAAGCATTTGGGTTTGGTGGAGGATGTGGATGCCCTT

GTATGCCGCAACCATGTATTCCACAACCACCTCCAATTGCTTTACCTTCT

CTATGTTTCCCTCAAATCCAATTGCCCTGTCCCCCTCCATCTTGTGGATG

TTGTGGTAGAAGAAAAAGAGAAAGTGGAGCTTCAGCATTATTAACAGCAG

TTTCAACAAAGTCGGGAATTAAAAGAATTGGAGAAGAAAAAAATCATTGT

AATAATCCACACATTAAAAGAATTATTTTAAAGAATTTAATTATTGGAGA

TTGGGTTGGTACAAGAAATGCAATATATTCAGAATTAAGAGCTAAATTAG

GGGGGAATTATATAATTAATTGTGCTCATGCCCCCTCATTTGCGTATTCT

GGTGATTCTGTGATTGATTATTGTGTGGATGGACATCAGGCAATAACTTG

TGCAGTCTTCAAAATTCAATGAGAATAAAATCAGAATGAATTCTAinTT

TTAATAAATATAAAAATTITTATAATATATTTTGAGCATTATAAATATTT

ATAAATTAG’rrrrriTrGATAAATTAATTTGAAAATGTATAAATTAGTTT TTACTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID N0:8) 73 WO 2006/044480 PCT/US2005/036711 >4P03>msp28»bankit47921 4>»AY1 35364_

GACGCAATTCCATTTTGCGTTCAATCAATTTAGAAAAAGG CTG G AAAT AA

TGATTCATCAACAACTTTATTATTAGCGCTTAGTGTTCCGGGCTTTCATT

TCAACAGAGAAATTTCAAATTCACCTATTTGGAGCTTGGATCAGTTCTTC

TATCGACTTGTAGTCCTAACCTATACTAAAAATTTTTTAAATTAACCACA ATGGAACTTGCTATTAACAGTCGATTGTTATCAI “ΓΓΓΓΘΤΟΤΤΤΑΤΤΟΟΤ

ATTCATATTTCCTTTAAATGTTGTTGCTCAACGGCATCGTTACCCACACA

ATCAAGGAAATTATTTCAGCAGACAAAAGCTGCAAGAAATACAGAAGGAG

GAAAATGAGGCTGAAAATTCTTTACCAAAAATCmGCGCGCATGGAGC

TTCAGTAGCCGGCCGTTGCGTATGTGATCATGGTTGGGCCGGTACTAATT

GCCAGCGGGAAATGCATTGTGCTACTTTTGAGCGAAATGCTAATGGAAGC

TGCCCAGTCTGTCAGCCCAATTTTCAAGGGGATAAGTGCGAATATATTGA

ATGCCAAAATGGAGGCCAAGAATCATTGGAAACTCAGAATTGTAACTGCC

CAAAGCCTTATTCTGGCCG TITrrGTGATGAATTACTCACAGGAAATGTC

TACTACTACTATAACTCTAAAGTAGCAACCCTTGGTCCTCTTGGACTTAT

TTCTGTTATACCAATGATTTGTCTTTATGTTTTATGTGAAAAGATTTGCA

AGGAAAAGACAAGTGAGACGGATTGAGAAAACTTGGAATTTACAGAGCAG

TAAAACTGTGAATCCTGCTCATATTGAATTTCTATTAAGGGAAAAAAAAA AAAAAAAAAAAAAA (SEQ ID NO:9) >5G05>msp26»bankit478498>»AY135362_

G ACCTT AATC AAT AAAAAATATTTTTT ACAT AAAAATGTTTTATTT ATTT

TATTTTAAATTAI “rrrrATTTTCCTTAATTTCTTTAAATAAAGTTAATGG

ATTTTGTATGAAGACTATTTGTTCTGCGGACACCGATTCTCGACACCCTG

TAAATCGAGTAATCGGTATTGGTTCTGATGGAATTAGTGGGGAATATAAA

GCTTTGAGACGAAATGATCAAATTGTTGAAGCTGTAGATTTAAGTTGTAG

AGAAGGAAGTTTTGTTTATTCTCCCTTTGAAGGGGAAATTTCTGCTTGGA

GACCTTTTGATGGAAATGGGCAAGATGAAATTAGAACGGATGAAAATAAG

AAGAATACAGATGGATGCAGACCTGACCGAGGAGTTAGAATTGATGGAAA

AGGACAATGGCAGGGATATCACGTCCTTATTGGCTCTGTTCGTTTATTCC

GTTACAGTGGACATGTTAATGCTGGACAAAAAATTGGTGTATCTTTGGAT

ATTGAATGTGAATTGAAATTAAATAAACAAAAGATGAATAAACGTCCTCG

AAAAGAAGAAAATTTTGTCAGAGTTTATTTACACAAGGAAGGACGTCCAA

TTGATCCAACACATCATTTAATTGATTGTATGTGTATAAACCAAGTCTGT

GAGACAAACAGAATTAATGCTTTGGAAGGACCGTTATTTAAATTTGACAG

TCGTTTTAACGGTGTTAGAGGATGGGAAATTAAATGTCCAGATATTCAAC

AAATTGAAGAAGAAAATTCTTCAGAAGAAGAGGAAGAAAAGAAAAAAGAA

GAAAATAATTTAAATGAAGAATGGGGAACTCCAAAATTTATTCACCTATA

GAAGGGGAATTGGTTGGAAGAATTAGAGTTAATAGTGAACCTGGGGCACA

GACTTATACTGGATGTACTAATGAAGGAATATTTATGGTTGGGGCTGGAA AGTGGAATGATTATGAAGTTCGAATTT (SEQ ID NO: 10) 74 WO 2006/044480 PCT/US2005/036711 >6F06>msp4»banklt482257>»AF531 163

GTTCATTTAAAAAT I I I I ICCTAAAAAACTTCAAAAAAGCAACTTTTATT

GCGTAAATGAAAGAAAATCTGTTTAAAAAGAGCCTTATAGGCCTATTTTT

GTTGTTAGCATTCAATTTTACTGAAGCTAAGGACTCTGGAGAGAATACTA

GTCTTGAAGCTAGTTTGAAACCAACTAAAAGTATTGAAAATGCTTCCCTA

GAAGAAAAGAATCAAAAAGAAGAAAATGGAGTAACATTCCCGGCAGAAGG

TCATGAAATTGTCGAAACAAAAAAAGAAATCAACTCACCAGAAGAGGTGA

CAGATTCAACTAAAGGACAGGAAAATTCCGAGGATCGTAAAGTGACAATG

AATGGTGATGAGTCTGAGGCCGATAAATTAAACAATGAAAATGTTGAGGG

TGAAGAAAAGAAAGCAACTGAAAACAAGAATGAAGTTGAGGAAAAAGAAG

TTTTAGAGGATGAGAAGACAAAAGAAGAGGAAGATAAAATTAGCGATGAG

CCTGTGAAGACAAAGGAAATGAAATCAACAAACAATGATAAGGAAGTTGA

AGATTTGAAAGAAGAGGAAGAGAAAGTCGAGGTAAAAGGTAACAAGGATG

AAGAAGAAAATAAGGAAGAGAAGAAGGAAGATAAGAAGACAAAGGATGAA

AAAAAG GTTCC AG AGGTTATTGAG GG AGAG AAG AAAAC ACCC AAG GAAAA

GGAACACAAAAGCCATTGGTTTATGGACAAATTTAAACATGCTTTCTGTT

TCATAACTCATTACTTCTTTTGTCCATCTAACTCTGCAGAAAAAGGCAAA

GAATCCCATCATGAAGGAAAAGAATCACACCGTGGAAAGCGTCTTAACTC

TGATTTTAGTTCrrTAAGCAGTGATGAGGAAATGATTGAGAATTTTGAAA

ATGCCCACGAATTTAGTGAAGAAATTGAAGAAAATGGGGAATTTAAAGCT

AAAATGAATGTTGGTGCAACATACTTCAAAGCTGAGACAGATAATTCTGG

AAAGATGCGCGGCAAAATTGAAAAATTTAATGCTGAAATGCATAATTGAA

AAGATTGTAAGGATGGTGGGTGTGCTGATGAGTAAAACAAAAAAAAGCAA

TCCGATTTTATTCTAAATTTTAl ΤΓΓΤΓΑΑΑΘΤΘΑΤΤΟΟΑΑΟ AAGTG ATT

CCATTAACCCCTCAAATTTATTTAAAAAAACGAAATTTTAAAAGTT CTGG

ATTTATGTCCCAAAAATTGTACAAATTATTCAAACAAACTCAATGGTTTT

GGACATTATAI “ΓΓΓΓΓΓΑΤΤΑΤΤΤΤΟΤΑΑΟΑΑ ΤΓΓΓΓ ATTAATGTTGAAG TAAAAGATTAATTCAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:11) >6G07>msp5»bankit482261 >»AF531 164

ATTCTTAATTTATTTAAAGAATTTATTCTGCATGATGAAATTAATTAATA

TTTTArrrriAl-rrrrrGTTATTTTACTGAATTCTATGGCTTTCGGAAGG

TTTTCTTTATTTTTGGAAAAATCAAAATTTCAATTGAATATTTTGTTTTC

ATTCCAAGATTTTCTCACACAGATCCCGCTTAGTGTGAGATATCGGATAA

AGCTTCATAACCTCTACAATTTTAAGATATCCACATTACTCCGTCCAAAT

TCCCTTATATCTCCTTTGATCCCTACAAATCTACCGATATCCCCTCCACC

GATATTTTCCTTTTCCGAACCTTAACTTCCGATCAATCCGCTATCTGGAC

AAATC GTTATTCCTCTAAAC AAG AATTTATG CUT AAATGT AT AAAAC C

AATCTTTAATATTCTTCAAAAAAATITCAGTCCTTCTCTCAATTCAGTG

CGTGCTAAACGTCAAGGCTGGGGAGGATGGGGTTGGAACCCTCAAGTTCA

AACAGATATTGATCGTCTTCGTATTGATAAAGACAAACTGCGATTAGATA

TGGACCGTTTAAGACTAGATCAGGATAGCTCTTGGGGATGGGGAAAATGA

GAGAATCAAAC GACTAATTTAAGTG TAAC G ATTTTT AATT AACG ATTT AT

AAATTAATAAATACTTGATTGATACACAATTTAGATAATTTAAAATAAAT

TTTATTAAATGATAAAATTAAATTGCCGTTTTAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA (SEQ ID ΝΟ.Ί2) 75 WO 2006/044480 PCT/US2005/036711 >7A01 >msp6»bankit482263»>AF531165 gaccaTcaaaTCaTcTcctcatcaactaaaaatccctaaaaacaccccaa

AACATCCATAAAAACAACCACGAAAATGGCCACCTTriTCACTTTTACCC

TTCTAATCATTTCAATTATTGCCACAACTGAGGGAATGAATACTAATCGA

AGTGCTTCAACCTCCGATTCTCTCAAAGCCCAAAAGGATTGTAAAGTGAT

ATATGGCATGTTTGTGCCTGTAGCAGGGTCAAAAATGCATGGAGACGCCA

AAAGCGCAATGAAGCCAAACAATCCAAGTCTCTCCAATAAATTAATTGTA

TCAGGTGGCAACTCAAAATATTCAGTGACTTTACAGGTTGAAAACCAGCC

GAAGTGTGTTGCCCAAAATGACGGAAACCCTGTAGAATGCCAAATTCAAG

GAGACAAACTTTCAGGAAAATTGATTTATGATATTGAAAACGGCCCTTCT

GTCAACGTTCCCTTCAAAGACACCCCAATCTTTGTTGGAAATAAATGCGA

AATTGTriTrGTAGACTACGATAAGGACCACAAATTAACTCnCTTATGA

ATAAAGTAAAGCTGATGATTGAGCCGACTGAAAAGCAAATTGTAAAGGCT

TGTGGAGTGAAAAATTAGATGGAAAAATGATATATGAATGAATGAATGTG

AGAGGGAGGGAAAGAAAAATATTTTTAAAATTGAAGAAAGCATTCAAAAA

AATTAAAAAAAAACAATTCTTCAAATAATATAACCTTAAAATTTCTGATA AATTATGTTTTTACAAAAAAAAAAAAAA(SEQ ID NO:13) >7E12>msp7»bankit478534>»AF531166

