US20030175767A1

US20030175767A1 - Chemoreceptors in plant parasitic nematodes

Info

Publication number: US20030175767A1
Application number: US10/324,415
Authority: US
Inventors: Eric Davis; Yitang Yan
Original assignee: Individual
Current assignee: Individual
Priority date: 1999-11-05
Filing date: 2002-12-20
Publication date: 2003-09-18
Also published as: WO2001034791A3; US6521438B1; AU1359401A; WO2001034791A2

Abstract

Isolated DNA encoding a nematode guanylyl cyclase chemoreceptor is disclosed. Preferably, the encoded nematode guanylyl cyclase chemoreceptor is selected from the group consisting of order Tylenchida and order Aphelenchida chemoreceptors. Also disclosed are vectors and cells containing the DNA, the encoded proteins, oligonucleotides that bind thereto, and methods of using the same.

Description

FIELD OF THE INVENTION

The present invention concerns isolated DNA encoding chemoreceptors of plant parasitic nematodes, cells that express such DNA, the proteins so expressed, and methods of use thereof.

BACKGROUND OF THE INVENTION

Annual crop losses to plant-parasitic nematodes (soil-dwelling microscopic worms) are estimated to exceed 70 billion dollars world-wide. The soybean cyst nematode (SCN), Heterodera glycines, causes about one billion in annual soybean losses in the United States alone. Environmental restrictions in the use of toxic nematicides and limitations in available plant resistance schemes to nematodes have prompted an urgent need for alternative management strategies to reduce nematode-related damage in agriculture.

One way to control nematodes is by understanding and specifically interfering with the nematode's ability to locate and feed from plant roots. Like most plant-parasitic nematodes, infective juveniles of SCN migrate in the soil and use their neurosensory organs to follow chemical signals emanating from host roots that they will attack

Chemoreceptor molecules have been identified in the model nematode, Caenorhabditis elegans, as described in S. Yu et al., Proc. Natl. Acad. Sci. USA 94, 3384-3387 (1997). However, essentially nothing is known about putative chemoreceptors in plant-parasitic nematodes. Accordingly, there is a continued need for more information about the chemoreceptors of plant-parasitic nematodes.

SUMMARY OF THE INVENTION

A first aspect of the present invention is an isolated DNA encoding a nematode guanylyl cyclase chemoreceptor selected from the group consisting of: (a) isolated DNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 6; (b) isolated DNA that hybridizes to DNA of (a) above under stringent conditions and encodes a nematode guanylyl cyclase chemoreceptor; and (c) isolated DNA that differs from the DNA of (a) or (b) above due to the degeneracy of the genetic code, and encodes a nematode guanylyl cyclase chemoreceptor encoded by (a) or (b) above.

In one particular embodiment of the invention, the isolated DNA is selected from the group consisting of: (a) isolated DNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6; (b) isolated DNA that hybridizes to DNA of (a) above under stringent conditions in which said isolated DNA does not hybridize to DNA having a nucleotide sequence of SEQ ID NO: 1, and encodes a nematode guanylyl cyclase chemoreceptor; and (c) isolated DNA that differs from the DNA of (a) or (b) above due to the degeneracy of the genetic code, and encodes a nematode guanylyl cyclase chemoreceptor encoded by (a) or (b) above.

In another particular embodiment of the invention, the isolated DNA is selected from the group consisting of: (a) isolated DNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3; (b) isolated DNA that hybridizes to DNA of (a) above under stringent conditions and encodes a nematode guanylyl cyclase chemoreceptor; and (c) isolated DNA that differs from the DNA of (a) or (b) above due to the degeneracy of the genetic code, and encodes a nematode guanylyl cyclase chemoreceptor encoded by (a) or (b) above.

In another particular embodiment of the invention, the isolated DNA is selected from the group consisting of: (a) isolated DNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 4; (b) isolated DNA that hybridizes to DNA of (a) above under stringent conditions and encodes a nematode guanylyl cyclase chemoreceptor; and (c) isolated DNA that differs from the DNA of (a) or (b) above due to the degeneracy of the genetic code, and encodes a nematode guanylyl cyclase chemoreceptor encoded by (a) or (b) above.

In still another particular embodiment of the invention, the isolated DNA is selected from the group consisting of: (a) isolated DNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 6; (b) isolated DNA that hybridizes to DNA of (a) above under stringent conditions and encodes a nematode guanylyl cyclase chemoreceptor; and (c) isolated DNA that differs from the DNA of (a) or (b) above due to the degeneracy of the genetic code, and encodes a nematode guanylyl cyclase chemoreceptor encoded by (a) or (b) above.

Preferably, the encoded nematode guanylyl cyclase chemoreceptor is selected from the group consisting of order Tylenchida and order Aphelenchida chemoreceptors.

A second aspect of the invention is an oligonucleotide that specifically binds to isolated DNA as described above is a further aspect of the invention. Such an oligonucleotide may comprise DNA or RNA, or may be a synthetic oligonucleotide.

A third aspect of the invention is an antisense oligonucleotide that specifically binds to an mRNA transcript of a DNA as described above, along with DNAs that encode such antisense oligonucleotides.

A fourth aspect of the invention double-stranded RNA that is complementary to a DNA as described above and interferes with the expression thereof in a cell that expresses the encoded protein.

A fifth aspect of the invention is an expression cassette comprising a DNA as described above and a heterologous promoter operatively associated therewith, along with cells that contain such expression cassettes and express the encoded nematode guanylyl cyclase chemoreceptor (e.g., yeast cells, plant cells, insect cells).

A sixth aspect of the invention is an isolated nematode guanylyl cyclase chemoreceptor protein encoded by a DNA as described above (a protein of the invention), along with proteins or peptides (e.g., antibodies) that specifically bind to such nematode guanylyl cyclase chemoreceptor proteins.

A seventh aspect of the present invention is a method of screening a compound for the ability to disrupt plant parasitic nematode feeding or chemotaxis, said method comprising: determining whether or not said compound selectively binds to a nematode guanylyl cyclase chemoreceptor protein encoded by a DNA as described above. The presence of such binding indicating said compound is useful in disrupting plant parasitic nematode feeding or chemotaxis.