GCCATCAAATAATCTCCTCAACAACTAAAAAACTCAAAAAAACACCCCAA

AACAACTCTAAAATGGCGGCTCTCOTCTTCACTTCTACCCTTCTAATCAT

TTCATTGGCTTTTATTGCCATAGCTGAGGGAGCAGGCGATCGAAATGCAT

CAGCTTCAAGCCCTGGTTGTATGCAGGTTGCAACCCTTATTCATATAGGG

GAAATTCGCCCAGCAAAAGCAAACAAACCAGGTGTACAAAATACTCTAAA

AATGTCTGGAAATGTTCAAACATTCAAAACTACTCAAGTGACATTACAAG

TAGCTGGGCAAGAGCCTTGTACCGTTAAAATTAATAATGGCGAAACCAAA

TGTAAAATAACCGGAGATGAATTAAATGGAAAATTAATTTTCAAAACTGA

AAAAGGAACTGAAATTTCTGCTTATTTCGAACTGGTTCCATTATTTTCTG

AAAATAAGTGTGTTATTGAACTTGACACTTATAACAAGGAAACCCATGAA

ACTAAACTTAAAATTAATGGAAATAATTTTATGATTAAAAAGAAGGAAGG

TAATGTGTCAATTAAGTGTGGTGGAAGAGCTAATACTGTTTAAATTTTAA

AAGTGTGAATTGAAAGAGGAAGAGAATAAACAAATGTGAAGATGAGAAAA

AAATAT ntGAAGAAAGCATTATAAAAATATTAAAAAAAATTAATTCTTC

AAATT riTATTTGAI “rTTrGAATAAATTATTTT ATT AAAAAAAAAAAAAA AAAAAAA(SEQ ID NO: 14) 76 WO 2006/044480 PCT/US2005/036711 >7H08>msp8»bankit482285>»AF531168 gctcattaattagttaaaaaatttaaaaaatkatttaaaaaatgaaaatt ΤΑΤΤΤΤΑΑΤΤΤΑΑΤΤσΓΓΓΓΓΟΤΑΤΤΤΑΤΤΤΤΑΑΑ ΓΓΓΓΪΑΤΤΤΤΘΤΟΘΑ

ATTGGCAAAAAGGAAGGCAACGGATACTGAGATTCCTGAGCAAAATAAAA

AGCAAAATACAAGCAACCATGCCCATCAACAATTAACTCCTTCTTCTTCA

AATGCTGATAATGAGAAGCAAGGAAATCTTTCCTCTGAAGCTTCAAATAT

TCGAGGAAAAAATATTCTGCATGATCAGTCTGCTATTAAAAACAATTCGT

TAACTAATCAACAATTAGGAGCCTCCTCTTCTAATGCTGGGCAACAGAGA

AATAATAATTCGGATCTTTTAAAATTAACAATTATAAATCATTTGTTATC

CCATCGCCAATTTAATGCCTCTTCTTCAAATGCTGGTCAACACAAAAATA

TTCCCTCCGAAAATCTAAATTTTCATCAAAAAACTATTCCAATTGCTACT

AAAAATAATTTGTTCCCCAATCAGCAATTTATTGCATCTTCTTCAAATGA

TCTTGATTTTCAACAAAAAAATATTCCATATGGAACTAAAAAGAAGGTGT

TACATCAATTTATGCCATCTTCTTCCAATGCTAATAAACGCAAAAATAGT

TCCACGGAATATTTAAAATATGCAATTAAAAATAGATrrrrATCTAATCA

GCCATTTGATGACGACATTTATGGTAAAAAGAAAAATGTTTCCCCGGAAT

ATCAAAATATTCAACAAAAAAATCTTCCATATGTCCAATATGCTATTGAT

AATAATTTGAAATTGCCAATTCCAAAAAATCCTAAAGCACTTCCATATGA

TTTGTCTAAATACGCATTTAACTTCCCCAATATGAACAAGAAAAATATTT

ATGAAGGAGCATATGATCCTTATTATATTAATTTTCAACAATAACAGATT

TGGCTAATAAAACGTTGGAAAACGACTAAGAAGTTATACATTTGACATAA

ATTAAATAAATAAAATTAAATTACTATTATAAAATTGTTAATTATCGTAA TAAAATTTTTTAACTCAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:15) >8D05>msp9»bankit478548>»AF531 169_

CTAGTCAGTCATTTAAAATAATTTAATATTCCTCTAAAAATCCCTAAATT

AATTTAMTATTTCTTTAATCAATTTTTCTTCAAAAAATTTAAAGAAGGA

AATGTrTrrACAAAAACAATTATTGrrrnGGTTGTTCTTCTATTAGCCT

TTTCTCTTGTAAAGGGAGTAACCGAGAATAAGAATAAAAGCGAAATAAAA

AATGAAACAACCACAAAAGTAATTCAAACATCAACTGGAGGTTATGATGA

TAACGAAAAAGCAGACTATGGCGATTTGGCTGCAGAATTGGCTAAACTTG

TTGAGGAGGAAGATGAATTAAATAAAAAGAAGAATGCTTTGAGTTCGGAG

AATGGAAATAAAAATAGCACAGGAAAGCCTTATATTCAAAAAGATAAAAG

TAAAAAATATTTGGAAGAAGATAAAGGAAAATATGAGGAAAGAAATTCTA

GAAATAAATATGAAAACTCGGATGAAACCCATGAAAGTGAATCAGGTTCA

AGTTCGGATGAGGATTTAGATGAAGATAATTTAGAAAGATTGCCAGGGCC

TTCGCGACACAATGAAGGAATTTCTAGGCGAAGAGTTGAAAAGGAAAAAG

GTGGAGAAGATGAGGAGGAGGAAGAAAAAGAGCAAGAAAATTCTAATGAT

AAAGAAGAAAGAAAGAAGAAAAGGAACACCAAATATAATCCAAAAGATGA

GAGTGAGGAAGATATTTCTTTTGATGGTCAAATACCTAAAAGTGTACGTA

AATTACTTAAACAATTAGCAGCTGGTGGAAAGAATCCTGTAATTATACCT

TTAATTATAAATAACAACAATATACCGAATCGAAGAGAAGATGAGTCTGA

GGAATGGAATAAAAAAAGACATGGGAGACCTCATAGATTAAATGATTGGA

ATAATCCGTTTCCTCCATTCTTTCAATCTTCAATGTTTCAACCACCAATG

TTTCAACCACCTATGTTTCCACCACAACAGCCACCTTTTGGTGGCCCTCC

AACATTTGCTCAGCACTTAATCTTCCTGGAGGGCCTCTCGGAGGAGGTCT

TGCTGGCAGTCTTCCCAACACAAATCCATTTTTATCACAACTAAATCGTG

GTGTAAGTCCTAATCAATTTCCCAATCCTCCCTCTAATCACGTTCCACCT

TTTGGGCAACAAAATCAATTCTATCCTCCTCAACAACAACAACAAAATCA

AGTCAACCCACAGGGAGCAGATGGCAATGATGTGAAAAAAGTGAATTAAA CAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 16) 77 WO 2006/044480 PCT/US2005/036711 >8H1 1>msp1 Q»bankit478550»>AF531 170_

GATCGTCATTCTTGTAAACTAAAAATCTTCAAACTTCAAAAAATATTCCT

TAAACTTCTTCACAAAAAGAATTGAAAAATGTTATTAAAATTCTTTCTCC

CATTATTGCTTTTGGTTACCCTTATCTATTTGGGGTGTTCTGAGGAGGAT

AAGGAAGACATTGCAAATGGTCCTCAGGAATCTGAGAATCAGGTTGATCA

AGAATTGGTTAGATTGAAAAGAGATGATGAAGAAGAGGAGGGAGAGAAGG

CTGAAGATGAAGAGAAGCCTGAAGAGGAGGGAGAAAAGGCCGAAGATGCT

GAGAATGCAGAAGGAGATGCTGATAAAGGAGATGCTGATGAGGAAGAAAA

AAAAGAAAGTGAAGATGAAGAGAAAAAGAGTGAAGGTGAAGAAGAAAAAG

CGGAAGGTGAAGAGGAAGAAAAAAAGGATGGAATTGAGGAAGAAAAGAAG

GATGAAGATGAAGAAGAGAAGAAAGATGATGATGAAGAAAAAAACGAGGA

AGAAGGAAAAAAGGATGATGAAGAAGAAAACGTAGACAAAGAAGAAAAGA

AAGATGATACGGAAGAGAAAGAGGATAAACATTCAAAGGATAAAAGTAAG

AAGGATAGTAAGTCCGTTCAAAAGGATAAAAAGGAGGAGAAGGAGAAAAA

GGATAAAAGTTCAAGTGGTGATAATTCTAAAACAGATAAATCAGATAAAT

CACATAGTAATCAAAAACAAGACAGCAAAGAACCATGTAATGGGGATACT

GCTTACAACTGTCCTAAACTATCAGGTCTTTGTGAATCAAAAATTCAAGT

ACAACAAGACTTCATGGGTGAAAAATGTTGTGCTACATGCAAAAATTCGG

TTCCTGTCGCGAAGAAAGATATACCCTTATGCACTGATTTGGCTGATAAT

TGTGATCAAATAGCATCCACCTGTGGGGAAGAGGCGTGGCAACCGACTAT

GATTTCTGATTGTGCTCAGACATGCGATAAGTGTGAATTACATTTTCAAA

TGTTGGAAAAGAAACTTGCAGCAGCTGCTGCTTAAAAATTTTGAAAGGAA

AAGMTTTTTATCAAAAATATATGTATCAAAAATATATTTTCTTTGATTT

TCACACCCTTAATACTAAAATTTCAATTTATTCATCAGTGTTTCTCGTAA

TTATATTTTATTAATTTGTTTCGAGATTTAGTAAAGATGCTTTAAACCAA AAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 17) >9H1 0>msp1 1»bankit478544>»AF531 167_

GGAATTTTTCAAAAAAAGTAGGCTGGAGAATAAATTTATTGAAAAACCAG

AATTCTTAAAGTTTCAACCATTTAAAAAATGTCAAACAATTTTAAAACTT

GCCCAGCTTTATTATATTTATTGCTTCTGTTGGGAAAAGCAAGTTGCAAT

TATnTGAATCAGAATTAAGCTTAGCTAATGACAAAACTTCTATAGTTCG

CAAATGTTGTCCTAAGGAGAAGATTAGACACCATCGGAGACCGTTGCATT

GCTGCCAGGATGGGTTATTCCGTGATGAAGTTGATGGTTATTTATTAAAA

GAATGTGCAGATCAAGGTGATTCCATAGTOAAAACAATTAGATGTGCTCA

ACAAGAAATACATGGTGAAAATGCAGTGGAGATTTGCAAAGCCTATTGCT

GCGAATTATTCAGAGATAATAATTGTTCCAAAATATGCCTAACAAACATT

ACCAAAGTAAACATGTCTATTGAAATATTATTTGAGCTGTTAAAAAAATG

CAGGAATCATGAGAATTATGGGGAAGTCCATGACTGTATCCATTC AAAAA

GACCAAAAAACATGGATGCCGCAGAGTTGGAAATTTATTGTAAAAGGGCT

ATTAATATGGTTTAAATCTGGAATTTAI“rrrrrAATTTATTCTACTCGAT

CTCCT nTATCTATTTAATTATTAATTTA TrrTIGGCAATAAAATTTAAT AAAAAATGTAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 18) 78 WO 2006/044480 PCT/IJS2005/036711 >10A08>msp34»bankit487923»>AY142117_