The foregoing and other objects and aspects of the present invention are explained in detail in the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a comparison of the guanylyl cyclase (gcy) domains of HG-gcy-1, HG-gcy-2, and HG-gcy-3 to various other guanylyl cyclases and proteins.[0018]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Amino acid sequences disclosed herein are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence. Nucleotide sequences are presented herein by single strand only, in the 5′ to 3′ direction, from left to right. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission; or (for amino acids) by three letter code, in accordance with 37 CFR § 1.822 and established usage. [0019]
The present invention may be carried out with plant parasitic, or plant feeding, nematodes. That is, chemoreceptors proteins of the present invention may be those of such nematodes, and isolated DNA may be isolated, directly or indirectly, from such nematodes. Examples of plant parasitic nematodes include, but are not limited to, cyst nematodes (Heteroderidae spp.), root knot nematodes (Meloidogyne spp.), lesion nematodes (Pratylenchus spp.), and reniform nematodes (Rotylenchulus spp.). In general, nematodes of the orders Tylenchida and Aphelenchida are preferred, particularly nematodes of the order Tylenchida. Nematodes of the Heteroderoidea superfamily are particularly preferred, the Heterodae family more preferred, and the genus Heterodera most preferred. [0020]
The production of cloned genes, recombinant DNA, vectors, transformed host cells, proteins and protein fragments by genetic -engineering is discussed in greater detail below. It will be appreciated, however, that the techniques employed in carrying out the instant invention are well known. See, e.g., U.S. Pat. No. 4,761,371 to Bell et al. at Col. 6 [0021] line 3 to Col. 9 line 65; U.S. Pat. No. 4,877,729 to Clark et al. at Col. 4 line 38 to Col. 7 line 6; U.S. Pat. No. 4,912,038 to Schilling at Col. 3 line 26 to Col. 14 line 12; and U.S. Pat. No. 4,879,224 to Wallner at Col. 6 line 8 to Col. 8 line 59. (Applicant specifically intends that the disclosure of all patent references cited herein be incorporated herein in their entirety by reference).
1. Isolated Nucleic Acids. [0022]
Isolated DNA of the present invention can be of any species of origin, but is preferably isolated either directly or indirectly from plant parasitic nematodes as described above. Thus, polynucleotides that hybridize to DNA disclosed herein as SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 6 (or fragments or derivatives thereof which serve as hybridization probes as discussed below) and which code on expression for a protein of the present invention (e.g., a protein according to SEQ ID NO:2), are also an aspect of the present invention. [0023]
Conditions which will permit other polynucleotides that code on expression for a protein of the present invention to hybridize to the aforesaid DNA can be determined in accordance with known techniques. For example, hybridization of such sequences may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35-40% Formamide with 5× Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.; conditions represented by a wash stringency of 40-45% Formamide with 5× Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and conditions represented by a wash stringency of 50% Formamide with 5× Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C., respectively) to the aforesaid DNA in a standard hybridization assay. See, e.g., J. Sambrook et al., [0024] Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory). In general, sequences which code for proteins of the present invention and which hybridize to the DNAs disclosed herein will be at least 60% homologous, 70% homologous, 80% homologous and even 90% homologous or more with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO: 4, or SEQ ID NO: 6, respectively.
As noted above, in one particular embodiment of the invention, the isolated DNA is selected from the group consisting of: (a) isolated DNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6; (b) isolated DNA that hybridizes to DNA of (a) above under stringent conditions in which said isolated DNA does not hybridize to DNA having a nucleotide sequence of SEQ ID NO: 1, and encodes a nematode guanylyl cyclase chemoreceptor; and (c) isolated DNA that differs from the DNA of (a) or (b) above due to the degeneracy of the genetic code, and encodes a nematode guanylyl cyclase chemoreceptor encoded by (a) or (b) above. In this embodiment, the stringency of the wash conditions is routinely determined by adjusting wash stringency upward until the stringency meets the test of excluding hybridization to a nucleotide sequence of SEQ ID NO: 1. [0025]
Further, polynucleotides that code for proteins of the present invention, or polynucleotides that hybridize to nucleotides having a sequence as given in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO: 4, and/or SEQ ID NO: 6, as described above, but which differ in codon sequence from the given coding sequence due to the degeneracy of the genetic code, are also an aspect of this invention. The degeneracy of the genetic code, which allows different nucleic acid sequences to code for the same protein or peptide, is well known in the literature. See, e.g., U.S. Pat. No. 4,757,006 to Toole et al. at Col. 2, Table 1. [0026]
The invention also encompasses production of DNA sequences, or fragments thereof, which encode proteins of the invention, entirely by synthetic chemistry. Where modeled after the natural protein or DNA, such sequences may be considered to have been indirectly isolated from the species carrying the naturally occurring nucleotide or protein. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art. [0027]
In general, those skilled in the art will appreciate that minor deletions or substitutions may be made to the amino acid sequences of peptides of the present invention without unduly adversely affecting the activity thereof. Thus, peptides containing such deletions or substitutions are a further aspect of the present invention. In peptides containing substitutions or replacements of amino acids, one or more amino acids of a peptide sequence may be replaced by one or more other amino acids wherein such replacement does not affect the function of that sequence. Such changes can be guided by known similarities between amino acids in physical features such as charge density, hydrophobicity/hydrophilicity, size and configuration, so that amino acids are substituted with other amino acids having essentially the same functional properties. [0028]
2. Oligonucleotides. [0029]
The term “oligonucleotide” refers to a nucleic acid sequence of at least about 6 nucleotides to about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about −20 to 25 nucleotides, which can be used in PCR amplification or a hybridization assay, or a microarray. As used herein, oligonucleotide includes “amplifiers”, “primers”, “oligomers”, and “probes”, as commonly defined in the art. [0030]
Knowledge of the nucleotide sequences disclosed herein can be used to generate hybridization probes which specifically bind to the DNA of the present invention or to mRNA produced by the transcription of such nucleotides to determine the presence of, amplify, or determine the overexpression of the proteins of the present invention. The oligonucleotides may also be used as active agents for the control of plant feeding nematodes as described above. [0031]
A label or detectable group may be conjugated to the oligonucleotide, if desired. A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to proteins of the invention oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding proteins of the invention, or any fragments thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio)). Suitable reporter molecules or labels, which may be used for ease of detection, include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like. [0032]
Assays for detecting the nucleotides of the invention, or the extent of amplification thereof, typically involve, first, contacting the cells or extracts of the cells containing nucleic acids therefrom with an oligonucleotide that specifically binds to proteins of the invention under conditions that permit access of the oligonucleotide to intracellular material, and then detecting the presence or absence of binding of the oligonucleotide thereto. Any suitable assay format may be employed (see, e.g., U.S. Pat. No. 4,358,535 to Falkow et al.; U.S. Pat. Nos. 4,302,204 to Wahl et al.; 4,994,373 to Stavrianopoulos et al; 4,486,539 to Ranki et al.; 4,563,419 to Ranki et al.; and 4,868,104 to Kurn et al.) (the disclosures of which applicant specifically intends be incorporated herein by reference). [0033]
3. Expression Vectors and Transgenic Cell Lines. [0034]
A vector is a replicable DNA construct. Vectors are used herein either to amplify DNA encoding the proteins of the present invention or to express the proteins of the present invention. An expression vector is a replicable DNA construct in which a DNA sequence encoding the proteins of the present invention is operably linked to suitable control sequences capable of effecting the expression of proteins of the present invention in a suitable host. The need for such control sequences will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. [0035]
Vectors comprise plasmids, viruses (e.g., adenovirus, cytomegalovirus), phage, retroviruses and integratable DNA fragments (i.e., fragments integratable into the host genome by recombination). The vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself. Expression vectors should contain a promoter and RNA binding sites which are operably linked to the gene to be expressed and are operable in the host organism. [0036]
DNA regions are operably linked or operably associated when they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of leader sequences, contiguous and in reading phase. [0037]
Transformed host cells are cells which have been transformed or transfected with vectors containing DNA coding for proteins of the present invention need not express protein. [0038]
Suitable host cells include prokaryotes, yeast cells, or higher eukaryotic organism cells. Prokaryote host cells include gram negative or gram positive organisms, for example [0039] Escherichia coli (E. coli) or Bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Exemplary host cells are E. coli W3110 (ATCC 27,325), E. coli B, E. coli X1776 (ATCC 31,537), E. coli 294 (ATCC 31,446). A broad variety of suitable prokaryotic and microbial vectors are available. E. coli is typically transformed using pBR322. See Bolivar et al., Gene 2, 95 (1977). Promoters most commonly used in recombinant microbial expression vectors include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature 275, 615 (1978); and Goeddel et al., Nature 281, 544 (1979), a tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980) and EPO App. Publ. No. 36,776) and the tac promoter (H. De Boer et al., Proc. Natl. Acad. Sci. USA 80, 21 (1983). The promoter and Shine-Dalgarno sequence (for prokaryotic host expression) are operably linked to the DNA of the present invention, i.e., they are positioned so as to promote transcription of the messenger RNA from the DNA.
Expression vectors should contain a promoter which is recognized by the host organism. This generally means a promoter obtained from the intended host. Promoters most commonly used in recombinant microbial expression vectors include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al., [0040] Nature 275, 615 (1978); and Goeddel et al., Nature 281, 544 (1979), a tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980) and EPO App. Publ. No. 36,776) and the tac promoter (H. De Boer et al., Proc. Natl. Acad. Sci. USA 80, 21 (1983). While these are commonly used, other microbial promoters are suitable. Details concerning nucleotide sequences of many have been published, enabling a skilled worker to operably ligate them to DNA encoding the protein in plasmid or viral vectors (Siebenlist et al., Cell 20, 269 (1980). The promoter and Shine-Dalgarno sequence (for prokaryotic host expression) are operably linked to the DNA encoding the desired protein, i.e., they are positioned so as to promote transcription of the protein messenger RNA from the DNA.
Eukaryotic microbes such as yeast cultures may be transformed with suitable protein-encoding vectors. See e.g., U.S. Pat. No. 4,745,057. [0041] Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available. Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding the desired protein, sequences for polyadenylation and transcription termination, and a selection gene. An exemplary plasmid is YRp7, (Stinchcomb et al., Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschemper et al., Gene 10, 157 (1980). This plasmid contains the trp1 gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12 (1977). The presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3-phospho-glycerate kinase (Hitzeman et al., [0042] J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7, 149 (1968); and Holland et al., Biochemistry 17, 4900 (1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPO Publn. No. 73,657.
Cultures of cells derived from multicellular organisms are a desirable host for recombinant protein synthesis. In principal, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture, including insect cells. Propagation of such cells in cell culture has become a routine procedure. See Tissue Culture, Academic Press, Kruse and Patterson, editors (1973). Examples of useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, BHK, COS-7, CV, and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the gene to be expressed, along with a ribosome binding site, RNA splice site (if intron-containing genomic DNA is used), a polyadenylation site, and a transcriptional termination sequence. [0043]
The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources. For example, commonly used promoters are derived from polyoma, [0044] Adenovirus 2, and Simian Virus 40 (SV40). See, e.g., U.S. Pat. No. 4,599,308. The early and late promoters are useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. See Fiers et al., Nature 273, 113 (1978). Further, the protein promoter, control and/or signal sequences, may also be used, provided such control sequences are compatible with the host cell chosen.
An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV), or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient. [0045]
Host cells such as insect cells (e.g., cultured [0046] Spodoptera frugiperda cells) and expression vectors such as the baculorivus expression vector (e.g., vectors derived from Autographa californica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed to make proteins usefull in carrying out the present invention, as described in U.S. Pat. Nos. 4,745,051 and 4,879,236 to Smith et al. In general, a baculovirus expression vector comprises a baculovirus genome containing the gene to be expressed inserted into the polyhedrin gene at a position ranging from the polyhedrin transcriptional start signal to the ATG start site and under the transcriptional control of a baculovirus polyhedrin promoter.
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding proteins of the invention may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing proteins of the invention in infected host cells (Logan, J. and Shenk, T. (1984) [0047] Proc. Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
Rather than using vectors which contain viral origins of replication, one can transform mammalian cells by the method of cotransformation with a selectable marker and the chimeric protein DNA. An example of a suitable selectable marker is dihydrofolate reductase (DHFR) or thymidine kinase. See U.S. Pat. No. 4,399,216. Such markers are proteins, generally enzymes, that enable the identification of transformant cells, i.e., cells which are competent to take up exogenous DNA. Generally, identification is by survival or transformants in culture medium that is toxic, or from which the cells cannot obtain critical nutrition without having taken up the marker protein. [0048]
4. Antibodies and Other Binding Proteins and Peptides. [0049]
As used herein, the term “antibody” refers to intact molecules as well as fragments thereof, such as Fa, F(ab′)[0050] ₂, and Fc, which are capable of binding the epitopic determinant. Antibodies that bind proteins of the invention can be prepared using intact proteins or fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal can be derived from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit) from which antibodies or spleen cells are collected.
Antibodies that specifically bind to the proteins of the present invention (i.e., antibodies which bind to a single antigenic site or epitope on the proteins) are useful for a variety of diagnostic purposes. [0051]
Antibodies to proteins of the invention may be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies, (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use. [0052]
For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with a protein of the invention or any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable. [0053]
Monoclonal antibodies to proteins of the invention may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) [0054] Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120).
Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) [0055] Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991) Nature 349:293-299).
Antibody fragments which contain specific binding sites for proteins of the invention may also be generated. For example, such fragments include, but are not limited to, the F(ab′)[0056] ₂fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).
Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between a protein of the invention and its specific antibody. [0057]
Antibodies may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation. Antibodies may likewise be conjugated to detectable groups such as radiolabels (e.g., [0058] ³⁵S, ¹²⁵I, ¹³¹I, enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
Kits for determining if a sample contains proteins of the present invention will include at least one reagent specific for detecting the presence or absence of the protein. Diagnostic kits for carrying out antibody assays may be produced in a number of ways. In one embodiment, the diagnostic kit comprises (a) an antibody which binds proteins of the present invention conjugated to a solid support and (b) a second antibody which binds proteins of the present invention conjugated to a detectable group. The reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like. The diagnostic kit may further include, where necessary, other members of the signal-producing system of which system the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like. A second embodiment of a test kit comprises (a) an antibody as above, and (b) a specific binding partner for the -antibody conjugated to a detectable group. Ancillary agents as described above may likewise be included. The test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test. [0059]
Binding proteins or peptides other than antibodies, and binding compounds other than proteins or peptides, can be identified by screening combinatorial libraries of such compounds with the screening assays described below. [0060]
5. Screening Assays. [0061]
As noted above, the present invention provides methods of screening a compound for the ability to disrupt plant parasitic nematode feeding and/or chemotaxis. The methods comprise determining whether or not that compound selectively binds to a nematode guanylyl cyclase chemoreceptor protein encoded by a DNA as described herein. The presence of such binding indicates the compound is useful in disrupting plant parasitic nematode feeding and/or chemotaxis. The determining step may be carried out in vitro with a cell membrane preparation containing the proteins produced from recombinant cells as described above, or even in a membrane-free preparation. Alternatively, the determining step may be carried out in vivo in a cell culture comprising cells that express the protein, in accordance with known techniques. [0062]
The compound screened may be a member of a combinatorial library, which generally are comprised of non-oligomers, oligomers, or combinations thereof. Non-oligomer combinatorial libraries include a wide variety of organic molecules, such as heterocyclics, aromatics, alicyclics, aliphatics and combinations thereof, comprising steroids, antibiotics, enzyme inhibitors, ligands, hormones, drugs, alkaloids, opioids, terpenes, porphyrins, toxins, catalysts, as well as combinations thereof. [0063]
Oligomer combinatorial libraries include oligopeptides, oligonucleotides, oligosaccharides, polylipids, polyesters, polyamides, polyurethanes, polyureas, polyethers, and poly (phosphorus derivatives), e.g. phosphates, phosphonates, phosphoramides, phosphonamides, phosphites, phosphinamides, etc., poly (sulfur derivatives) e.g., sulfones, sulfonates, sulfites, sulfonamides, sulfenamides, etc., where for the phosphorous and sulfur derivatives the indicated heteroatom for the most part will be bonded to C,H,N,O or S, and combinations thereof. [0064]
When the compound to be screened is a member of a combinatorial library, the screening step may be incorporated into a high throughput screening procedure in accordance with known techniques. In this case, the members of the combinatorial library may be immobilized on solid supports, which solid supports may be separate from one another (e.g., particles or beads) as described in U.S. Pat. No. 5,656,324 to Still et al., or may be discrete regions on a surface portion of a unitary substrate. Such “chip-type” or “pin-type” solid supports are known. See, e.g., U.S. Pat. No. 5,288,514 to Ellman (pin-based support); U.S. Pat. No. 5,510,270 to Fodor et al. (chip-based support). In addition, the screening step may be carried out with any other suitable combinatorial library technique, including but not limited to phage display. See, e.g., U.S. Pat. No. 5,812,047 to Garrard et al.; U.S. Pat. No. 5,223,409 to Ladner et al.; U.S. Pat. No. 5,498,538 to Kay & Fowlkes; U.S. Pat. No. 4,953,002 to Dulbecco. [0065]
One of the principal assays to determine efficacy of potential inhibitors of chemosensory receptors will be to transform plant cells and tissues with genes encoding chemosensory inhibitor molecules (like those mentioned in this application, preferably peptides) and test their effect on nematode chemotaxis. Expression of cassettes producing encoded inhibitory molecules that may be retained, or preferably designed to be exuded from plant tissues (i.e. roots), may be under the control of constitutive promoters such as CaMV 35S, Ro1A-D, nopaline synthase, gamma-TIP, T-cyt, and TR2′, or inducible promoters such as those derived from expressed plant genes like TobRB7, cdc2At, and wun1 (H. Atkinson et al., in The Physiology and Biochemistry of Free-Living and Plant-Parasitic Nematodes, pp. 382-413 (ed. R N Perry, D J Wright, 1998); G. Gheysen et al., in [0066] Cellular and Molecular Aspects of Plant-Nematode Interactions, pp. 120-132 (ed. C Fenoll, F M W Grundler, S A Ohl, 1997); A. Goverse et al., Physiol. Mol. Plant Pathol. 52:275-284 (1998)). Transformation of plant cells and tissues may be conducted using Agrobacterium tumefascians, Agrobacterium rhizogenes, or biolistic approaches (Atkinson et al., 1998). Transformation of plant roots via A. rhizogenes is preferable for screening purposes since these systems work in many plant species and have been demonstrated to produce a reproducible and scorable plant-nematode interaction (D. Cai et al., Science 275, 832-834 (1997); M. Savka et al., Phytopathology 80, 503-508 (1990)). A variety of agar and soil-based assays may be utilized to assess the effect of transgenic expression of chemosensory inhibitors in plants on nematode chemotactic ability (C. Bargmann and I. Mori. Chemotaxis and thermotaxis. Pp. 717-737 In Riddle, D. L., T. Blumenthal, B. J. Meyer, and J. R. Priess, eds., C. elegans II, (Cold Spring Harbor Laboratory Press, NY 1997)).
6. Antisense Oligonucleotides and Double-stranded RNA. [0067]
Antisense oligonucleotides. The term “antisense”, as used herein, refers to any composition containing nucleotide sequences which are complementary to a specific DNA or RNA sequence. The term “antisense strand” is used in reference to a nucleic acid strand that is complementary to the “sense” strand. Antisense molecules include peptide nucleic acids and may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and block either transcription or translation. The designation “negative” is sometimes used in reference to the antisense strand, and “positive” is sometimes used in reference to the sense strand. [0068]
Antisense oligonucleotides and nucleic acids that express the same may be made in accordance with conventional techniques. See, e.g., U.S. Pat. No. 5,023,243 to Tullis; U.S. Pat. No. 5,149,797 to Pederson et al. The length of the antisense oligonucleotide (i.e., the number of nucleotides therein) is not critical so long as it binds selectively to the intended location, and can be determined in accordance with routine procedures. In general, the antisense oligonucleotide will be from 8, 10 or 12 nucleotides in length up to 20, 30, or 50 nucleotides in length. Such antisense oligonucleotides may be oligonucleotides wherein at least one, or all, or the internucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphonothioates, phosphoromorpholidates, phosphoro-piperazidates and phosphoramidates. For example, every other one of the internucleotide bridging phosphate residues may be modified as described. In another non-limiting example, such antisense oligonucleotides are oligonucleotides wherein at least one, or all, of the nucleotides contain a 2′ loweralkyl moiety (e.g., C[0069] ₁-C₄, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, every other one of the nucleotides may be modified as described. See also P. Furdon et al., Nucleic Acids Res. 17, 9193-9204 (1989); S. Agrawal et al., Proc. Natl. Acad Sci. USA 87, 1401-1405 (1990); C.
Baker et al., [0070] Nucleic Acids Res. 18, 3537-3543 (1990); B. Sproat et al., Nucleic Acids Res. 17, 3373-3386 (1989); R. Walder and J. Walder, Proc. Natl. Acad. Sci. USA 85, 5011-5015 (1988).
Antisense oligonucleotides may be used as biological control agents per se, or DNA encoding such antisense oligonucleotides may be provided in an expression cassette which is capable of infecting a host nematode and transforming cells of the same, which expression cassette may in turn be used as a biological control agent. [0071]
RNA interference (RNAi). RNAi is a methodology to directly inhibit gene activity that is both powerful and efficient is the double-stranded (ds) RNA-mediated interference (RNAi) of gene expression as demonstrated in [0072] C. elegans (A. Fire et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature 391, 806-11 (1998); L. Timmons and A. Fire, Specific interference by ingested dsRNA. Nature 395, 854 (1998)). In this methodology, dsRNA complementary to a gene-of-interest (in this case, a DNA as described above) is administered to (e.g., injected into or ingested by) the target nematode. As a consequence, activity of the gene-of-interest is transiently abolished in the treated animal. Thus such agents are useful in the control of nematodes as described above. Two distinct advantages provided by RNAi analyses include; the dsRNA does not have to be injected into the nematode germ line to exert inhibitory effects in tissues distal to the injection site (i. e. RNAi does not require successful transformation); and the inhibitory effects of injected dsRNA can be realized in one or more subsequent nematode generations derived from the treated parent. RNAi designed to knock-out gene function in nematodes can be assayed directly for its effects on chemosensory behavior. It also will be important to monitor the effects RNAi by mRNA in situ hybridization and/or antibody probes to the target gene product to confirm inhibition. The dsRNA is, in general, from 8, 10 or 12 nucleotides in length up to 20, 30, or 50 nucleotides in length, each strand (although the strands do not have to be identical in length).
7. Control of Plant Parasitic Nematodes. [0073]
The present invention provides a variety of means for controlling plant parasitic nematodes, as described above. [0074]
Nematodes may be administered an expression cassette that contains and expresses a DNA as described thereof, or a fragment thereof of a length sufficient to induce silencing (e.g., at least partial silencing) of expression of the guanylyl cyclase chemoreceptor in the nematode, and thereby disrupt feeding or chemotaxis of the nematode. [0075]
Nematodes may be administered an antisense oligonucleotide as described above, either per se or through a vector that expresses the antisense oligonucleotide, in an amount sufficient to disrupt feeding or chemotaxis of the nematode. [0076]
Nematodes may be administered an oligonucleotide as described above (such as an RNAi oligonucleotide as described above) in an amount sufficient to disrupt feeding or chemotaxis of the nematode. [0077]
Nematodes may be administered a protein or peptide (e.g., an antibody) that specifically binds to the guanylyl cyclase chemoreceptor proteins disclosed hereinabove, in an amount sufficient to disrupt the feeding or chemotaxis of the nematode. Such proteins and peptides, including antibodies, are readily produced in the manner described above. [0078]
Administration of the active compounds described above may be by any suitable means, such as by spraying crops or plants with the active agents described above, by treating soil with the active agents described above, etc. The active agents may be combined with a suitable agricultural carrier, including aqueous carriers, nonaqueous carriers, emulsions, dry powders, etc., which may optionally include stickers, adjuvants and the like, all in accordance with standard techniques, with the active agent being included in any suitable amount (e.g., from 0.001 to 99 percent by weight of the total composition). [0079]
The present invention is explained in greater detail in the following non-limiting Examples. [0080]