GGTCGTCATTCTTGTAAACTAAAAATCTTCAAACTTCACAAAAATATTCC

TT AAACTTCTTC AC AAAAAG AATTGAAAAATGTTATTAAAATTCTTTCTC

CCATTATTGCTnTGGTTACCCTTATCTATTTGGGGTGTTCTGAGGAGGA

TAAGGAAGACATTGCAAATGGTCCTCAGGAATCTGAGAATCAGGTTGATC

AAGAATTGGTTAGATTGAAAAGAGATGATGAAGAAGAGGAGGGAGAGAAG

GCTGAAGATGAAGAGAAGCCTGAAGAGGAGGGAGAAAAGGCCGAAGATGC

TGAGAATGCAGAAGGAGATGCTGATAAAGGAGATGCTGATGAGGAAGAAA

AAAAAGAAAGTGAAGATGAAGAGAAAAAGAGTGAAGGTGAAGAAGAAAAA

GCGGAAGGTGAAGAGGAAGAAAAAAAGGATGGAATTGAGGAAGAAAAGAA

GGATGAAGATGAAGAAGAGAAGAAAGGTGATGATGAAGAAAAAAACGAGG

AAGAAGGAAAAAAGGATGATGAAGAAGAAAACGTAGACAAAGAAGAAAAG

AAAGATGATACGGAAGAGAAAGAGGAATAAACATTCAAAGGATAAAAGTA

AGAAGGATAGTAAGTCC GTTCAAAAG GAC AAA AA AAAAAAAAAAAA AAAA AAAAAA (SEQ ID NO: 19) 79 WO 2006/044480 PCT/US2005/036711 >l0G02>msp29»bankit479222>»AY1 35365__

ATATTTAI I I I I IAATTTAACAAAAATA I I I I IAATTAAAATTATTTATT

TAATGTTTAAATTGTTGrrrrrCATmGTTTGCCTTATTAAATTCTGTT

GATTGTCTTTTAAAATTACGAACACTGGATAAAGAACATCTTCTGGTTGA GGAGAGATATGCCAAGGAAGATACGCTTTATC ΤΓΓΓΓΘ'ΓΤΤΓΓΟΟΤΑΘΑΑ

CATCAAATGCCCCATATTTTGGAGCAATGTGTCTTTATGTTGAAGCTGTT

TTAACTTGGAAAGGAATTCCTTTTCATAGAATAAGTAACCAATTCTTTCT

TGGTTCAAAAACTGATGGAGCAATTCCTTTTGCTATTTATAACGGGAAAT

ATTTGGATGGAGCAGAAAAAATAATTGAAGAAGTTAGAAAAAAGGGAAAT

AAAAAATTGAGTGATGAACATGATGATAATATTAGAAAATTTGCAACTAG

AACCTTGCTAAAGACTCTAATTGCTGATAGAACATTTCGGAGAGATCTTC

CCCATGCAACAATTCCAAAAAATAATTCCGAAACACAAATAGCCTCTTCT

TCATTATCAAATAGTGCACCAGCAACTCCCAAGGGTGGAATCCCTACAAG

AAAGAGATTTAGTCCAATTGATATTAAAATCCCTCATACTAAAAATGAAG

AAATAATAATGGCAAAATCTGAGGGGCATTCTCCTGGAAGTTCTTTCTTT

TCTAGAACTATTGCTCATTTAAAATTACATAATAATAATTCTCCAAAGAA

AGGTCCGGGTGGTCTTGATTGGATGTTAAAAGATGAAGGAGTTCGTGAAC

AATTAATTCCAGTTATTCCAGAGGCT TnTTAGAAGAAAGTATGAGTGAT

GAATAHTTGATTCCCCGGTAAAAGATAAAAATGAAAAGAAATCAAAAAG AGAGGAGGAAGATGAAAGTGATGAAACAAAAATATCTAAAATTAAATATT CCATTAAATTGACGTTAAGTCCAGAATTGTGGAAAGATTATTTTAATATT TTAAATAAAATAAAAATAAATGGAAGGGAAAATAGAGAAGAAATTAATTT ATTGAAAATAAATTTTCTTCAAGAATATTTCGGATTCTTAGCAAGAATTG ATGATGATTGGGAACGTGTAAATTCTATTCTGAAAAATACAATTAACGAT ATTTTAAAGAAATTAATTGTTGATAGCCAAATACC TrrrrGTTGGGAAAA AAGGTTGAGAGAGATTAATGGGAAAAATATTAATGAAGTTGAAGTATTTA ATGAATTTAAAGATAAAATAAAATCGTTGGGTATAATAAAAAGTTGACTG AGGCAGAGACTAAAAATAAT TrrnGCATGGAAATAATCCAACTTTGGCT GATTTTGCCCTTTTTGCTTTTCTCAATCAATTTTTTGAATTTCCTTTAAA T ATTC C AG AATTT AAAG AATT ATTT ACC CC AG AAAAG CTC AGT AATG AGG AAAAAGAATTAATTGCG AAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NQ:20) 80 WO 2006/044480 PCT/US2005/U36711 >11A01>msp12>>bankit478790)>>AY134431

GGCATCAAACAATCTCCTCAACAACTAATAAACTCAAAAAACACCCCAAA

ACAACCCTAAAAACAACCCAAAAATGTCCATCTTCCCTACTTCTGCTCTT

CT AGTC ATTTC AATAATCG CTATG ACCG AG GGTGGAGGCG ATC GAAG CGC

TTCAACCTCTACTGGTTGTACAACCTATTTTGGCATGCTTGATCATGCGG

ATACCAAGGAAAATAACAAAAGAAAAACTTTCAAACCCAACGATAAAACC

AAATCCAACACCTTGCAAGTGACTGGTGGGGCAATGTTCAGCAATACCTC

GGTGGCGTTGGTTGTCGGTGATAAGGCGTTATGTATGGCTAAGACAGGAA

GTTCAGACGATTGCGGAATGCGCTACGATGCTTTGACTGGAACAATGAAA

TTTATCATTTCTGATAATATTACTGTTGAAGTGGGTGTGGGTTTATATAA

TTGGTGCCAGACAAATGGAAAGGCCCCTGTCACTAACATACCATCCGGAG

CGTTCATGTTGCCCCGGAAGTAACTGGTGGCCCACAAAAGGGCAATCACT

ATTGATTACAACCCAAATATCTATAGGATTTTGACATTTTCTGGCATAAT TTAGGTATIITCTGACAI “ΓΓΓΓΟΤΘΑΟΑΓΓΓΓΙΑΑΟΤΑΘΑΑΤΤΑΑΤΤΟΑΑ

TTGAAAACAAAATAATAGGATTGACCTAAATGAGCGTTTCTTG GAT ATCC

TTTTAACAGGAGCAGTCTCTAATTTTGTAAGAGCTCCTAATGTTTACCCT

CCTCCATCTCCCTCCCCCTCTATGCTCCTACCAATGACTGATTAAGTTAA

AAATCGTACATAAAATGGAGAGTGTATAAATCTGGGTGTATATACAATCA

GGATTCGACTTTATAACATTTGAAGGTTCCATTTGAAGACGTrrrrrrCT

TCACCGACAACAAGTGTGTCATCCAGCTTGTAAGCTACGATAATAAAACT

AATAAAACTCTTCTCAAAATTAATGATGTCGACTTCAAAATTATCCCTAC

TGATAAGAAAATTTCCCCGAAGGCTTGTACTATGAAAATGTGAGCTTGTA

CTATGAAAATGTGAGGGAAAAAAGTAAAGAAAAGAATAACAAAAAGTGTA

AATATGGAAGGATAAAAACGAAACAAAAATGAATGTGAAGTAAAAAATAA

AAAGAAATTCAAGTAGATTTAAAAAAAATGTTAAGCTTCACAATATCTGT

CTC CTTTTGTTTATGTTTTTCG AAT AAATCGC ATTACC AAAAAAAAAAAA AAAAAAAAAAAAAAAA (SEQ ID NO:21) >i2H03>msp13»bankit482577>»AY1 34432

GAATCACAAAAATGGCCACCTTTTTCACTTTTACCCTTCTAATCATTTCA

ATTATTGCCACAACTGAGGGAATGAATACTAATCGAAGTGCTTCAACCTC

CGATTCTCTCAAAGACCAAAAGGATTGTAAAGTGATATATGGCATGTTTG

TGCCTGTAGCAGGGTCAAAAATGCATGGAGACGCCAAAAGCGCAATGAAG

CCAAACAATCCAAGTCTCCCCAATAAATTAATTGTATCAGGTGGCAACTC

AAAATATTCAGTGACTTTACAGGTTGAAAACCAGCCGAAGTGTGTTGCCC

AAAATGACGGAAACCCTGTAGAATGCCAAATTCAAGGAGACAAACTTTCA

GGAAAATTGATTTATGATATTGAAAACGGCCCTTCTGTCAACGTTCCCTT

CAAAGACACCCCAATCTTTGTTGGAAATAAATGCGAAATTGTnTTGTAG

CCTACGATAAGGACCACAAATTAACTCTTCTTATGAATAAAGTAAAGCTG

ATGATTGAGCCGACAAATAAGCAAATTGTAAAGGCTTGTGGAGCGAAAAA

TTATATGGAAAAATGATGAATGAATGAATGTGGGAGGGAAGGAAATGAAA

AATATTTTTAAAATTGAAGAAAGCATTCAAAATTTAAAAAAAAAACAATT

CTTCAAATAATATATAACTTTAATATrrrrGATAAATTTTATTTCATAAA AAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:22) 81 WO 2006/044480 PCT/US2005/036711 >13A1 2s»msp14»ban klt478806>»AY1 34433

GGCATCAAACAATCTCCTCAACAACTAATAAACTCAAAAAACACCCCAAA

ACAACCCTAAAAACAACCCAAAAATGTCCATCTTCCTTACTTCTGCTCTT

CTAATCATTTCAATAATCGCTATGACCGAGGGTGCAGGCGATCGAAGCGC

TTCAACCTCTACTGGTTGTACAACCTATTTTGGCATGCTTGATCATGCGG

ATACCAAGGAAAATAACAAAAGAAAAACTTTCAAACCCAACGATAAAACC

AAATCCAACACCTTGCAAGTGACTGGTGGGGCAATGTTCAGCAATACCTC

GGTGGCGTTGGTTGTCGGTGATAAGGCGTTATGTATGGCTAAGACAGGAA

GTCCAGACGATTGCGGAATGCGCTACGATGCTTTGACTGGAACAATGAAA

TTTATCATTTCTGATAATATTACTGTTGAAGTTCCATTTGAAGACGTTTT

TTTCTTCACCGACAACAAGTGTGTCATCCAGCTTGTAAGCTACGATAATA

AAACTAATAAAACTCTTCTCAAAATTAATGATGTCGACTTCAAAAUATC

CCTACTGATAAGAAAATTTCCCCGAAGGCTTGTACTATGAAAATGTGAGC

TTGTACTATGAAAATGTGAGGGAAAAAAGTAAAGAAAAGAATAACAAAAA

GTGTAAATATGGAAGGATAAAAACGAAACAAAAATGAATGTGAAGTAAAA

AATAAAAAGAAATTCAAGTAG ATTTAAAAAAAATGTTAAGCTTCACAATA

TCTGTCTCCTTTTGTTTATG TCrnCGAATAAATCGCATTAGCAGCAAAA AAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:23) >14E06>msp15»bankit478812»>AY134434_