EXAMPLE 1

Identification of HG-gcy-1

The soybean cyst nematode (SCN), [0081] Heterodera glycines guanylyl cyclase −1 (HG-gcy-1) coding sequence was first located by the instant inventors about 1 kb upstream of β-1,4-endoglucanase-1 precursor during a study of the organization of β-1,4-endoglucanase gene family in the soybean cyst nematode. The full-length chemosensory guanylyl cyclase gene HG-gcy-1 was generated by further Lambda genomic clone mapping , oligo(dT) cDNA library screening and 5′RACE. A partial cDNA clone of Hg-gcy-1 was obtained by screening a SCN cDNA library and the partial HG-gcy-1 cDNA sequence was released in GenBank on Nov. 9, 1998 (GenBank accession number AF095746). The HG-gcy-1 full-length cDNA sequence (3762 bp) (SEQ ID NO: 1) was released in GenBank on Mar. 5, 1999 with an accession number (AF095746).
By comparing the HG-gcy-1 genomic sequence (SEQ ID NO: 3) and cDNA sequence, 24 introns were identified in HG-gcy-1 gene. The predicted protein of HG-gcy-1 (SEQ ID NO: 2) had strong homology to a family of guanylyl cyclase chemoreceptors reported in the nematode [0082] Caenorhabditis elegans. We have recently localized transcripts of HG-gcy-1 in SCN chemosensory cells by mRNA in situ hybridization (data not reported).

EXAMPLE 2

Identification of HG-gcy-2 and HG-gcy-3

A DNA DIG labeled probe was synthesized based on the guanylyl cyclase catalytic domain sequence. The primers used to synthesize the probe was cycleExp3 and cycleExp4. The cycleExp3 primer sequence is: [0083]
AGCGGATCCCGTCCGCGCATGGACATTGTG (SEQ ID NO: 8). [0084]
The cycleExp4 prime sequence is: [0085]
CCGCTCGAGCGTTGCGGCACTCGCATTTCT (SEQ ID NO: 9) [0086]
The thus synthesized DNA probe was used to screen the SCN oligo(dT) cDNA library with both hybridization and washing temperature at 65° C. This endeavor lead to the identification of two additional guanylyl cyclase genes, namely HG-gcy-2 (3499 bps) (SEQ ID NO: 4) (the protein fragment being given as SEQ ID NO: 5) and HG-gcy-3 (3007 bps) (SEQ ID NO: 6) (the protein fragment being given as SEQ ID NO: 7). These sequences may be elongated in accordance with known techniques to provide the full length sequences thereof. [0087]
The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. [0088]
1 9 1 3762 DNA Heterodera glycines CDS (78)..(3533) 1 gggatttgaa tcccgacaaa tcgccttttt aatgaattca tttcatttta aaatttcctt 60 gcccaaaatc tctcaaa atg gaa atg ccg tcc tgt ttc ttc ctc ctt ttc 110 Met Glu Met Pro Ser Cys Phe Phe Leu Leu Phe 1 5 10 ttt ctt atg ctt ttt gtc agc cct tct cgg cac caa tta gtc act gtt 158 Phe Leu Met Leu Phe Val Ser Pro Ser Arg His Gln Leu Val Thr Val 15 20 25 agc aac tca tcg tct tcg ccc att ggc acc acc gtc gct ttt ggc act 206 Ser Asn Ser Ser Ser Ser Pro Ile Gly Thr Thr Val Ala Phe Gly Thr 30 35 40 cct tcg ccg atc atc cca atc tct act gca ccc tcc acg aat ggc act 254 Pro Ser Pro Ile Ile Pro Ile Ser Thr Ala Pro Ser Thr Asn Gly Thr 45 50 55 gcc act ttt ggc gtc cct ccg ccg atc agt ccg ccg att aat tct tcg 302 Ala Thr Phe Gly Val Pro Pro Pro Ile Ser Pro Pro Ile Asn Ser Ser 60 65 70 75 tcc tct ctc cca tca act ggt cct ttg gaa gca tcg gtt cag tta aaa 350 Ser Ser Leu Pro Ser Thr Gly Pro Leu Glu Ala Ser Val Gln Leu Lys 80 85 90 atc ggc ttc ctc ttt gct aac ggc acc caa cgg ttg cga atg ctt ttc 398 Ile Gly Phe Leu Phe Ala Asn Gly Thr Gln Arg Leu Arg Met Leu Phe 95 100 105 ggc ttt ggc caa tcc gcg ccc gcc gtc act ttg gca ctc gaa cgg gcg 446 Gly Phe Gly Gln Ser Ala Pro Ala Val Thr Leu Ala Leu Glu Arg Ala 110 115 120 agg cag gag cac ctc atc gac agc atc aac ttc act tac acg tgg cga 494 Arg Gln Glu His Leu Ile Asp Ser Ile Asn Phe Thr Tyr Thr Trp Arg 125 130 135 atg tgc ggc tgc ttt cag cct tgg gct gtc ggc tac gcc act caa ctg 542 Met Cys Gly Cys Phe Gln Pro Trp Ala Val Gly Tyr Ala Thr Gln Leu 140 145 150 155 gtt ctg acg gaa aat gtg gac gct ttg atc ggt ccg cct tgt gcc atc 590 Val Leu Thr Glu Asn Val Asp Ala Leu Ile Gly Pro Pro Cys Ala Ile 160 165 170 gcc gcg gga tac gtg gcc tcc ttc tac aac att cca ctg tat ttg tgg 638 Ala Ala Gly Tyr Val Ala Ser Phe Tyr Asn Ile Pro Leu Tyr Leu Trp 175 180 185 ggt gct act gtg gcc tcg gaa ttt tac aac act acc gta tac cct aca 686 Gly Ala Thr Val Ala Ser Glu Phe Tyr Asn Thr Thr Val Tyr Pro Thr 190 195 200 ctg aac aac gtg aac gtt aac tcg gac atg ttg gcg ttg gcc tta caa 734 Leu Asn Asn Val Asn Val Asn Ser Asp Met Leu Ala Leu Ala Leu Gln 205 210 215 agt gtg ttg gtg caa ttc aat tgg aca gaa gtg tcc ttc gtg tac act 782 Ser Val Leu Val Gln Phe Asn Trp Thr Glu Val Ser Phe Val Tyr Thr 220 225 230 235 ccg gac aat gag cga atg gtc tgt aac tcg gtg aaa cag agt ctc aca 830 Pro Asp Asn Glu Arg Met Val Cys Asn Ser Val Lys Gln Ser Leu Thr 240 245 250 aat gtg ctc aac gtg acc aat gtg acc att gtt ttc cag cat cag atg 878 Asn Val Leu Asn Val Thr Asn Val Thr Ile Val Phe Gln His Gln Met 255 260 265 gag tcc aat gtg gac agt atg aag gcg acg ctg aga aat ctg cgc agc 926 Glu Ser Asn Val Asp Ser Met Lys Ala Thr Leu Arg Asn Leu Arg Ser 270 275 280 cga tcg cga att gtg ctt tcc tgt ttc gat gtc gag gtt gac cgt cgc 974 Arg Ser Arg Ile Val Leu Ser Cys Phe Asp Val Glu Val Asp Arg Arg 285 290 295 aac ttt ctg ttg tcc att ttc gac act ggt ctt gct gcg gac aac gaa 1022 Asn Phe Leu Leu Ser Ile Phe Asp Thr Gly Leu Ala Ala Asp Asn Glu 300 305 310 315 ttt gtg ttc atc atg gga tcc ctg cgc aac cag ggc atg ctc cag cag 1070 Phe Val Phe Ile Met Gly Ser Leu Arg Asn Gln Gly Met Leu Gln Gln 320 325 330 gtt gcg tcg cgt gag gac ggc agt gtc aaa tat gtg aac aat tgg atg 1118 Val Ala Ser Arg Glu Asp Gly Ser Val Lys Tyr Val Asn Asn Trp Met 335 340 345 gac aaa aac agc cca ggc gat ggc cgc gac tcg gac gca ctc gcc gcg 1166 Asp Lys Asn Ser Pro Gly Asp Gly Arg Asp Ser Asp Ala Leu Ala Ala 350 355 360 aca aaa cac gtc ata atg att gac ctg gaa aac caa tcg agt gat cat 1214 Thr Lys His Val Ile Met Ile Asp Leu Glu Asn Gln Ser Ser Asp His 365 370 375 ctt aac gaa ttc aac cga gat ttg agt gcg aaa ttc ggc act tat ccc 1262 Leu Asn Glu Phe Asn Arg Asp Leu Ser Ala Lys Phe Gly Thr Tyr Pro 380 385 390 395 ttt ttc tgc aac gga agt tgc atg ggc ggc gca gca gaa caa tcg ccg 1310 Phe Phe Cys Asn Gly Ser Cys Met Gly Gly Ala Ala Glu Gln Ser Pro 400 405 410 tcg caa tac gcc agg gct ttg ttc gac aca aca tac gca tat ttt aga 1358 Ser Gln Tyr Ala Arg Ala Leu Phe Asp Thr Thr Tyr Ala Tyr Phe Arg 415 420 425 gca ttg aat cgc aca atg gaa aag cgc aaa tcg aat ggg agg gat ttg 1406 Ala Leu Asn Arg Thr Met Glu Lys Arg Lys Ser Asn Gly Arg Asp Leu 430 435 440 ttg cgc aac ggc acg gaa ttg aac gca gaa act gcc ggg acg acc ttt 1454 Leu Arg Asn Gly Thr Glu Leu Asn Ala Glu Thr Ala Gly Thr Thr Phe 445 450 455 cag ggc gag acc gga cgc atc act ttt gac gcc cac ggc aac cgc cag 1502 Gln Gly Glu Thr Gly Arg Ile Thr Phe Asp Ala His Gly Asn Arg Gln 460 465 470 475 ccg acc ttt ttt gtg acg atg cta aac gca ctg aat gtg ccc act gtt 1550 Pro Thr Phe Phe Val Thr Met Leu Asn Ala Leu Asn Val Pro Thr Val 480 485 490 atg gtg aaa gtg aac att acc aac gga gta ttg aaa atg gaa cgg ctg 1598 Met Val Lys Val Asn Ile Thr Asn Gly Val Leu Lys Met Glu Arg Leu 495 500 505 tac ggc agt gag gcg tcg ctg tgg gtc aat tgg ggc ggc ttt cgg ccg 1646 Tyr Gly Ser Glu Ala Ser Leu Trp Val Asn Trp Gly Gly Phe Arg Pro 510 515 520 atg acc acg ccg ttg tgc ggc tac aac ggc aca atg tgt ggc caa aat 1694 Met Thr Thr Pro Leu Cys Gly Tyr Asn Gly Thr Met Cys Gly Gln Asn 525 530 535 gtg acg gtg tac att ctg atc ggc gtt acg ctt atg ttg ctg ttg ctg 1742 Val Thr Val Tyr Ile Leu Ile Gly Val Thr Leu Met Leu Leu Leu Leu 540 545 550 555 gtc gcc gct ttg ctt ggc atc gga tac gca att cgg gag aaa atg cgc 1790 Val Ala Ala Leu Leu Gly Ile Gly Tyr Ala Ile Arg Glu Lys Met Arg 560 565 570 gag aag cag cgc ctg aca cgc gag tgt ttg atc cca ttt gca gag ctg 1838 Glu Lys Gln Arg Leu Thr Arg Glu Cys Leu Ile Pro Phe Ala Glu Leu 575 580 585 cgc aac ctg aaa gag ctg cgc agt tcg gag gaa ctg aag tcg gag acg 1886 Arg Asn Leu Lys Glu Leu Arg Ser Ser Glu Glu Leu Lys Ser Glu Thr 590 595 600 gag aag agc atg cgg agc atg cgt agc agt cag tcg gga agc aca cgg 1934 Glu Lys Ser Met Arg Ser Met Arg Ser Ser Gln Ser Gly Ser Thr Arg 605 610 615 ctt acg gtc ggc agc cac aaa gcg cag cgg gag acg gcc aat tgc gcg 1982 Leu Thr Val Gly Ser His Lys Ala Gln Arg Glu Thr Ala Asn Cys Ala 620 625 630 635 ttc ttt gtg ttc aac cgg gaa att gtg ctg gcc gtg aaa tac cac gtc 2030 Phe Phe Val Phe Asn Arg Glu Ile Val Leu Ala Val Lys Tyr His Val 640 645 650 agg gtg cga att atg tcc gaa gat ttg gcc ttc att agg aag ctg cgg 2078 Arg Val Arg Ile Met Ser Glu Asp Leu Ala Phe Ile Arg Lys Leu Arg 655 660 665 cag ttg gac cac gac aac atg aac aag ttg tac ggc gtg tgc acc gat 2126 Gln Leu Asp His Asp Asn Met Asn Lys Leu Tyr Gly Val Cys Thr Asp 670 675 680 ggg ccc ctt ttg ttc gca att tgg cgc aat tgt cag cga ggg aca tta 2174 Gly Pro Leu Leu Phe Ala Ile Trp Arg Asn Cys Gln Arg Gly Thr Leu 685 690 695 aaa gaa ctg atc gcc aag gag caa tac gtt ggg gac aat tgt gtg atg 2222 Lys Glu Leu Ile Ala Lys Glu Gln Tyr Val Gly Asp Asn Cys Val Met 700 705 710 715 ttt gct ctg atg cgg gac att gca aat ggt ctg ctc gcc atc cat caa 2270 Phe Ala Leu Met Arg Asp Ile Ala Asn Gly Leu Leu Ala Ile His Gln 720 725 730 tcg ttc atc gga gcc cac ggg ctg ctc tcc tct gaa aat tgt ctg atc 2318 Ser Phe Ile Gly Ala His Gly Leu Leu Ser Ser Glu Asn Cys Leu Ile 735 740 745 aat gac cgg tgg caa gtg aaa atc agc gac ttt ggc ctg aat atg atc 2366 Asn Asp Arg Trp Gln Val Lys Ile Ser Asp Phe Gly Leu Asn Met Ile 750 755 760 aga gaa agt caa acg ctg tcg aag aaa gca ctt ttg tgg acg gcg cct 2414 Arg Glu Ser Gln Thr Leu Ser Lys Lys Ala Leu Leu Trp Thr Ala Pro 765 770 775 gaa ctt ttg cga gaa aac aat cgg aag gga gca aaa gag ggc gat gtg 2462 Glu Leu Leu Arg Glu Asn Asn Arg Lys Gly Ala Lys Glu Gly Asp Val 780 785 790 795 ttc agt ttt gcg atc att tgt gtg gaa atg atg aac aga gag acg gtg 2510 Phe Ser Phe Ala Ile Ile Cys Val Glu Met Met Asn Arg Glu Thr Val 800 805 810 tgg aac gga gtg gaa agg gac caa gac atc gat gaa atc ctt tat cgg 2558 Trp Asn Gly Val Glu Arg Asp Gln Asp Ile Asp Glu Ile Leu Tyr Arg 815 820 825 ctc aga cgc acc aac acc aca atc