GAAATAATCTCCTCAACAACTAAAAAAACTCAAAAAAACACTCCAAAACA

ACTCTAAATGGCTTTCCTCTTCACTTCTACCCTTCTAATCATTTCATTGG

CTTTTATTGCCATAGCTGAGGGAGCAGGCGATCGAAATGCATCAGCTTCA

AGCCCTGGTTGTATGCAGGTTGCAACCCTTATTCATATAGGGGAAATTCG

CCCAGCAAAAGCAAACAAACCAGGTGTACAAAATACTCTAAAAATGTCTG

GAAATGTTCAAACATTCAAAACTACTCAAGTGACATTACAAGTAGCTGGG

CAAGAGCCTTGTACCGTTAAAATTAATAATGGCGAAACCAAATGTAAAAT

AACCGGAGATGAATTAAATGGAAAATTA/OTCAAAACTGAAAAAGGAA

CTGAAATTTCTGCTTATTTCGAACTGGTTCCATTATTTTCTGAAAATAAG

TGTGTTATTGAACTTGACACTTATAACAAGGAAACCCATGAAACTAAACT

TATAATTAATGGAAATAATTTTATGATTAAAAAGAAGGAAGGTAGTGTTT

CAACTAAGTGTGGTGGAAGAGCTAATACTGTTTAA/YTAAAAGTGTGA

ATTGAAAGAGGAAGAG’AATATAAACAAATGTGAGGATGAGAAAAAAATAT TTTTGAAGAAAGCATTACAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:24) >16D1 0>msp1 6»bankit47881 4>»AY1 34435_ GAGAAAATAAAATATAAATTATTCCTCAAAAATACCATAAAGTTAATTAT TCTTCAATCAAAAAAATGTTTACTAATTCAATTAAAAATTTAATTATTTA TTTAATGCCTTTAATGGTTACTTTAATGCTTTTGTCTGTCTCATTTGTGG ATGCAGGCAAAAAGCCTAGTGGGCCAAATCCTGGAGGAAATAATTGAAGA AAAATGATTGAAGAAAAACGTTTAAATTAAACGATAAATGGGAAATAATG GAATTTAAATTAAGCTAATTTTGATGGTTTCCTTTGTTAATTTCAACATA AAATTAATTGAATTTACTG AATAAAATTATATCTGAAAAAAAAAAAAAAA AAAAAAAAAAAAAA (SEQ ID N0:2) 82 WO 2006/044480

PCT/US2005/0367H >l6E05>msp1 7»banklt482587>»AY134436_

GATTCAAAAAATATTATTTAAAAATTCTTTACCATTTAATTAACAAATTG

TAATAAAAGAAAGACAATTAAAAAATGAGTCCTTCCTCATTCACCTTAAC

GGCAGTACTTCTTGAGGCGATTGT1TTCTTTACAACCGTCAAGTAGCGG

CAATGCTTTCCATGCATCCGAGCTGTTCTGGCCGTTCATCAACCATTGAG

AATAAATTGAAAATGAGCGGGGGTGGTAACGGCATCAATAAATTTACACC

GGGAAATGTTTCATTCCCGGTAGCATGCCAATACCATTCAAAGAATCTCA

AAGCAACAAATAAAAAGGAATATAAAATCTCAGAAGATTTGCCTATGAAT caagaaaagcttacaaAcagtaaggaagatgatctcattcataaggtaaa

AAAGATAGATAAGGGCAATGGAGCTGCTGTTCCTTATAAAACAAACAAGA

ACAATGAAATTGGAGATGGAGCCGAGAATGGAAAAGCTGTCAAAATTAGA

GAAATTATnTTACTGAAGAGCAAAAGAAAATGACTAGCGAAGAATTTGA

GCATTATTTGTATAGTGTTCCATATGACAAAAACAAGAAAAACAAAATTG

GAAAAAACGAAAATGGTGAAAAAGTTGATAAACCAAGCAAAGAAGGAGGA

GATACAATGTTTTATTCAAAAGCTGGGATAATTGCTAAAAAGATAAAAGA

ATATGTCCCCACTAATGGCGAATTCAAGATCCAGACTGGACTTGTATATC

GTAACAATAGTTTTAATGCTTCCCAAGATGATAGTAAAAATTTACTAAAT

ATTTCGCATATTTTAATGGCTTTAAATGAAAATGAGAGGGATTCTCAAGA

AAATTTGAGAAATGCTGCTGATTTGTTTGTGGCACTTCATGAGTGTTACC

AACTCTTTTCGGCAATTCCTCTAG'Iiri&AAGTAGAAATGGTTTTGAAA

AAACTTG AGG AAG AGGGAAAC AAAG ACG ATCC AAT AAAATTACTCGAATA

TTTCCGTTTGCC AAC AATTAAATATCC ATTATTGGATTTG ATTAAAATTG

AGAACTCAACTGTGTCTCCAGATGAGTTGATTGAAAACGTCACTAAAACT

ATTCACAAAGCAGACAATTTTATTGCTAAAAACATCCATGCATTCTTCAT

AAATGACAACGAAACATTTTTTAATGAAATAATTTCTCGTCTTGAAACAG

CTGATATGGTTTTGGCCAGTATCAAAAAAATTCTTAAAATGTTCAATAAC

TTTAATGAGAAAATTCCCGAAAACTTTTCGATGCTAAAACGTTTAAAACC

AATTG AAATGC ACG ATTTATTCGAAAATTCTAAACTG CTTC AAAAG CTTC

ATGCAGCAATTTTGCCTGGAGATGAAATGAAAmTGAAATGAGAGTTAA

ATATnTTAAAAAAATTTTGCGAACACAAAAACAACAACAAATTAGAAGA

ATATTAAAATTATTAATGAAACAAGAGTTGCCGCGGCTGATCGGAAATAT

TAATTAAATCCAATTTAGCTGACGTTGCCTGCTCAATCACCAAATAAATC

AATTTATGATTTTGCCCATTCTCTATCATTACCTTATTTCCTATTTGTAC

Α'ΓΓΓΓΓΓΤΤΤΟΤΤΤΤΤ TAAAAATTA TTTTTAGTTTTGTTCTTGAATGTTC GCTTAAATAAATTCTAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:25) 83 WO 2006/044480 PCT/US2005/036711 >l7H02>msp1 8»bankit482591 >»AY1 34437

GATCAAACAATCTCCTTAACCACTAAAAAACTCAAAAAACCCCCTAAAAG

CAGCCCAAAAAATACCCAAAAATGGCCATCCTCTTTACTTCTACCCTTCT

AATCATTTCCCTTTTGGGAATTACCGAGGGAGTGAATACAGGCATTCCGA

GCGGATCTTCTCCACCCTCTTCTGCTTGTGAGACTTACAAGGGCAAAATT

GAGCACATGCCAGAAACCGCCAGAAAAATTGAATGGAAGGAAAATACTCC

CGGAGGAAAGCATTTAATCCTTAAAAAGTCTATTCAAGGTCTAGACAAAG

TAACCCTCAAAATTGAAGGCAAAGAATGTAGTGCTTCCCTCAACAACCCT

GGAACATGTCAAGTCGATGGACAGTCCCATGCCGGTCAATTAGTCTTTGT

AACTTCAAAGGCTAAAATTGAGGTTGACTTTGGGGAAGCTCAAATCTTCT

CTGGGAACAAGTGCGAGATTGAAATTGAGAAGTATGACCGTGCTACCTAC

GTAACTCTAATCAAAATTAATGGGGGTGACTTCAAAATTACGCCTGATTC

GCCACCTATGCCGATGCCATGCAAAAATATGATGAACTAAAAAGTGAGGA

GGAGGGAAGGAAAAGTGAAGGAAGAGACCATGTAAAATTGAAATATTGAA

G AAAG C ATTC AAAAATTTAATTTTTAAAAAATCTGTTTGTTTAGTAACTA

AATATAGCTTTCTATTGTTCTTATATTTTGTTTATCTTTATCAAATTAAA ATGAAAAACTCAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:26) >19F07>msp32»banklt484054»>AY142116

GACATTCATTTAAACATTCATTAATTACCTAAAATTGrrrrrCAAATTGT

TTGCCTCTGAGTTTTGCTCAACTGAGAAGAAAAATGCTTCCTTACTCAAT

TCTATTTCAATTGGGAATAGTTTCGTTGCTTCTACCTCATGCAAATGGAA

TGCAGTCTGGCAGTAGCAAAATTATGAACAAAGCATCTGAAAAGAAATAT

GCTTTGGTTGTTGCTCCAAACTTTCTTAAAGTTCATTTTAAAATGAACAG

TGTCTTTGCCAATGCGTTGACCAAAAAGTTrTTTGTGCACTTTCTAATTC

TGAACACCAAAAATGAAGAAATTGGAGATAATTTCGACTATGGAATTGAT

CTCGAAAAATTTGAAGAAGGAACGGGAAATACATATCAAGTTGTAAATTT

TCCAGATGATTATCCCGAAAAATTGAACGAAGGCGTGAAGAATTTAGAGA

ACAAATTCATTAAGAGAGGTTACGAACAGAGTAGTCAAATTCTGAAAAAT

G AAG CTTT C AC CG TTT AT AAAG ATTT ATTTG AAAAC AATG G AG CT ATTG T

TCATTACTTGAAGGAGGCAAAGTTTGATTTAGGGGrrrrrGACACTTGGG ACACTGGAGCTCTCTTCATTCTCCATGCAGCAGGAATTAAAAATGrrrrr

GGCATTAACAACATTCAACTTAATGCTTATCAATTTAAATATGCTGGGAA

AGAATTTCCAAAAAATATTCCAGAAATTTAATTCGGCACAAACAGGCGAT AATGAATTATCACCAACAAAGGAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:27) 84 WO 2006/044480 PCT/US2005/036711 >21E02>msp19»bankit482599>»AY1 34438

GGCATCAAACAATCTCCTCTCCTCAACAACTAAAAAACTCAAAAAACACC

CCAAAACAACTCTAAAATGTCGGCTCTCCTCTTCACTTCTACCCTTCTAA

TCATATCATTGGCTTTTATTGCCATAGCTGAGGGAACAGGCGATCGAAAT

GCATCAGCTTCAAGCCCTGGTTGTATGCAGGTTGCAACCCTTATTCATAT

AGGGGAAATTCGCCCAGCAAAAGCAAACAAACCAGGTGTACAAAATACTC

TTAAAATGTCTGGAAATGCTCAAATATTCAAAACTACTCAAGTGACATTA

CAAGTAGCTGGGCAAGAGCCTTGTACCGTTAAAATTAATAATGGTGAAAC

CAAATGTAAAATAACCGGAGATGAATTAAATGGAAAATTAATTTTCAAAA

CTGAAAAAGGAACTGAAATTTCTGCTTCTTTCGAACAGGCTAAATTGTTT

TCTGAAAATAAGTGTGTTATTGAACTTGACACTTATAACAAGGAAACCCA

TGAAACTAAACTTAAAATTAATGGAAATAATTTTATGATTAAAAAGAAGG

AAGGTAGTGTGTCAATAAAGTGTGGTGGAAGAGCTAATACTGTTTAAATT

TTAAAAATGTGAATTGAAAGAGGAAGAGAATATAAACAAATGTGAAGATG

TGAAAAAATATTTTGAAGAAAGCATTCCAAAAAAAAAAAAAAAAAAAAAA AAAA(SEQ ID NO.28) >25B1 Q>msp33»bankit487909»AY1 421 18_

GACATTCATTTAAACATTCATTAATTACCTAAAATTGTTTTTCAAATTGT

G ATTTTATTTATTTCTATTTAATATCTTTAAATGCGGAGTGCTTTAAAAA

CTTTAATTGTTTTGTGGCCTCCTTTGCTTGGACATTTTATTTTGTTAATT

CCTAGTGGAGTAGCTTTTGTAGTTAAAGAGAATGTTCAAGAAGTATCGCC

TGTTATTCCTGATAAACCCGGAGTAATTGGAGGTGATGTTATTGATAAAA

GCGCAAAAACTAGTCAACTAAAAAAGGGAAGTGAAAGTCTGATTTCTGGA

ATTGAACGTAGCCATGTTGAGGAATTAAAGGAGGAAATTAAAGGAGAAGG

TAAGAAAGTACCCAAAATGAATGGACAGGATAATGAAAGCCTTGAAACTA AAATTGTTGAAAAG(SEQ ID NO:29) >28B04>msp35»bankit484130»>AY142119 _

GATCAACCAATCCCCTCAACAACTAAAAGACTCAAAAACACCCCAAAACA

ACTCTAATATGGCTCTCCTCTTCAGTTCTACCCTTCTAATCATTTCATTT

ATTGCCATAGCTGAGGGAGCAGGCGATCGAAATGCATCAGCTTCAAGCCC

TGGTTGTATGCAGGTTGCAACCCTTATTCATATAGGGGAAATTCGCCCAG caaaagcaaacaaaccaggtgtacAaaatactctaaaaatgtctggaaat