cct cac cgt ccg cag ctt cat ccc 2606 Leu Arg Arg Thr Asn Thr Thr Ile Pro His Arg Pro Gln Leu His Pro 830 835 840 cgc gca gag att aac caa agt ttg ctt cat ctg atc aga gac tgt tgg 2654 Arg Ala Glu Ile Asn Gln Ser Leu Leu His Leu Ile Arg Asp Cys Trp 845 850 855 tcc gaa gtg ccg tcc gaa cgt ccg cgc atg gac att gtg cga acg atg 2702 Ser Glu Val Pro Ser Glu Arg Pro Arg Met Asp Ile Val Arg Thr Met 860 865 870 875 ctc aaa cag atg gtc cag gac ggc agt caa aat ctg atg gat tac gtg 2750 Leu Lys Gln Met Val Gln Asp Gly Ser Gln Asn Leu Met Asp Tyr Val 880 885 890 ttc ggc atg ttg gag cag tac gcg agt tcg ctg gag cag gag gtg gag 2798 Phe Gly Met Leu Glu Gln Tyr Ala Ser Ser Leu Glu Gln Glu Val Glu 895 900 905 gaa cgg acc aaa gag ttg gtg gag gag aag cgc aag agc gac att ctt 2846 Glu Arg Thr Lys Glu Leu Val Glu Glu Lys Arg Lys Ser Asp Ile Leu 910 915 920 ctc tac cgg atg ttg ccg cgg cag gtg gcg gac aaa ctg aag ata ggc 2894 Leu Tyr Arg Met Leu Pro Arg Gln Val Ala Asp Lys Leu Lys Ile Gly 925 930 935 gag tct gtg gag cca gaa tcc ttc caa atg gcc acc att ttc ttc tcc 2942 Glu Ser Val Glu Pro Glu Ser Phe Gln Met Ala Thr Ile Phe Phe Ser 940 945 950 955 gac gtc gtc tcc ttc acc act ttg gcc ggc aaa tgc tcg cca ttg caa 2990 Asp Val Val Ser Phe Thr Thr Leu Ala Gly Lys Cys Ser Pro Leu Gln 960 965 970 gtt gtg aat ctg ctc aac ggt ctg ttc aca gcc ttt gac ggg atc att 3038 Val Val Asn Leu Leu Asn Gly Leu Phe Thr Ala Phe Asp Gly Ile Ile 975 980 985 gac act cat gac tgc tac aaa gtt gaa acc att ggc gat ggc tat ttg 3086 Asp Thr His Asp Cys Tyr Lys Val Glu Thr Ile Gly Asp Gly Tyr Leu 990 995 1000 gtc tgt tcg ggc att ccg aag cgc aac ggc gac caa cac gcg aaa 3131 Val Cys Ser Gly Ile Pro Lys Arg Asn Gly Asp Gln His Ala Lys 1005 1010 1015 gaa ata gcc gaa ctt tcg ttc gcc ttc ctt cgc act gtg tcc agc 3176 Glu Ile Ala Glu Leu Ser Phe Ala Phe Leu Arg Thr Val Ser Ser 1020 1025 1030 ttc cgt gtc gat cac ctc ccc tcc gaa cgg gtc aac ctt cgc att 3221 Phe Arg Val Asp His Leu Pro Ser Glu Arg Val Asn Leu Arg Ile 1035 1040 1045 ggc ttc cat tcc gga cca gcg gtc gct ggc gtc gtc gga ctg aca 3266 Gly Phe His Ser Gly Pro Ala Val Ala Gly Val Val Gly Leu Thr 1050 1055 1060 atg ccg cgc tat tgt ctc ttt ggg gac tca gtg aac acg gcc agc 3311 Met Pro Arg Tyr Cys Leu Phe Gly Asp Ser Val Asn Thr Ala Ser 1065 1070 1075 cga atg gag tca aac gga aag gca ggc cga gtg cac att tca tca 3356 Arg Met Glu Ser Asn Gly Lys Ala Gly Arg Val His Ile Ser Ser 1080 1085 1090 agt gcc aac cac ttt ttg acc agt gta atc ggc gga tat gtg aca 3401 Ser Ala Asn His Phe Leu Thr Ser Val Ile Gly Gly Tyr Val Thr 1095 1100 1105 gag cca aga ggc gaa gtg att ata aag ggc aaa gga gtg atg gag 3446 Glu Pro Arg Gly Glu Val Ile Ile Lys Gly Lys Gly Val Met Glu 1110 1115 1120 acc ttt tgg ctg tta ggg cga att gga gag gca cat ttg tcg gag 3491 Thr Phe Trp Leu Leu Gly Arg Ile Gly Glu Ala His Leu Ser Glu 1125 1130 1135 ggc aca gcg gaa aga aat gcg agt gcc gca acg aga aaa tga 3533 Gly Thr Ala Glu Arg Asn Ala Ser Ala Ala Thr Arg Lys 1140 1145 1150 agaaacatca cacggcattc cctctgatca ctcattttaa tgactcgaaa tcattgacca 3593 attttaatga attttaatct cttttattat tatgatagcg caatttttgc gcacatttaa 3653 gcgataacaa tttttatatt aaagttcccc ttaacaaatt tactattgta aatactgtct 3713 cgaatacaaa aaatgtataa tttactatta aaaaaaaaaa aaaaaaaaa 3762 2 1151 PRT Heterodera glycines 2 Met Glu Met Pro Ser Cys Phe Phe Leu Leu Phe Phe Leu Met Leu Phe 1 5 10 15 Val Ser Pro Ser Arg His Gln Leu Val Thr Val Ser Asn Ser Ser Ser 20 25 30 Ser Pro Ile Gly Thr Thr Val Ala Phe Gly Thr Pro Ser Pro Ile Ile 35 40 45 Pro Ile Ser Thr Ala Pro Ser Thr Asn Gly Thr Ala Thr Phe Gly Val 50 55 60 Pro Pro Pro Ile Ser Pro Pro Ile Asn Ser Ser Ser Ser Leu Pro Ser 65 70 75 80 Thr Gly Pro Leu Glu Ala Ser Val Gln Leu Lys Ile Gly Phe Leu Phe 85 90 95 Ala Asn Gly Thr Gln Arg Leu Arg Met Leu Phe Gly Phe Gly Gln Ser 100 105 110 Ala Pro Ala Val Thr Leu Ala Leu Glu Arg Ala Arg Gln Glu His Leu 115 120 125 Ile Asp Ser Ile Asn Phe Thr Tyr Thr Trp Arg Met Cys Gly Cys Phe 130 135 140 Gln Pro Trp Ala Val Gly Tyr Ala Thr Gln Leu Val Leu Thr Glu Asn 145 150 155 160 Val Asp Ala Leu Ile Gly Pro Pro Cys Ala Ile Ala Ala Gly Tyr Val 165 170 175 Ala Ser Phe Tyr Asn Ile Pro Leu Tyr Leu Trp Gly Ala Thr Val Ala 180 185 190 Ser Glu Phe Tyr Asn Thr Thr Val Tyr Pro Thr Leu Asn Asn Val Asn 195 200 205 Val Asn Ser Asp Met Leu Ala Leu Ala Leu Gln Ser Val Leu Val Gln 210 215 220 Phe Asn Trp Thr Glu Val Ser Phe Val Tyr Thr Pro Asp Asn Glu Arg 225 230 235 240 Met Val Cys Asn Ser Val Lys Gln Ser Leu Thr Asn Val Leu Asn Val 245 250 255 Thr Asn Val Thr Ile Val Phe Gln His Gln Met Glu Ser Asn Val Asp 260 265 270 Ser Met Lys Ala Thr Leu Arg Asn Leu Arg Ser Arg Ser Arg Ile Val 275 280 285 Leu Ser Cys Phe Asp Val Glu Val Asp Arg Arg Asn Phe Leu Leu Ser 290 295 300 Ile Phe Asp Thr Gly Leu Ala Ala Asp Asn Glu Phe Val Phe Ile Met 305 310 315 320 Gly Ser Leu Arg Asn Gln Gly Met Leu Gln Gln Val Ala Ser Arg Glu 325 330 335 Asp Gly Ser Val Lys Tyr Val Asn Asn Trp Met Asp Lys Asn Ser Pro 340 345 350 Gly Asp Gly Arg Asp Ser Asp Ala Leu Ala Ala Thr Lys His Val Ile 355 360 365 Met Ile Asp Leu Glu Asn Gln Ser Ser Asp His Leu Asn Glu Phe Asn 370 375 380 Arg Asp Leu Ser Ala Lys Phe Gly Thr Tyr Pro Phe Phe Cys Asn Gly 385 390 395 400 Ser Cys Met Gly Gly Ala Ala Glu Gln Ser Pro Ser Gln Tyr Ala Arg 405 410 415 Ala Leu Phe Asp Thr Thr Tyr Ala Tyr Phe Arg Ala Leu Asn Arg Thr 420 425 430 Met Glu Lys Arg Lys Ser Asn Gly Arg Asp Leu Leu Arg Asn Gly Thr 435 440 445 Glu Leu Asn Ala Glu Thr Ala Gly Thr Thr Phe Gln Gly Glu Thr Gly 450 455 460 Arg Ile Thr Phe Asp Ala His Gly Asn Arg Gln Pro Thr Phe Phe Val 465 470 475 480 Thr Met Leu Asn Ala Leu Asn Val Pro Thr Val Met Val Lys Val Asn 485 490 495 Ile Thr Asn Gly Val Leu Lys Met Glu Arg Leu Tyr Gly Ser Glu Ala 500 505 510 Ser Leu Trp Val Asn Trp Gly Gly Phe Arg Pro Met Thr Thr Pro Leu 515 520 525 Cys Gly Tyr Asn Gly Thr Met Cys Gly Gln Asn Val Thr Val Tyr Ile 530 535 540 Leu Ile Gly Val Thr Leu Met Leu Leu Leu Leu Val Ala Ala Leu Leu 545 550 555 560 Gly Ile Gly Tyr Ala Ile Arg Glu Lys Met Arg Glu Lys Gln Arg Leu 565 570 575 Thr Arg Glu Cys Leu Ile Pro Phe Ala Glu Leu Arg Asn Leu Lys Glu 580 585 590 Leu Arg Ser Ser Glu Glu Leu Lys Ser Glu Thr Glu Lys Ser Met Arg 595 600 605 Ser Met Arg Ser Ser Gln Ser Gly Ser Thr Arg Leu Thr Val Gly Ser 610 615 620 His Lys Ala Gln Arg Glu Thr Ala Asn Cys Ala Phe Phe Val Phe Asn 625 630 635 640 Arg Glu Ile Val Leu Ala Val Lys Tyr His Val Arg Val Arg Ile Met 645 650 655 Ser Glu Asp Leu Ala Phe Ile Arg Lys Leu Arg Gln Leu Asp His Asp 660 665 670 Asn Met Asn Lys Leu Tyr Gly Val Cys Thr Asp Gly Pro Leu Leu Phe 675 680 685 Ala Ile Trp Arg Asn Cys Gln Arg Gly Thr Leu Lys Glu Leu Ile Ala 690 695 700 Lys Glu Gln Tyr Val Gly Asp Asn Cys Val Met Phe Ala Leu Met Arg 705 710 715 720 Asp Ile Ala Asn Gly Leu Leu Ala Ile His Gln Ser Phe Ile Gly Ala 725 730 735 His Gly Leu Leu Ser Ser Glu Asn Cys Leu Ile Asn Asp Arg Trp Gln 740 745 750 Val Lys Ile Ser Asp Phe Gly Leu Asn Met Ile Arg Glu Ser Gln Thr 755 760 765 Leu Ser Lys Lys Ala Leu Leu Trp Thr Ala Pro Glu Leu Leu Arg Glu 770 775 780 Asn Asn Arg Lys Gly Ala Lys Glu Gly Asp Val Phe Ser Phe Ala Ile 785 790 795 800 Ile Cys Val Glu Met Met Asn Arg Glu Thr Val Trp Asn Gly Val Glu 805 810 815 Arg Asp Gln Asp Ile Asp Glu Ile Leu Tyr Arg Leu Arg Arg Thr Asn 820 825 830 Thr Thr Ile Pro His Arg Pro Gln Leu His Pro Arg Ala Glu Ile Asn 835 840 845 Gln Ser Leu Leu His Leu Ile Arg Asp Cys Trp Ser Glu Val Pro Ser 850 855 860 Glu Arg Pro Arg Met Asp Ile Val Arg Thr Met Leu Lys Gln Met Val 865 870 875 880 Gln Asp Gly Ser Gln Asn Leu Met Asp Tyr Val Phe Gly Met Leu Glu 885 890 895 Gln Tyr Ala Ser Ser Leu Glu Gln Glu Val Glu Glu Arg Thr Lys Glu 900 905 910 Leu Val Glu Glu Lys Arg Lys Ser Asp Ile Leu Leu Tyr Arg Met Leu 915 920 925 Pro Arg Gln Val Ala Asp Lys Leu Lys Ile Gly Glu Ser Val Glu Pro 930 935 940 Glu Ser Phe Gln Met Ala Thr Ile Phe Phe Ser Asp Val Val Ser Phe 945 950 955 960 Thr Thr Leu Ala Gly Lys Cys Ser Pro Leu Gln Val Val Asn Leu Leu 965 970 975 Asn Gly Leu Phe Thr Ala Phe Asp Gly Ile Ile Asp Thr His Asp Cys 980 985 990 Tyr Lys Val Glu Thr Ile Gly Asp Gly Tyr Leu Val Cys Ser Gly Ile 995 1000 1005 Pro Lys Arg Asn Gly Asp Gln His Ala Lys Glu Ile Ala Glu Leu 1010 1015 1020 Ser Phe Ala Phe Leu Arg Thr Val Ser Ser Phe Arg Val Asp His 1025 1030 1035 Leu Pro Ser Glu Arg Val Asn Leu Arg Ile Gly Phe His Ser Gly 1040 1045 1050 Pro Ala Val Ala Gly Val Val Gly Leu Thr Met Pro Arg Tyr Cys 1055 1060 1065 Leu Phe Gly Asp Ser Val Asn Thr Ala Ser Arg Met Glu Ser Asn 1070 1075 1080 Gly Lys Ala Gly Arg Val His Ile Ser Ser Ser Ala Asn His Phe 1085 1090 1095 Leu Thr Ser Val Ile Gly Gly Tyr Val Thr Glu Pro Arg Gly Glu 1100 1105 1110 Val Ile Ile Lys Gly Lys Gly Val Met Glu Thr Phe Trp Leu Leu 1115 1120 1125 Gly Arg Ile Gly Glu Ala His Leu Ser Glu Gly Thr Ala Glu Arg 1130 1135 1140 Asn Ala Ser Ala Ala Thr Arg Lys 1145 1150 3 7127 DNA Heterodera glycines 3 gaatggcgaa cagtcatttg ccgatattta tatgccaatc tccacctcta agccgaccga 60 actcatccgc cggttcttgg ctcactttgc cctttcattt ggctatattt catttccata 120 gctttaaatt tattcagacg tcaattgttg aattcgtttc gccattagct tttgtttatg 180 acttttccac atctgttcaa ataatttgaa caccaaaaac ttggcttttt tgattgctta 240 accattcatt ttgttaaatt tgccgttcat tttgtttgat cacctaaaaa atttggcgta 300 gtttttgtga tcacattagg agttgggacc aagttagcca atttttaggg gtgtattttt 360 cgttttttta aatcccgagc taggtggtgg attttccaaa atttttagcc gttttcattt 420 tttacttatt taatgggaaa atgtaaaacg gaaaaaatta aacgcatgga actgaaaaaa 480 ggaaatgaaa gatatttcaa acggaagagg aaaagaaaac tatcaaaaat taaagaaaat 540 tcttaagaaa aagcaccgat tatttatata tatattttta ttttttgctg aatatatttt 600 ttcattttta cttgtattat tataattttt tgtttattga taaggatttt aaaaatttcg 660 atatttctta gaattaaaat ttgtcactct gtcaggactt tgaactttaa aaaatttgaa 720 ctttggcggt aaaattattt aacgatgccc tcccaaacaa agctgaagag gaagtggatg 780 gaatcacttc aaaagaacga attcattttg gaattttgaa atgcccttta agggattatg 840 cgactgccgc aaaacgaccg taccgaatca gcttgaaaca ttttgtaagg tttctccgct 900 gggatttgaa tcccgacaaa tcgccttttt aatgaattca tttcatttta aaatttcctt 960 gcccaaaatc tctcaaaatg gaaatgccgt cctgtttctt cctccttttc tttcttatgc 1020 tttttgtcag cccttctcgg caccaattag tcactgttag caactcatcg tcttcgccca 1080 ttggcaccac cgtcgctttt ggcactcctt cgccgatcat cccaatctct actgcaccct 1140 ccacgaatgg cactgccact tttggcgtcc ctccgccgat cagtccgccg attaattctt 1200 cgtcctctct cccatcaact ggtcctttgg aagcatcggt tcagttaaaa atcggcttcc 1260 tctttgctaa cggcacccaa cggttgcgaa tgcttttcgg ctttggccaa tccgcgcccg 1320 ccgtcacttt ggcactcgaa cgggcgaggc aggagcacct catcgacagc atcaacttca 1380 cgtgggcaat tggaatgaat ttagaaactc acaattttca aatcactttt tgcaaattta 1440 aaaatctcca agcgagcgga aataattggc cgtaaatgcc aatttcagtt acacgtggcg 1500 aatgtgcggc tgctttcagc cttgggctgt cggctacgcc actcaactgg ttctgacgga 1560 aaatgtggac gctttgatcg gtccgccttg tgtgaccagt aatgcctttt tgtttgacac 1620 tttgccaaaa attgcgtaaa tgaaaaaggt gccatcgccg cgggatacgt ggcctccttc 1680 tacaacattc cactgtattt gtggggtgct actgtggcct cggaatttta caacactacc 1740 gtatacccta cactgaacaa cgtgaacgtt aactcggaca tgtcagtgga gaaaatattc 1800 ttcgccttcc atcccctaaa attattattt ctactaataa aataaactaa aatggatttg 1860 ctttacgagc cgtcaccaaa tcaaatgacc aatgatttct tattaatttc aacaattatt 1920 gcaggttggc gttggcctta caaagtgtgt tggtgcaatt caattggaca gaagtgtcct 1980 tcgtgtacac tccggacaat gagcgaatgt aagaattatt ttgaataatt aattaattaa 2040 ttagctatta atataattta attagggtct gtaactcggt gaaacagagt ctcacaaatg 2100 tgctcaacgt gaccaatgtg accattgttt tccagcatca gatggagtcc aatgtggaca 2160 gtatgaaggc gacgctgaga aatctgcgca accgatcgcg aagtgatggg ataattaatt 2220 tgatagcatc gctaattacc atcaattagt tgtgctttcc tgtttcgatg tcgaagttga 2280 acgtcgcaac tttctgttgt ccattttcga cactggtctt gctgcggaca acgaatttgt 2340 gttcatcatg ggatccctgc gcaaccaggg catgctccag cagggtaatt aggcaaatgg 2400 ccaaattagg ggagggataa ttaaaggggc aattgattag acctaacagt gcttcagttg 2460 cgtcgcgtga ggacggcagt gtcaaatatg tgaacaattg gatggacaaa aacagcccag 2520 gcgatggccg cgactcggac gcactcgccg cgacaaaaca cgtcataatg gtcaatccac 2580 ggcgaagcca agcaaattta agtgccttag tgtcgttcag attgacctgg aaaaccaatc 2640 gagtgatcag cttaacgaat tcaaccgaaa tttgagtgcg aaattcggca cttatccctt 2700 tttctgcaac ggaagttgca tgggcggcgc aacagaacaa tcggttgcaa tttgcacgaa 2760 aaatggaaat tataaaaaaa gataatgctt tttagccgtc gcaatacgcc agggctttgt 2820 tcgacacaac atacgcatat tttagagcat tgaatcgcac aatggaaaag cgcaaatcga 2880 atgggaggga ttgttgcgca acggcacgga attgaacgca gaaactgccg ggacgacctt 2940 tcagggtgag gtggagagga agaaaaggag gggggggggt agaagcgaat ttgggagagg 3000 aaccaatgga taaagcctgg caaaatgatg gagcattaag gcgagaccgg acgcatcact 3060 tttgacgccc acggcaaccg ccagccgacc ttttttgtga cgatgctaaa cgcactgaat 3120 gtgcccactg ttatggtgaa agtgaacatt accaacggag tattggtgcg aatggattaa 3180 gcggcggcgg attcttattt ttgaaattca acagaaaatg gaacggctgt acggcagtga 3240 ggcgtcgctg tgggtcaatt ggggcggctt tcggccgatg accacgccgt tgtgcggcta 3300 caacggcaca atgtgtggcc aaaatgtgac ggtgtacatt ctgatcggcg ttacgcttat 3360 gttgctgttg ctggtcgccg ctttgcttgg catcggatac gcaattcggt aaacagagga 3420 aaagcatcgg actgtgccga tcaaaaattg atttaaaagc aaagccaatg gtcgataatt 3480 ggaccaaaag gaatgttaac gccaaaatca ctcattaatt ataaatttta agcttttaat 3540 gcattaattg gaccgaattt ctccgaattg gcacaactta agcccattaa tccgccgatt 3600 ttgccatttg cagggagaaa atgcgcgaga agcagcgcct gacacgcgag tgtttgatcc 3660 catttgcaga gctgcgcaac ctgaaagagc tgcgcagttc ggaggaactg aagtcggaga 3720 cggagaagag catgcggagc atgcgtagca gtcagtcggg tgagcgtacg gtagcgacca 3780 ttcgctcaaa tatggcatat ggtaacgcag gaagcacacg gcttacggtc ggcagccaca 3840 aagcgcagcg ggagacggcc aattgcgcgt tctttgtgtt caaccgggaa attgtgctgg 3900 ccgtgaaata ccacgtcagg gtgcgaatta tgtccgaaga tttggccttc attaggaagg 3960 taaatgggac aacggcagga gaccatgttg gaaaatgtga ctaattacct aaagatgggt 4020 caatcgttac catataatat atacttccgt cgttacggta cagctgcggc agttggacca 4080 cgacaacatg aacaagttgt acggcgtgtg caccgatggg ccccttttgt tcgcaatttg 4140 gcgcaattgt cagcgaggga cattaaaagt gcaatttgac agaaaataac atttgttcag 4200 taaaataaca tgtcatgtca ttaaggaact gatcgccaag gagcaatacg ttggggacaa 4260 ttgtgtgatg tttgctctga tgcgggacat tgcaaatgta aacactcaca tgggaagtgc 4320 tttggcattg gcacttccca tcagctgttt ctttcctaaa tcggggccat tttcggggat 4380 ctccgacctc gacattcttc tgtcataatt ggcggcttgc cgtttccaat ttcttctgca 4440 taaattcgaa tttcggtcac tccatcccca gggtctgctc gccatccatc aatcgttcat 4500 cggagcccac gggctgctct cctctgaaaa ttgtctgatc aatgaccggt ggcaagtgaa 4560 aatcagcgac tttggcctga atatgatcag agaaagtcaa acgctgtcga agaaaggttg 4620 gcattgggtt tacgaagtaa ttcgcgtaat ttgcgccatt taaagcactt ttgtggacgg 4680 cgcctgaact tttgcgagaa aacaatcgga agggaacaaa agagggcgat gtgttcagtt 4740 ttgcgatcat ttgtgtggaa atggtgaaca gagagacggt gtggaacgga gtggaaaggg 4800 accaagacat cgatggtggg agggcggagg aggggatttg gaggggaaat ttcggcactc 4860 ggcttttcct tttccatcgg tgccattgtc cgacctttct ttagaaatcc tttatcggct 4920 cagacgcacc aacaccacaa tccctcaccg tccgcagctt catccccgcg cagagattaa 4980 ccaaagtttg gtatgcgcac gcatttctca tggcattttg ctgctgctat tctgtcttat 5040 accatatccc cacttaccgt aagcttcatc tgatcagaga ctgttggtcc gaagtgccgt 5100 ccgaacgtcc gcgcatggac attgtgcgaa cgatgctcaa acagatggtc caggacgggt 5160 cagtaagtca acagcggagc aatcaatgga cacgcttgtg atgctcgaaa gtctcgagag 5220 agcagtctcg gggtttttta atgcccttga ccgggtcaaa gcttgagtat cggcgatctt 5280 aagtagaaca agcgcttttc cgatccgctt ttgccccccc ccccaatttt tgcccatttc 5340 ctttctcttc agcagtcaaa atctgatgga ttacgtgttc ggcatgttgg agcagtacgc 5400 gagttcgctg gagcaggagg tggaggaacg gaccaaagag ttggtggagg agaagcgcaa 5460 gagcgacatt cttctctacc ggatgttgcc gcggcaggtg gcggacaaac tgaagatagg 5520 cgagtctgtg gagccagaat ccttccaaat ggccaccatt ttcttctccg acgtcgtctc 5580 cttcaccact ttggccggca aatgctcgcc attgcaagtg ccgtaaaaaa agaaaattta 5640 ccgctacact tttggaaaaa taaattgtcg catatttttc agaccccaat taatcaatta 5700 atttaaatca aacaagattg atcaaaatgg gaaatactga tcaattacat tgatcaaaat 5760 ggggaggaat cgactgatca atcccatccg tccccacccc tctcttctcg tttaggttgt 5820 gaatctgctc aacggtctgt acacagcctt tgacgggatc attgacactc atgactgcta 5880 caaagttggt aagtgaccag cgaatacctc actaatcgtc ttcaactctc tctcctccta 5940 ttttattgct ttgtattagt tctaatttgc cattttaatt gccccccgcc acttctcccc 6000 tcagaaacca ttggcgatgg ctatttggtc tgttcgggca ttccgaagcg caacggcgac 6060 caacacgcga aagaaatagc cgaactttcg ttcgccttcc ttcgcactgt gtccagcttc 6120 cgtgtcgatc acctcccctc cgaacgggtc aaccttcgca ttggcttcca ttccggttcg 6180 ttttcgctat taccgaatca aaaagactcc caacggcacc ccggggcatt ccctggcttc 6240 ttcccaattt ggcatttctt tacgaatgcc atggttaatt aattaattag gaccagcggt 6300 cgctggcgtc gtcggactga caatgccgcg ctattgtctc tttggggact cagtgaacac 6360 ggccagccga atggagtcaa acggaaaggg taaataaacg ggagaaaaag cgaaacaaaa 6420 caaatcaaat taatttggca accattttca gcaggccgag tgcacatttc atcaagtgcc 6480 aaccactttt tgaccagtgt aatcggcgga tatgtgacag agccaagagg cgaagtgatt 6540 ataaaggtca ttaattaagg atgggggcaa tggctccaat tagtcggtta atcccattat 6600 tagggcaaag gagtgatgga gaccttttgg ctgttagggc gaattggaga ggcacatttg 6660 tcggagggca cagcggaaag aaatgcgagt gccgcaacga gaaaatgaag aaacatcaca 6720 cggcattccc tctgatcact cattttaatg actcgaaatc attgaccaat tttaatgaat 6780 tttaatctct tttattatta tgatagcgca atttttgcgc acatttaagc gataacaatt 6840 tttatattaa agttcccctt aacaaattta ctattgtaaa tactgtctcg aatacaaaaa 6900 atgtataatt tactattttt ctcacgatat tcatggcaaa aaggtcatcc ctaattatta 6960 aacgttactc tttcatgtgt tcattaacac acaataattt tttgtctcag atttactaat 7020 tacatataca ataagaacaa aaatattttt tggaaaaagt ttacaatata aagataatat 7080 taaaggagca attagtgaaa atgcatataa ttagaaatga tcgagtc 7127 4 3499 DNA Heterodera glycines CDS (3)..