GCTCAAATATTCAAAACTACTCAAGTGACATTACAAGTAGCTGGGCAAGA

GCCTTGTACCGTTAAAATTAATAATGGCGAAACCAAATGTAAAATAACCG

GAGATGAATTAAATGGAAAATTAATITTCAAAACTGAAAAGGGAACTGAA

ATTTCTGCTTCTTTCGAACAGGCTAAATTGTTTTCTGAAAATAAGTGTGT

TATTGAACTTGACACTTATAACAAGGAAACCCATGAAACTAAACTTAAAA

TTAATGGAAATAATTTTATGATTAAAAAGAAGGAAGGTAATGTGTCAATT

AAGTGTGGTGGAAGAGCTAATACTGTTTAAATTTTAAAAGAGTGAATTGA

AAGAGGAAGAAAATATAAACAAATGTGAAGATGAGAAAAAAATATTTTGA

AGAAAGCATTCCAAAAAATAAAAAAAAATTAATTCTTCAAATCCATTTAT TTTTTGAATAAAACATTTTACTAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQID NO:30) 85 WO 2006/044480 PCT/US2005/036711 >30G11>msp21 »bankit48261 1>»AY1 34440_

GATTTTTATTAATTTTAAAAATTATTAACTCTCCAAAATGAAGTGTTTGC

TCCCCTTCTTTTGGATTTTATTAACAATTTTTGTTTCTTGCACTAATGGC

ACTTCAAATGAGTATAGTGAACTTGTTTTGGTTCAAGCTTTGTGGAGACA

CG GTG ATCGTTC ACC CACAAAAAC CTTC AAAAC GG AT AAATATC AAG AAA

AGGATTGGCCTCAAGGATGGGGGCAATTAACACCTACAGGAATGGCTCAA

CATGTAGAGTTGGGAAGACGACTAAGACAGCGATATATAGAGGAATTGAA

ATTTGTTGGTCCTCGGTATAATAGCCATGAAATTTATGTTAGAAGTACTG

ACTGGAATAGAACATTMCTAGTGCTATATCGAATTTTATTGGCTTCTAC

GGCCCCGGAAATGATGATGAATACCCAAAGGATTTGGGCGCAAACAAATG

GCCAGGATGGTTnTCCCAATAGCGATACATTCACTCCCTGGAAACGAAG

ATTTTATGGCTCCTGGAGAATCGGAATGTAAACGATTTGAACAAATAAAA

GAACGGATAACTTTAACAGAAGAATACAACTCGACTTTGATTAAATACAA

ATGGCTACTCGATTrrrRGAGTGAAAAGACGGGACAGAATGTCGACCCTT

TCG ATATGTG G ATG ATT AACG ATGCTMTAT ATTG AG AAATT AAAAG GC

AAAAAATTGGTAGACTGGGCAGAGGGGAACCAAACACTTTTGGATACGAT

TGCTGAACTTGACAATTTACAAGAAAGATGGATGGTTGGGTTAGATTTAA

AACCTCTGGGTGATGCCAACTTTCGCGAAGAACTTCCAAAAATTTTGGGC

GGGCCAATCTTATGGAAATTTATAACAAATATGCAGGAGAAGTTGGCTTG

TTCAAAGCGAATGAATTCTGTAAAAGAAATTGACAGGGAAATAGAGGGAA

GAAAATCGCCAATGGGGACGCCCTTGTGTAAATGGATGAACAAAATGCGC

TATnTGCGTACTCTGCGCACGACAGCACAATTATTGCAATTITGCAGC

TTTGGGTTTAAACAAAACGAATTATGACGAGGATGGTTACCCGAAGTATT

CTACTTGTGTAACTTTTGAATTGTGGAGGGAGAAGAATACTGGTCAATTT

GATGTTAAGGTATTnTATGGAGACCTAACACCAACGAGACTTCCCCTAA

AGAAATAACGACAGATATTGAAGGCTGTCAAAGCAATTCAACTCTAGAAC

AATTTGTTGAAAGATCAAAAAATTATCAAATGCTGCCTTCACCCAAAGAC

TATTGTTCACAACTTCTACAACCCCTAAATAATGCTGCACGTATGTTAAT

TCAGTGGAAATTGGAAATGCTTATCTAAT3GGAATTCCTTCAATTGTTG

CTAATGTTGTATAGAGAA ΤΤΊΊΤΠ'Ο I I I 1 I GGAAATTATTTAGTTGCAC

CTATTCATCAAAAGAAGGGCAAAATAAATT TlrATCCCCTAAAAAAAAAA AAAAAAAAAAA {SEQ ro NO: 31) 86 WO 2006/044480 PCT/US2005/036711 >30H07>msp20»bankit478826>»AY134439__

G ATG CTTC AATTGAC ATTTAAAATT AAAAAGACCCAGGTTTCTTAATTAA

AGAATGTTTTTAAAAATTTTAACTTTCCTTTTAATTATAAACAAAATTAT

TGCGGATGATTCTAATAGCGGAGATAGTGGCAATGAAAATTCTAATAGTA

AGCCCAGTGATGAGCTTGCCGACTCTGTTGATGTTCGAGAGCATGATAAT

GAGCAACATCCATCCAATTCGATCGACAAGCAAAATCTTCAAGACCCACA

ATTTATTAAAGAAGATGTTACAAATGTATTGCCACTATTAAATAACGATG

AGAATAATCTCATTGATGAATACACAACAGAAAAAATTAAAGAAGATGAG

GAAGACCAACTAAATAATGAAGGATCTGGTATAGACAATGAATTTCCTGA

GGAAGATAATGATGTAAATGGATTGGATATTAATACAACTGCAAAATATG

CTAATGATGTAGATGATAATAATAACAATGAAGGTGATGGTCAATCATGC

GTTTATGAGGATGGTGTAATTAACGATAATGGTGACGAACGCACACCTAC

ATATGAAGAACAACAACAAATTGAAGAATATCTTCAAGAAATGCGTGAAT

TTGAAGAGCAAATGGTCAAAGACAGCGCTAATTTTATG AG AAATTTG GCA

CAATTTGTGATGAGTCAATTCGAAAACATTTTTGGTTCTTCTACCTCGTC

TCTATCAGGTAACAATAATAATTTGTTGGAGAAAAAACCTTTAGAAGCAC

CAACACCTCCTTGTTTATGCAAAAAATGTGACAGTATGACATTTATACAA

AATAAGCAAACCAAATATCTTAAAAATTTTGCTAATTAATTAACAAAAAT

TTGAAGAATAATTTAAATAATGTTTATCTCTTTCTTGAGATTTTCAAATT

ATTTAATCCATTTATATATAAATTTAAATTCATTTTCTTTTACAAAAAGC TGAAGAGATTAAATTTTAATGTTTGAAAAAAAAAAAAAAAAAAA(SEQ ID NO:32) >31 H06>msp22»bankit48261 5>»AY1 34441_

GGAATAAAMGCGGCGAAATTTTACTTTATTCATCAAAGTACTTTAAAAT

ATTCTATAATCTAAAATGAAATTCAGCCAATTATTTGTTCTCTTAATAAT

TGCTTTACAATTTGTGGTTGCTCAAGGGTTGATTTACGATGCGAAAGCAA

TAGCCAAAGGAAAAGGAAAACCGTTCAGGGCGCTGAATATTTGGTATTTG

TCTTGGGCAACTATGTAATGAAGAAACGATATCTTTATGTAATTGATAAA

TAAAAATAACCTAAGTATCAAAAAATGTTTATGGGAAATAAAAGATTTAT

CTTCATTTAAAATCTAATAAATTTGTCAATCCCAAAAAAAAAAAAAAAAA AAAAAAAAAAAA (SEQ ID NO:33) 87 WO 2006/044480 PCT/US2005/U36711 >34C04>pectlnase»bankit476418>»AF527788

GGTTTAATTACCCAAGTTTAAGGGGTAAAAAATGTTTTCAAGCAAAACTA

GCTTCAATTTCCTTCTTCTAATTTCTTCATTTGCTTTATGTAAGGCCGAC

TTTTGGCCTAAAGCAAGAAATAATATTACGGTATCCGAAACAATACAAAT

TACTMCC GTG ACTGTAATTTTG ATC GTT ATATTCCCGATCCG AGTAA AC

TTGGAAACGGAGGTCAGAACGAGCATCAAGGCTACGTTTTGAAATAAAA

AATGGTGGTTCTTTATCTAATTGTATAATTGGTGCTAGGCCTGGGACTAA

AGGCTCTGCTCATGGAGTTCTTTGTGATGGAGATTGCGATATAAACAATG

TTTGGTTTGAGGATGTTGGGGAAGATGCTATTAATTTTAATGGAGATTCT

GATGGTTGTGTTTATAATGTTAATGGTGGTGGTGCTAAGAATGGAGAAGA

CAAAGTTATGCAATTTGACGGAAAGGGGACACTGAATGTTAACAACTATT

ATGTAGACAATTATGTCCG TnTrGTCGCTCCTGTGGCGACTGCGGTGAC

C AAC ATC AACG CCATATC GTG ATTACTAATCTG AGAGCGGTTC ATG GCC A

AGCTGGTCAATTCGTTTGTGGAGTAAATAGCAATTATCAGGATACGTGTA

CCTTGCATGATATAAAAATGGAGAAGGGTATTCACCCCTGCAAGCnrT

GATGGCAATTCTGATGGATCTGAGCCAACTTCGAATAACGACGAAGAGGA

CCACGGAGACGGGAAATTTTGTATTTATAAGAAGGGCGATATTAAATATA

TTGGATCCAAACCAAAGCCGAAAAGCAAAAAGAGCGCAAAGAATTAAGTG

CCGGAAGTTAAAAAGCCTTGAAGTTAAAAACGTTTAAAGGGATAAATTGT

AGGGTTGTCGGTTCTGAACCGAACCGAGCCGAAGAACCGATGATTTTTCG

GTTCGGTTCCGGATATCCAAAGATTTTCCAAGAGCCGACAACCCTAGTAG

TATGAGTAGAATCTATTATTATTTGGAATACTAATTTAATTTTGTGAAAT

TTCrrrrrACTATATTAATCCTGTCCAATAAAATTATGAAATCGAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:34) >34D01 >msp23»bankit48261 9»>AY1 34442_

GGCATCAAACAATCTACTCAACAAATAAAAATCCCTAAAAACACCCCAAA

ACAACCCTAAAAGCATACCAAAAATGGCCACCTTTTTCACATTTACCCTT

CTAATCATTTCAATTATTGCCACAACTGAGGGAATGCATACTAATCGAAG

TGCTTCAACCTCCGATTCTCTCAAAGCCCAAAAGGATTGTAAAGTGATAT atggaatgtTtgtgcctgtagcagggtcagaaatgcatggagacgccaaa agcgcaatgaagccaaacaatccaagtgtccccaataaattaactgtatc aggtggcaactcaaaatattcagtgactttacaggttgaaaaccagccga agtgtgttgcccaaaatgacggaaaccctgtagaatgccaaattcaagga gacaaactttcaggaaaattgatttatgatattgaaaacggcccttctgt caacgttcccttcaaagacactccaatctttgttggaaataaatgcgaaa

TTGTrrrrGTAGACTACGATAAGGACCACAAATTAACTCTTTTCATGAAT

AAAGTAAAGCTTATGATTTCCCCGACTGATAAGCAAATTGTAAAGGCTTG

TGGGGTGAAAAATTAGAAAGAAAAATGATGAATGAATGAAGGTGAGAGGG

AAGGAAAGAAAAAATATTTTTAAAATTGAAGAAAGCATTCAAAAATTAAA

AAAAACAATTCTTCAAGATAATATATAACGTTTAACTCTTTTTGATAAAT TTTATTTCAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:35) 88 WO 2006/044480 PCT/US2005/036711 >34F06>msp24»bankit482623>»AY134443

ATTGAAGAAGATGATAGATTCAATGTTCCTGTCATGGGAGAAGAAAATCA

TAGAGATATTCCTGTCGAAGAAGCTAATTATCAAGTTCCTCCTTCTGCTG

ATTTCACTTTTACAAGCTCTGAGCATGGAAGACGTACATCATTTACAGTT

GGAACACCGCATCATCGATACATGCAACCAGGCACTCGAGAAGCATATTT

ATTG CCC CATC CAG GAG GGGAAGGTGC AACG CTTATACGC AATG AAGTTC

GTCGAGATGGAACGCAAATTTCCCAACAAGACACACTTCAAAACATTGAA

GGAGGGAGAGGTTATGTTTATTCTTCATCGTCCCACACTCAAAACGAATC

AAGTAGTAGTTCAAGAATAACTTCGAGAATTCGTTTTGGGAATAATGAAA

GACATGGGAAAAAATGAGGAATAAAGGGAAGATTTAGGAAGACATGGAAA

AAAGTGAGGAATGGAGGAAGAGATTTATACTAATTATAAAATGATAGAAA

AATTGAAGAGATATTCTCTTATTCTTTCCTATATCTTTACTTTCACATAC

AAAATTCTATAATG GC AATTTATG ATTT AAC ATTAAAATTGAATTTAGAA

ΑΤΑ I I I I I IAAAATTAI I I IAG I I I I CA I I I I IATCAAI I I I I IGATATT

TAAATACGTCTTGTATTTATCTTCATATAATTGTTGATTAAACTTTTCTT

T AT C ATTC TTTTG TAG GT ATT CT AAA ATT A AAT AATT AT ATG AAT ATTT

TTTAATTTTCAATTTGAATAAAATTTTCTGCAAAA AAA AAAAAAAAAAAA A {SEQ ID NO:36) 89 WO 2006/044480 PCT/US2005/036711 >35A02>msp25»bankit478844>»AY134444

GACATTCCTCAGCTTCATTACCCATCCATTTTTCATAGACAACATCCCCC

TTGCCAAACATTAAAAATTGAGTAACGCTGAATGAAGC TrrrrGTCCTGT

TAATTGGAGTTTTAGCCTTCACGGTTCTAAATGTCCATGGAGGAGTCAGC

CATTC0ACATTGACTCACAGAAACCCGCGAAGCAACGAAATCGAACAATT

AACTGATGTGTCGTTGGACGATACCCCATCCTC'GCCTCCTCAAGCTGTGT

TGGACATTGGAATGTCAGGACAGCGAAATTTGCAACGTCGAGAAGCTCCA ATGTCGATTGGGAAAAAAGTGGTGGCTGTAAI “ΓΓΓΓΓΓΑΤΤΤΟΤΤΟΤΘΤΟ

TnTACATCGTTATATCTATTGGCCGTGCCAAAACAACAAATTCAACAAG

TTGAATACAAACAATTTCCACAACCCTATAAATTTGT.TCCGATTAGTAAC

ATTGTCAAGTGCGACAGAAAAACACGGCAATGTTCAATAAAGTTAGAGAA

TTTGGATCCGACAAACAACTATAGCCTCTATACTGCAAAGAATGACAAGG

GGAGAGGAGATAAAGTAAAGTTAACTAAAGTTGCAGATATAGATTTGGAC

AAATGTCAACTCGACAAGAATGTAAAACCAGAAGTAAATGGG GAAG AAAT

TTGTAATCAGATTGTCAAAGGAATTGATGATAACGCAAAAGCCGAAACTA

TTGAGGTTAACAGTGGAGAAATAGAATTTGGTTCGGAATTAGAAGGAACG

G AGG ATTATGC G ATAGTTG A AAAAG CAATGAATG AG AAG AATG AACATAA

AAATCAACAAGCGGTTGAGCATGTTCATATCCCTGGGCCAGGGGAACAAC

CAGTTGAACACAATCAGCCGACAATAGAATATCCAACAAATTCCAAACAA

GTTCATCCAGCTGACAAATATCAACATAAACTAGAAGAGCGCGCCAAAAA

ATTTGGGCTTAGCGACTTCAAACATGGAGATTTATATGAGGATTATCGCC

AACAAAAAACGGTCCAAGAAGATGAAAAGGATAAACGATATCAAAAGGTT

CTAGGAACACTAGGAGACCATAAACATCCATCGCTAGTTGATCAATATAA

CGAAGATAAAGGAAAATTCAATCAACGTGTTAAAAGTGACCCCACAGGCA

ATAAAGTTGAAAAGGCAAAAAATTCTGATTCTAATGGACTTGAACAAAAA

TTAGAAAAACTGGCACTGAGTGACTTCAAACATGGAGATTTATATAAAGA

TTATCAGCAACAAATCACGGTCAGAGATGATGAAAAGGATAAACGATATC

AAAAGGTTCTAGGAACACTAGGAGACCATAAACATCCATCGCTAGTTGAA

CAATATAACAGAGATAAAGGAAAATTCAATCAACGCGTTAAAAGTGACCC

C ACTAGC AATTG GC ATG AAG ATTTATTCGGAAAGGATTACCGACGTGCTA

TGAGCGATTTCGATCATTTAAAGGCTAAACAACGTGAAAAGATCCTTGGA

ACACTAGAAGATCATAAGCATCCATCGCTAATTGATCAATATAACAAAGG aagcttaaatcaacgcgctaaaagtgaccccacXggcaat/vktattggaa aggcaaaaaattctaattttaatgggtctgaacaaaaattagaaaaactg gcactgagtgacttcaaacatggagatttattaggtcgaaaaggaggaat taaacaacgcactataaatgttctcgctggcaaaaaaata'gAAcatgaaa mggaagtgattttaatgcaaacgttgaagaaatgataggggcagaaaac ggcaaggctaatcaagtgaatcccaaattaactggacgcaaactagctga

AfTTAATCATATTCCAGCTGTTGACAGAATTCTTGGiifrAAACGTGGAG

GTCATGCGCTAGAGGAGCCTCATAAAAATTGAGATATTTTGCCTGAAGAG

TTGGATTGAACGATGTATATAAGATTTTTTAACCATGTAAATATTTTTAA AAAAGATTTTATTAGAGCCAGGAAATTACGATACTGAATCCCGAAAAATA,

TCGTAATGGCTCTTAAI "ΓΓΓΓΙΑΙ’TnTTAACTTTTCCATTGCAAAGATT

TTTTTAAAATTTTTCCCGATTGTCTGGTAAACTTGTGATGAGATAAACTG

AT nTGATTGATAATAATCGTCCATTITCCAAAAAAAAAAAAAAAAAAAA AAAAAAAAAA(SEQ ID NO:37) 90 WO 201)6/04448(( PCT/US2005/036711 >35E04»bankit487B71 >»AY142121

TACCTAAAATTG "rrrrfAAATTGTTTGCCTCTGAGTTTTGCTCAACTGAG

AAGAAAAATGCTTCCTTACTCAATTCTATTTCAATTGGGAATAGTTTCGT

TGCTTCTACCTCATGCAAATGGAATGCAGTCTGGCAGTAGCAAAATTATG

AACAAAGCATCTGAAAAGAAATATGCTTTGGTTGTTGCTCCAAACTTTCT

TAAAGTTCATTTTAAAATGAACAGTGTCTTTGCCAATGCGTTGACCAAAA

AGTrrrrrGTGCACTTTCTAATTCTGAACACCAAAAATGAAGAAATTGGA

GATAATTTCGACTATGGAATTGATCTCGAAAAATTTGAAGAAGGAACGGG

AAATACATATCAAGTTGTAAATTTTCCAGATGATTATCCCGAAAAATTGA

ACGAAGGCGTGAAGAATTTAGAGAACAAATTCATTAAGAGAGGTTACGAA

CAGAGTAGTCAAATTCTGAAAAATGAAGCTTTCACCGTTTATAAAGGTTA

AAATCCAAAATATTTTGCCTTCTAAAATTGTT ATTTGATT AATAATATAT

AAAATATTTAAGATTTATTTGAAAACAATGGAGCTATTGTTCATTACTTG

AAGGAGGCAAAGTTTGATTTAGGGGTTTTGACACTTGGGACACTGGAGC

TCTCTTCATTCTCCATGCAGCAGGAATTAAAAATG 'ΓΓΓΓΓ GGCATTAACA

ACATTCAACTTAATGCTTATCAATTTAAATATGCTGGGAAAGAATTTCCA

AAAAATATTCCAGAAATTTATTCGGCACAAACAGGCGATAATGAATTATC ACCACCAAGGGAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:38) >35F03»bankit487855»>AY142120_

GACATTTAATTTTTTAAATTTCTTAACATTAAATAAATTCAAAAAGAAAA

TTGAGAAAAAAAATCTTTTAATTTAAAAAAAAAGAAAAAAGAAAAATGTA

TCCTTGGACAATTTTTCTTTTATTAATTATTTTGTTGGCTATGGCCATTG

AAATAATTGGAGGAAAAGGTCGAAAGTTAAGGAAGAGAGACAAAGAGGAA

AAAG GTCATG CCTCAATTTTCTGTTGGG C ATTC ATCTAGGGAAGGTTTCG

AGGAAAAGCTTGATGAAATGGTTGAATCAACTTCAAATATGTTAATAAAT

CTTGGTAAAAAAGTAAAGAAAGGAGGGAAGAAAGTTGTAAAAGGAGTTGT AGAAACTGCGCAGCTGATCAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:39) 91 WO 2006/044480 PCT/US2005/036711 >42G06>cbp>»AF049139 _ _ _ _ ____

CAAGTTTG'AGCGTCAGCAATTTTAAATTAAAAAAAGACAAACTATAAAAT

CTCTCTTATTTAAAATAAGCAGTATACCCTTCAATCTATCCACAATCCAA

TAAAACTTTCTAATAAAAATCCTCCACTAAAATGGCATCCTrrrrrrATT

TCTTATTTATTTCTGTTAGTCTTTTGATTCTAGCTAATGCTGATGATGCT

GGTAGATATCCTTCAGGAGATGATTTAGTTGAAGGTACTACTGCTGCTCG

GCTTCATTCGTCTTCTGACCTACCAGACGATGATGAAGAAGAATGCGAGT

GCGAAGATGACGACGAGACAACAGTCGCAACTCACATTTCTACACGCAGC

AATGGTTACCCTTCTAATAACGGAGCCCCCACTAGCACTAAACGCCCTTC

AAACAACGGAAGCTCAAACAATGGAGGCTCAAGCTCTGTCACAGGATCTG

TTATATTGAGAGATAAATGGGTAAATGGCGCCAATTGTAT nTAGCTTTC aagaataatggaaacgctagagcatgtggtgtcaAgttcgagctgactct

CGGTGATAATCAAAGAATCCAAAGCATTTGGAACGTTGAGAAAGTCGGAG

ATAAAGTTTACAGAATTCCGGACTACATCCAACTTGGTCCAGGAGTCGAA

AACAGAGATATTGGAGTTGTTTACAATGATGTGCCAGAACTCTTCCACAA

TCAAGGTCTTGGACAAGAAGAAGGATGCAACATTATTGAATAAAAAATAT

G G ATATAAAAATATTTAAAAAAG ATTAAATAAGTATTATTAAAGCTTGTG

AATATAAACTTTTTCGAAAATTAAAATAATGGCAGAAAAAAAAAAAAAAA AAAAAAAAAA {SEQ ID NO:40) > 1 C05B>msp36»AY422829_

TATTAAAAAAATAACAATTTCTTTTAAAAATAAAATGTATTCCCGTTCAT

CTTTAATTTCrrrrrrrCTnTAATTAATTTAATTTTGACTCCAATGATT

TTGGCTACTAATAATGATGGTGTTGCTGCTCCGGTTGTTGCTAATAAAGA

TGCTGGGAAAGTTAGAGCGACGGAAATTATAAGAGCACTCCGTGGATTTT

GGAAAGGAGTGGCAGGTGGAGCATTGGTAGGAGGAGGTGCTGTTTTAGCT

GCACGGATATTTCGGAACGCTGGCCGGCGCGGATCGCCCCGACCCATCTG

GATGAGGTCCATCTGGATAATGGAGATGCAGCATGAGCGGTCCTCACAGA

CTTTCCTCGGTCGAACTGGACGCCGCGACGCTGCCGGCGGCGACCGCAGA

GATCGAGCATGAGCGCCGCGTGGCCATCTTCGATCTGGTCGAAAAGAACA

GTTTCGAGCCGGTCGGCGCCGAGGGCGGCCCGTATCAGCTGAAGCTGTCG

CTGCAGGACAACCGGCTGGTGTCCGGCTAAATTCGCATTTAAGGAAATTC

GATGTrrrrAATAATTTMTTTAATAAATTTGTTTTATCTTTAAAAAAAA AAAAAAAAAAAAAAAAA (SEQ ID NO:41) 92 WO 2006/044480 PCT/US2005/036711 >1C 11B>eng-1 »AFT 00549 _

ATTAATTTTAAAAATCTAATTAAAAATGAATTCTCTCTTATTAATAGCAT

TTTTATCCCTCTCATnTGTGTTCCAATAAAGGCTGCTCCTCCATATGGG

CAATTATCTGTGAAAGGAAGTCAATTAGTGGGCAGTAATGGACAACCAGT

TCAACTTGTTGGAATGTCACTTTTCTGGTCGAGTTGTGGTGAAGGAGAAG TT nTTATAATAAAGGAACAGTAAATAGTCTTAAATGCTCTTGGAATTCA .

AATGTAGTTAGGGCTGCAATGGGTGTAGAGTATTCAGGGTGCCAACGACC

AGGTTATTTGGATGCCCCAAATGTTGAGCTGGGCAAGGTTGAAGCTGTTG

TTAAGGCCGCAATAGAGTTGGATATGTATGTTATCCTTGACTTTCACGAC

CACAATGCTCAACAACATGTGAAACAAGCTATCGAATTCTTCACATATTT

TGCCCAAAACTACGGATCTAAATACCCTAACATAATCTATGAGACTTTCA

ATGAGCCACTACAAGTAGACTGGAGTGGTGTAAAGTCATATCATGAGCAA

GTTGTTGC AG AAATT AG AAAAT ATG ACAC AAAG AATGTCATCGTTCTCGG

TACAACAACATGGTCTCAG’GATGTCGATACTGCTGCTAACAATCCTGTAA

GCGGCACAAACCTTTGCTACACTCTACACTTCTACGCAGCAACTCATAAA

C AAAAC AT AAG AG AC AAGG CG C AAGCTGC AATG AATAAAG GAGCTTGTAT

CTTTGTAACTGAATACGGAACTGTTGATGCAAGTGGAGGTGGTGGAGTGG

ATGAAGGTTCGACAAAAGAATGGTATAACTTCATGGATAGTAACAAGATT

TCTAACCTCAACTGGGCTATCTCAAACAAGGCAGAAGGTGCTTCAGCACT

CACATCTGGAACGAGTGCTTCTCAAGTTGGCAATGATGACCGATTGACTG

CCTCCGGTGTTCTAGTGAAGAAGTATATTAAATCAAAGAATACTGGTGTC

AGTTGCAATGGTGCATCACCAGGCAGTGGTTCAGGAAGTAACCCCTCAGG

AAATAAACCGAGCAACTCACAAACCAGCACTGCCAAAACATCAAGCAATT

CAGGAAATAAAGGCGGTAATTCTAACACAGGGAATAATGCAAATAACTCA

GGAAGTAAACCGGGCAACTCCGGAAGTAATACAGGAAATACGGGTAGCAA

TGCCGGAGCCAGTTCAGGAAATACGGGGACCAGTACAAGCGGTAGTTCTG

TTACAGCTTCAGTACAAGTTCCCGATAAATGGGATAATGGCGCAAGATTC

C AATT AGT ATTTAAAAACAATGCAAGTACAAAAAAGTGTGCAGTG AAATT

TTCATTGACTTTTGCCTCTGGACAACAAATTACTGGCATTTGGAACGTTC

AAAATGTAACAGGAAATAGTTTTGTTCTTCCAGACTACGTTACAATTGAG

GCAGGGAAACAATATACAGATGCAGGAATGAATATAAATGGGCCAGCAAC

TCCTCCACAAATTAAGGTGCTCGGCGATGGAAAATGCGTTTTTTGAAATT

AAAGACTCCGTCTTAATTGTTGAATTATTTTAATCTTATGATTGTTTAAA

TTGGAAAAAAATATATGTATAATTTGCTTCTGTTAATTTTGTTTATTTTA AATATACGATAAAAATTA(SEQ ID NO:42) 1D08B>msp37»AY422830_

TAAAAAATGATTTTTATTTCCTTAATTATCCTCGTATTGGCTGCTGAATC

TAATGAAGCAAGCACAAACTGCAAGGATGGTGAAGGCGCGGTAACCTTCT TGTCCAACCAGCTCGGTAACATACAGGGAATAAAAGGAAATAGTTATTAT.

AACAAAACTTGTTCCAACAAAAATACTGCAAAACGTTGCTACCCAAATGA

TGAATCAAATATTAGCGTTTTTAAAATTGTTTGCCCCACAAATATTTGTA

TTTGTGGTAATGTTGATAATCAATGTTACTCTGCAAAAACAGTTAATCCT

GGAGATTTAGACTATATGTTCTATTCTCATAGTGGCAGCATGTTTGTTAA

CCCAAATGTTGGTTCAATTTCATTATCGTCACCTGATAATCATTATTTTG

ATCCAAAGACTAGTGCCCCAAAATTCATGGAATTAACCCCAGGCACAAAA

TCATATCTTAATGGGAATGAGCTTTCTGTTGCTTGTACATCTTGTGCTAA CTTTAAGCAGCTAACGTGTTG AAC AATAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:43) 93 WO 2006/044480 PCT/US2005/036711 2B02B>pel2»AY327873_

GAAAATAAATTATTCTTTTTAAAATTATCAAAAAATGCCACATTTTTATT

TAAAArrrrLAATTAATTTAATTTTATTAAATTTATTCCCATTACTTATA

AAAAGCGATTTGTGTAAATTTCCAACGGCTAAGGGGAACCAAACTGTTGA

TG AAAC AATACC ATT AAAT AAAG ATAAAGATTTTGGGTTTATTCGTCTG A

TAGCTTCTCCAAAGTTGGGAAGTTGTACAATTGACTTTAGTAAGAAAATG

TCGCCAATATTATGGTTATCCGATGGGGTGACTGTTAGTAATTTAATTAT

TGGAACTGAATCTTCTTCAGGCATTTGGTGTAGTGGAAGTTGTACCTTGA

AGAATGTCTATTTTGAACGTGTTTGTACTCACGCCGCAG.CTTTTAATGCA

ACAACAGACTTTACAAAAACTGATAGACGTTCATTTACATATACAGTTGA

GGGGGGCGCTGGACTCCATGCTTTAGATAAAATGTTTGTACAATCTGGCC

CCGGAAAGACAATAATTAATAATnfGTGGGGATGGATTCCAXAAAGTT

TGGCGATCGTGTGGGACGTGTAATGATGAAGTGAGTCAAAATTCTAAACA

AAGAACTGTTACTATAACAAATTCAAATTTTACTGGCAAAGGACATGTAA

TTGCATCTGGAAATGCCCCTTATAAAGACAAAGTTTCCTTCAATAATGTC

AAAATATTTGGTTATAAAAATCGTTCAACAAGAGTTGTTTATGCCTGTGG

GGAAGTTAAACCAGAAATAAGTGAAGATCATTTAGATACAGGAGCTTCAA

ATTGGTATATACCTGGACGTGCTGGTACTGGAACTGTTTGTAATTATCCC

GCTTCAGCAGTTAAAATTGTTAATTAAACATTAAAAGCTTGATATTTAGA

AAATAGTAATAATAAATGTTATTTATTGTGAATAAAGTTTTATAATTAAA AAAAAAAAAAAAA (SEQ ID NO:44) 2G06B>cm1 »AY422834_

GGTTTAATTACCCAAGTTTAAGACAATAAACTTTTTAATAAAAATATTTA

ATTTTG AATGTCTTTG AATTG GCTTTATTGCAATTTATTTATTGTAATAC

TCCTTTTCAACATTGTAAAGAGTGATACCGATACTAATGCTGATATTGAT

CGATTTGTTGAAATTGCAGACGATCGTTTAACTCTTTCTGATTATGTTGC

TTTATATAAAATTGTTAATAACAAAAGTATTACTGATCCAAAACGAGAAG

AAAAACTTTTGAACGATATGAGAAGTAAG GG AAAG AATCTTTCGTTAAAT

GAGGATTATGTTACTTTAATATTCCAAGACCAAATAAATGCTAGTAAATA

TTTTCAGAATTATTTGGTTAATTTATGGAATCAATCAGGCATTCCACCTA

TTAAAGTTCGAGATTTAAATACAGACTTACGCCCAGCAATTGATCAAATA

AATACAGAAATGCTGCAATTGCTAGTTAAAATACAAAAACTTCCCTCCAA

AGATTGTTTAAAAAAAGTAGATAAGTCTGTAAATAATTTTATTATGAGAG

TTAATCAAATTGATGAACAAAATGATGCTTTGAAAATGGCTGTGAAAGGC

AAAGACCTCTGCCCTGCATGTAAACATAATTAACGTTTAGTTAATTATAA

AGGGAAAAGAAATTATAATTTTGAAAAAATTTTGGGTTTCACCAAAAAAA AAAAAAAAAAAAAAAAAAAA (SEQ ID NO:45) 94 WO 2U06/044480 PCT/US2005/036711 4F05B>msp38»AY422B31

AATACACAAAAACTATTTTAAAAAAGGCACTAACTTAAATAATGACTTGT

AGTATTAATATTTTTATTATTTTATTTATTACATTAATTATTGGAATATG

CACGGAGGCAAAAATCCGTAAACAATTTGTTGACTCTCCACAGGAACCAC

AAGCTAAATCGGTTGATTTGAATTTGCAAGTTTTAATCTTTATAAAAAGA

TGCAAATCACAATTATGGGCAGTTGGGTTAAATAATTATAAAACACAATT

TCCAAACTGCTCATTAATTGAGGAAATATATTCTCGTCATTATCCTTTTG

GAATGTTAAAAACTACACAATGGTTATTACAAACACTTCTTTTAinTCT

GCAATGTATTTTCCATATTTTGAAGTTCATGATATATCTTTGGTTGTTTT

TTTCACCCTGCAATTTTCAGTTTTATTCACTGGCTTTTATATTATTGCGC

AGTTCATGAAAGTCAAAATAATCCAAAACCAATTAATTTGTCTACTCTCT

TCTTTTCTGATATAATCATTTCATATTGCTTCACTATATTATTTATTGTA

CAGTTTATATCATCAGGAAGATATGGGGCATATTTGTTTCTCTTTGGATT

AATTTTGTATGGTGGTTATTCTTTAATTTTMCTTTTGrn*ATTTACGTA

ATAATGAAGATGGATCCTTTAAATTCCCAATTTCAATAAAAATAAATGTT

GAAATTATTCAAAAATCGGATAAAGAATTAAAACAGG AAAAAAAAAAAAA AAAAAAAAAAAAA (SEQ ID NO:46) 95 WO 2006/044480 PCT/US2005/036711 5A12B>eng3» AY422836_

GACAACACAAATCAAATTAATTTTAAAAATCTAATTAAAAATGAATTCTC

TCTTATTAATAGCATTTTTATCCCTCTCATTTTGTGTTCCAATAAAGGCT

GCTCCTCCATATGGGCAATTATCTGTGAAAGGAAGTCAATTAGTGGGCAG

TAATGGACAACCAGTTCAACTTGTTGGAATGfCACirrrCTGGTCGAGTT

GTGGTGAAGGAGAAGTTTTTTATAATAAAGCAACAGTAAATAGTCTTAAA

TGCTCTTGGAATTCAAATGTAGTTAGGGCTGCAATGGGTGTAGAGTATTC

AGGGTGCCAACGACCAGGTTATTTGGATGCCCCAAATGTTGAGCTGGGCA

AGGTTGAAGCTGTTGTTAAGGCCGCAATAGAGTTGGATATGTATGTTATC

CTTGACTTTCACGACCACAATGCTCAACAACATGTGAAACAAGCTATCGA

ATTGTTCACATATTTTGCCCAAAACTACGGATCTAAATACCCTAACATAA

TTTATGAGACTTTCAATGAGCCACTACAAGTAGACTGGAGTGGTGTAAAG

TCATATCATGAGCAAGTTGTTGCAGAAATTAGAAAATATGACACAAAGAA tgtcatcgttctcggtacAacaacatggtctcaagatgtcgatactgctg ctaacaatcctgtaAgcggcacaaacctttgctacactctacacttctac gcagcaactcataaacaaaacttaagagacaaggctcaggctgcaatgaa taagggagcttgtatctttgtaactgaatacggaactgttgatgcaagtg gaggtggtggagtggatgaaggttcgacaaaagaatggtataacttcatg gatagtaacaagatttctaacctcaactgggctatctcaaacaaggcaga aggtgcttcagcactcacatctggaacgagtgcttctcaaattggcaatg atgaccgattgactgcctccggtcttatagtgaagaagtatattaaatca aagaatactggtgtcagttgcaatggtgcatcatcaggcagtggttccgg aaataacccctcaggaaatgaaccgagcaactcacaaaccagcactgcca aaacatcaagcaattcaggaaataaaggcggtaattctaacacagggaat aatgcaaataactcaggaAgtaaAcggggcaactccggaagtaatacagg aaatacgggcagcaatgctggggcaaattcaggaaatacggggaccagta caggcagtagttctgttacagcttctgtgcaagttcccgataaatgggat aatggcgcaagattccaattagtatttaaaaacaatgcgagtacaaaaaa

GTGTGCAGTGAAATTTTCATTGACTnTGCCTCTGGACAACAAATTACTG gcatttggaatgcccaaaatgtaacaggaaataattttgttcttccagac

TACGTTACAATTGGAGCAGGGAAACAATATACAGATGCAGGAATGAATAT

AAATGGGCCAGCAACTCCTCCACAAATTAAGGTGCTCGGCGATGGAAAAT

G C GTTTTTTG AA ATT AAA G AC TCCG TCT AAATT G TTG AATT ATTTA AT CT

TATGATTGTTTAAATTGGAAAATAAATATATGTATAATTTGCTTCTGTTA

ATTTTGTTTATTrAAATATACGATAAAAATTAAAAAAAAAAAAAAAAAAA AAAAAA (SEQ ID NO:47) 5C03B>msp39»AY422832_:_

TAAATTTCTTCCCTAAAATTTATTTAAAATTTTATAACAAAAAAATGTTT

TCAATTCAAGGATTATCTTCTTTTCACTTCATTTTCCTCTCATTATTGAT

ATTATTGCAAAACTCTTCTACTGTATTTTCTCAACTTGGTTGTGATTATG

GATCAATGTATGGCGGGGGAATGAGTGGTTATGGCCAAGCAGGTTATGGA

AATGAAAGTACACACATCACTTCTGCCCACATTATATTGGCCAAAGTGAA

TCACATGGTTTCTCCTGACTTCAACAAGCAGGGCATGAATAATCTAACCT

CCCACAAAGAACACGACTAGGAAAGAAAATAGAATAATTGGCAAACACTA atgcaatctactacagaagtcaatggagaatttacctcctaaacaggaaa

ATG ATTTGTGCCTAAAAG G AAGG AAG AAGAACCTCCTCTTTGTTGAGGGG

AAAAGTCCATAACACAGGAGTGCTTGGACCCAAGTACACAAATATAAGAA

CCCTTCTAGGAAAACACGAGCTGGGGAAGCAGTTTCTCTTTGCTATTTTG TGAGAAAATAAATGGCAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:48) 96 WO 2006/044480 PCT/US2005/036711 6D09B>cm2»AY422835_

GGTTTAATTACCCAAGTTTAAGAAAATAAACTTTTTAATAAAAATATTTA

ATTTTGAATGTCTTTTAATTGGCTTTATTGCAATTTATTTATTGCAATAC

TCTrrprCAACATTGTAAAGAGTGATACCGATACTAATCCTGATATTGAT

CGATTTGTTGAAATTGCAGACGATCGTTTAACTCTTTCTGATTATGTTGC

TTTATATAAAGTTGTTAATAATCAAAGTATTACTGATCCAAAACGAGAAG

AAAAACTTTTGAACGATATGAGAAGTAAG GG AAAG AATTTTTCGTT AAAT

GAG GATTATGTTACTTTAATATTC CAAGAC C AAAT AAATGCTAGTAAATA

TTTTCAGAATTATTTAGTTAATTTATGGAATCAATCAGGCATACCACTTA

TT AAAGTTCG AAATTTAACAACAG ACTT ACG CCC AGC AATTG ATC AAATA

AATACAGAAATGCTGCAATTGCTAGTTAAAATACAAAAACTTCCCTCCAA

AGATTGTTTAAAAAAAGTAGATAAGTCTGTAAATAATTTTATTATGATAG

TT AATC AAATTG ATG AAC AAAATG ATG CTTTGAAAATG G CTGTG AAAG GC

AAAGACCTCTGCCCAGCATGTAAACATAATTAACGAAAAAAAAAAAAAAA AAAAAAAAAA (SEQ ID NO:49) 8E08B>eng4» AY422837_

ACGCGGGGAACACAAATCGAAATATTTTTAAAAATTTAATTAAATGTTTT

CCCTCTCATTAGTAGCAI "nTfATCCCTCACATTTTGTATTCAAATTAAT

GCTGCTCCTCCGTATGGGCAATTATCTGTGAAAGGAAGTCAATTAGTGGG

CAGTAATGGACAACCAGTTCAACTTGTTGGAATGTCACTTTTCTGGTCGA

GTTGTGGTGAAGGGGAAGGTTTCTATAACAGAGAAACTGTAAATAGTCTT

AAATGCTCTTGGAATTCAAATGTTGTTAGAGCTGCAATGGGTGTAGAATA

TTCTGGATGCCAACGACCAGGTTACCTTGATGCCCCAAATGTTGAG'CTGG

CAAAGGTtGAAGCTGTAGTGAAGGCGGCGATTGAGTTGGATATGTATGTT

ATTCTTGATntCACGACCACAATGCTCAGGGTCATGTGAAACAAGCTAA

ACAATTCTTCGCATATTTTGCCCAAAACTACGGATGTAAATACCCAAATA

TCATTTATGAGACTTTCAATGAGCCACTACAAGTAGACTGGAATGGTGTA

AAATCATATCATGAGGAAGTTGTTGCAGAAATTAGAAAATATG AC AATAA

GAATGTCATCGTTCTTGGTTCAACAACTTGGTCTCAAGATGTTGATACTG

CCGCTAATAATCCTGTACGAGGTTCAAACCTTTGCTATTCTTTACACTAC

TACGCAGCAACTCATAAACAAAACTTAAGAGACAAGGCACAGGCTGCAAT

TAATAAAGGAGCCTGTATCTTCGTAACTGAGTACGGAACCGTTGATGCAA

GTGGAGGTPGTGGAGTGGATGAAGGCTCGACAAAAGAGTGGTATAACTTC

TTGGATAGCAAGAAAATTTCTAACCTCAACTGGGCTATCTCGAACAAGGC

AGAAGGGGCTGCAGCACTCACCCCTGGAACGACTTCTTCTCAAGTTGGCA

ATGATGACCGATTGACTGCCTCCGGTCGTCTAGTGAAAAGTTATATTAAA

TCAAAGAATACTGGTGTCAGATGCAATGGAGGGGGTGCTGCAAAAAAAGG

CTCTT'CATCATCTAATACTGGTTCAAAAAAAGACAAACAAAAATTCAAAG

AACAAAAATTCAAAGAAAAAATCTAACAACGCCAAACTGCCGAAAAAAAG

GTCCCAAAAAGAACACTTAGACAAATATCAAGGAATTTAATGTTAAATGG AATATAATTGT ΠΤΑΑΑΤΤΑΑΑΑΑΑΑΑΑΑΑΑΑΑΑΑΑΑΑΑΑΑΑ (SEQ ID NO:50) 97 WO 2006/044480 PCT/US2005/036711 8E10B>msp40)>AY422833

GGTCATTCTTATAACTAAAAACCTTCAAACTTCAAAAAATATTCCTTAAA

CTTCTTC AG AAAAATAATTGAA AAATGTTATTAAAATTOTfTrCCCATT

ATTGCTTTTGCTTACCCTTATCTATTTGGGTTGTTCTGAGGAGGATAAGG

GAGACATTGCAAATGGTCCTCAGGAATCTGAGAATCAGGTTGATCAAGAA

TTGGTTAGATTGAAAAGAGATGATGAAGAAGAGGAGGGAGAGAAGGCCGA

AGATGAAGAGAAGGCTGAAGAGGATGGAGATAAAGCTGAAGATGCTGAGA

GTGCAGAGGAGGGAGATAAGGCTGAAGATGCTGATGAGGGAGAAAAAAAG agtgaagatgaagagaaaaagagtgaaggtgacgaagaaaaagcggaagg

TGAAGAGGAAGAAAAAAAGGATGGAACTGAGGAAGAAAAGGAGGATGAAG

ATGAAGAAGAGAAAAAAGATGATGATGAAGAAAAAAATGAGGAAGAAGAA

AAAAAGGATGACGAAGAAGAGAATGGAGATAAAGAAGAAAAGAAGGATGA

TACGGAAGAGAAAGAGGATAAACACACAAAGGATAAAAGTAAGAAGAAGG

ATAGTAAGTCCGTTCAAAAGGATAAAAAGGAGGAAGATGACAAGGAGAAA

AAGGAAAAAAGTTCAAGTGGTGXTAATTCTAAMCAGATAAATCACAAAA

TCAAAAACAAAGCAAAGAATCATGTAATGGGGATACTGCTTACAACTGTC

CTAAACTATCAGGTCTTTGTGAATCAAAAATTCAAGTACAACAAGACTTC

ATGGGTGAAAAATGTTGTGCTACGTGCAAAAATTCGGCTCCTGCTGCGAA

GAAAGATATACCCCTATGCACTGATTTGGCTGATAATTGTGATCAAATAG

CATCCACCTGTGGGGAAGAGGCGTGGCAACCGACTATGATTTCTG'ATTGT

GCTGAGACCTGCGATAAGTGTGAATTACATTTTCAAATGTTGGAGAAGAG

ACTTGCAGCAGCTGCTGCTTAAAATTTTGAAAGGAAAAGAATTTTATCAA

AAATATATGTGTATCATATTCACTAAGCAAGAAATTTTCTTTGATITCA

CACCTTTAATACGTAAAATTTCAATCTATTCATCCGTGTTTCTCGTAATT

ATGTTTTATTAAI "ΤΓΓΓΐ CGAAATTTAGTAAAAATGCCTCCAAAAAAAAA AAAAAAAAAAAAAAAAA (SEQ ID NO:51 ) 98 , crnxan rwzn ατπ iniat^a pnow pnszn irn πτ qaoa ,Ρ’ηη Txan naoana roavia nawmaa πρ’-ιοζ .zruwan rwaa mpnan ρηυ1? aixn-a □ιηπη bv

<img img-format="tif" img-content="drawing" file="IL182245AD00021.tif" id="idf0001" />

.(rnoia παΊηπ) cras&amp;amp;'an rwa