(3347) 4 at ttc gtt ccg atg ttt ttt ggg aca tcg gtt gct gtt gtt ctt tgt 47 Phe Val Pro Met Phe Phe Gly Thr Ser Val Ala Val Val Leu Cys 1 5 10 15 tgg ctt ttt tgc act ttc cca acg aca ttc ggc caa cag caa aat ggg 95 Trp Leu Phe Cys Thr Phe Pro Thr Thr Phe Gly Gln Gln Gln Asn Gly 20 25 30 act gcg ccg ctg atc aaa gtc ggg cta atg atg ccg cac aat cag tcg 143 Thr Ala Pro Leu Ile Lys Val Gly Leu Met Met Pro His Asn Gln Ser 35 40 45 tcc gat ttg tct ttt gcc cga tcc gcc ggt gcc atc tca gtg gcg ctg 191 Ser Asp Leu Ser Phe Ala Arg Ser Ala Gly Ala Ile Ser Val Ala Leu 50 55 60 aag cac att ttc aac gac aat ttg ttg cct ccc ggc acc aat ttc agt 239 Lys His Ile Phe Asn Asp Asn Leu Leu Pro Pro Gly Thr Asn Phe Ser 65 70 75 ttc att gtc cgt ttc gaa gag tgc cta atg tcc gtc gcc gcc ggg tac 287 Phe Ile Val Arg Phe Glu Glu Cys Leu Met Ser Val Ala Ala Gly Tyr 80 85 90 95 gcc ttc gat ttg ttg gat ggc cag caa gtg gac ctt ttc att gcg ccg 335 Ala Phe Asp Leu Leu Asp Gly Gln Gln Val Asp Leu Phe Ile Ala Pro 100 105 110 ccg tgc acc gac agt gcg caa gtt gca ctt ttc gtg tcc aca ttt tac 383 Pro Cys Thr Asp Ser Ala Gln Val Ala Leu Phe Val Ser Thr Phe Tyr 115 120 125 aac atc cct tcc atc aca tgg ggc cag aat tcg gac tcc tct ttc aat 431 Asn Ile Pro Ser Ile Thr Trp Gly Gln Asn Ser Asp Ser Ser Phe Asn 130 135 140 tcg cag agc aat tac ccc act ttg ctg agt gcg ctt ccc aat tac gcc 479 Ser Gln Ser Asn Tyr Pro Thr Leu Leu Ser Ala Leu Pro Asn Tyr Ala 145 150 155 gac ttt ggc caa att atc att tcg ctg tgc atc ttc ttc aag tgg tcc 527 Asp Phe Gly Gln Ile Ile Ile Ser Leu Cys Ile Phe Phe Lys Trp Ser 160 165 170 175 gtc atg gca ctg att tat cag ctc agc gag acg ggt caa tgc gcg tcg 575 Val Met Ala Leu Ile Tyr Gln Leu Ser Glu Thr Gly Gln Cys Ala Ser 180 185 190 ttc cag caa gac ttg cag atc gcg atc aat tcc aac gac aaa tgc gat 623 Phe Gln Gln Asp Leu Gln Ile Ala Ile Asn Ser Asn Asp Lys Cys Asp 195 200 205 atc agc tac aga gag gaa gtt aag atc agt tct gcg ggc acc agc gac 671 Ile Ser Tyr Arg Glu Glu Val Lys Ile Ser Ser Ala Gly Thr Ser Asp 210 215 220 gcc caa tac acc ata agt caa att cag agc agg gcg aga atc gtc att 719 Ala Gln Tyr Thr Ile Ser Gln Ile Gln Ser Arg Ala Arg Ile Val Ile 225 230 235 ctt tgc ttc gac gag ttt gtt cag ctg cgc aac ttt gcc gcc aaa ctt 767 Leu Cys Phe Asp Glu Phe Val Gln Leu Arg Asn Phe Ala Ala Lys Leu 240 245 250 255 cag gag ggt ggc ttg gac tcc gct gac tac gtt tat ctc atc ccc gga 815 Gln Glu Gly Gly Leu Asp Ser Ala Asp Tyr Val Tyr Leu Ile Pro Gly 260 265 270 ctc acc atg gat gat agt att gaa agt gtt aat tgt gtc ttt tat aaa 863 Leu Thr Met Asp Asp Ser Ile Glu Ser Val Asn Cys Val Phe Tyr Lys 275 280 285 att caa att tgc gtt tgc ttt ttc tct gtt ttt aat tta ctt ttt gtt 911 Ile Gln Ile Cys Val Cys Phe Phe Ser Val Phe Asn Leu Leu Phe Val 290 295 300 ttg ggt ggc tcc aag gcc acc gcg tgg tgg gtc gac ccg aac ccg acc 959 Leu Gly Gly Ser Lys Ala Thr Ala Trp Trp Val Asp Pro Asn Pro Thr 305 310 315 atc caa tca gcg gcc tac aga att gct cag cgc agt ctt tat ctg atg 1007 Ile Gln Ser Ala Ala Tyr Arg Ile Ala Gln Arg Ser Leu Tyr Leu Met 320 325 330 335 ttg gac atc ttc aac aaa gtc gca act tca ggt caa gtg ggc aac ggc 1055 Leu Asp Ile Phe Asn Lys Val Ala Thr Ser Gly Gln Val Gly Asn Gly 340 345 350 act tcg ttt gat cag gaa gtg atc aga cag gtc acc caa tgg ccc ttc 1103 Thr Ser Phe Asp Gln Glu Val Ile Arg Gln Val Thr Gln Trp Pro Phe 355 360 365 ttc tgt acc gat tgc gat cag tcg ttg cag gct tct tct tac gcc cct 1151 Phe Cys Thr Asp Cys Asp Gln Ser Leu Gln Ala Ser Ser Tyr Ala Pro 370 375 380 ttg ctc cac gac agt ttc tat ttg tat gcc atg gcc ctt tcc aaa gcg 1199 Leu Leu His Asp Ser Phe Tyr Leu Tyr Ala Met Ala Leu Ser Lys Ala 385 390 395 gca aaa att gcc ggc gca ttg tca cct tcc gtt tac cga aat ggc caa 1247 Ala Lys Ile Ala Gly Ala Leu Ser Pro Ser Val Tyr Arg Asn Gly Gln 400 405 410 415 ttg att cgc tcc caa acc gcc aat ttg tct ttt gaa gga atg acg ggg 1295 Leu Ile Arg Ser Gln Thr Ala Asn Leu Ser Phe Glu Gly Met Thr Gly 420 425 430 tca aac aaa ttt gga tct gat gga ctt cgt aat ttc att tac ctt gtc 1343 Ser Asn Lys Phe Gly Ser Asp Gly Leu Arg Asn Phe Ile Tyr Leu Val 435 440 445 tcc atg tat tcg agc ttg aac ggt gac ttg act tcg tat gtg tgg ctc 1391 Ser Met Tyr Ser Ser Leu Asn Gly Asp Leu Thr Ser Tyr Val Trp Leu 450 455 460 caa atg aac gat gcc gga gtg aat tct tca tgg att aat gcc acg gcc 1439 Gln Met Asn Asp Ala Gly Val Asn Ser Ser Trp Ile Asn Ala Thr Ala 465 470 475 gag aag ctg att tgg tcg agc cga aac ggc gtt aag cca ttg gcc gtg 1487 Glu Lys Leu Ile Trp Ser Ser Arg Asn Gly Val Lys Pro Leu Ala Val 480 485 490 495 ccg ttg tgc gga ttt gac ggc aac ggc tgt cac atg gac ttc ttc acg 1535 Pro Leu Cys Gly Phe Asp Gly Asn Gly Cys His Met Asp Phe Phe Thr 500 505 510 gag tac cgt ggg tat gtg ata gct gcc ggc tgt ctg ttg ctg ctc att 1583 Glu Tyr Arg Gly Tyr Val Ile Ala Ala Gly Cys Leu Leu Leu Leu Ile 515 520 525 ttg ggc tcg ttc gcc ttc ggc att tac tgg ctg ttc caa tcc aag gcg 1631 Leu Gly Ser Phe Ala Phe Gly Ile Tyr Trp Leu Phe Gln Ser Lys Ala 530 535 540 cgc gag atg gaa cgg caa aat cgc ctc tgg caa atc gcc tac agt act 1679 Arg Glu Met Glu Arg Gln Asn Arg Leu Trp Gln Ile Ala Tyr Ser Thr 545 550 555 ctg acg ccg gcg ggc acc aaa aag aaa atg atg gaa agt gtg cgc tct 1727 Leu Thr Pro Ala Gly Thr Lys Lys Lys Met Met Glu Ser Val Arg Ser 560 565 570 575 ctc cag tcg agc act tct tct cag ttc acg cgc gac tcc tcc cat tcc 1775 Leu Gln Ser Ser Thr Ser Ser Gln Phe Thr Arg Asp Ser Ser His Ser 580 585 590 cac gtt tcc atc aaa cac aac ttc aat ggc atc gtg tac att atg aac 1823 His Val Ser Ile Lys His Asn Phe Asn Gly Ile Val Tyr Ile Met Asn 595 600 605 ggc gag cgg gtg atc ggc att cag cat tcg gtt ggc att cga ctc agt 1871 Gly Glu Arg Val Ile Gly Ile Gln His Ser Val Gly Ile Arg Leu Ser 610 615 620 cca cag gac atg gcc gag ctg aga act atg cgc ctt ttg gat gga gac 1919 Pro Gln Asp Met Ala Glu Leu Arg Thr Met Arg Leu Leu Asp Gly Asp 625 630 635 aat gtg aac cga ttc atc ggc ctt tcc atc gat ggc gcc gcg ctt ctc 1967 Asn Val Asn Arg Phe Ile Gly Leu Ser Ile Asp Gly Ala Ala Leu Leu 640 645 650 655 tcc ctg tgg cgc tac tgc tcg cgt ggc ccc ctt tcg gac gtg atc tcg 2015 Ser Leu Trp Arg Tyr Cys Ser Arg Gly Pro Leu Ser Asp Val Ile Ser 660 665 670 ggc tct tcc tct ctg acc atg gac ggc ttc ttc att tat tcg ttg gtc 2063 Gly Ser Ser Ser Leu Thr Met Asp Gly Phe Phe Ile Tyr Ser Leu Val 675 680 685 cgc gac gtt gcc gaa gga ttg cgc ttc ctt cac gcg tcc tca att gga 2111 Arg Asp Val Ala Glu Gly Leu Arg Phe Leu His Ala Ser Ser Ile Gly 690 695 700 tgg tat ggc aat ttg cgt tcc acc aac tgt ttg atc gac gac cgt tgg 2159 Trp Tyr Gly Asn Leu Arg Ser Thr Asn Cys Leu Ile Asp Asp Arg Trp 705 710 715 caa ata aaa ctg tcc gag ttt ggt ctc cgc ttc ttt cgt gca cac gaa 2207 Gln Ile Lys Leu Ser Glu Phe Gly Leu Arg Phe Phe Arg Ala His Glu 720 725 730 735 aaa cgg gag gca aaa gat ttg gtt tgg aca gcg cca gaa ttg ttg cgc 2255 Lys Arg Glu Ala Lys Asp Leu Val Trp Thr Ala Pro Glu Leu Leu Arg 740 745 750 gat aat gac atc gtt ggc aac aaa ttt ggc gat gtt tac agc ttt tcc 2303 Asp Asn Asp Ile Val Gly Asn Lys Phe Gly Asp Val Tyr Ser Phe Ser 755 760 765 atc gtt tct tcc gaa att gtg aat atg aag cca att tgg gag cag gac 2351 Ile Val Ser Ser Glu Ile Val Asn Met Lys Pro Ile Trp Glu Gln Asp 770 775 780 gaa gcg aag gga aat gtt gaa agg gtc cga acc ggg ggg aag agg gca 2399 Glu Ala Lys Gly Asn Val Glu Arg Val Arg Thr Gly Gly Lys Arg Ala 785 790 795 ttt cgt ccc aaa ttg gag ccg agc agc cag gac ttg tcc ccg gca ctg 2447 Phe Arg Pro Lys Leu Glu Pro Ser Ser Gln Asp Leu Ser Pro Ala Leu 800 805 810 815 ctg cat ctg atc aaa gac tgc tgg gac gaa agc cct gca gaa cgg cca 2495 Leu His Leu Ile Lys Asp Cys Trp Asp Glu Ser Pro Ala Glu Arg Pro 820 825 830 aaa atg gag acg gtg acc gca ctt ttg cag tca atg aac acg gga agg 2543 Lys Met Glu Thr Val Thr Ala Leu Leu Gln Ser Met Asn Thr Gly Arg 835 840 845 agc acc aat ttg atg gac cac gtg ttc aat atg ctg gaa gtg tac gcc 2591 Ser Thr Asn Leu Met Asp His Val Phe Asn Met Leu Glu Val Tyr Ala 850 855 860 ggc tca ttg gag gag gaa gtt gag gaa cgg acc aaa gag ttg gtg gag 2639 Gly Ser Leu Glu Glu Glu Val Glu Glu Arg Thr Lys Glu Leu Val Glu 865 870 875 gag aag aag aag acg gac atc ctt ctc tac cga atg ctg ccc aaa caa 2687 Glu Lys Lys Lys Thr Asp Ile Leu Leu Tyr Arg Met Leu Pro Lys Gln 880 885 890 895 gtc gcc gac aaa ctc aaa ttg ggc caa tct gtg gag ccc gaa acc ttc 2735 Val Ala Asp Lys Leu Lys Leu Gly Gln Ser Val Glu Pro Glu Thr Phe 900 905 910 gac tgc gtt acc gta ttc ttc tcg gac gtc gtc tca ttc aca aca atc 2783 Asp Cys Val Thr Val Phe Phe Ser Asp Val Val Ser Phe Thr Thr Ile 915 920 925 gct tca aaa tgc tca cct ttg cag gtg gtc aat ttg ctg aac aat ctg 2831 Ala Ser Lys Cys Ser Pro Leu Gln Val Val Asn Leu Leu Asn Asn Leu 930 935 940 tac act ctg ttg gac tca atc atc gcc gaa ttt gac gtg tac aaa gtt 2879 Tyr Thr Leu Leu Asp Ser Ile Ile Ala Glu Phe Asp Val Tyr Lys Val 945 950 955 gag aca att ggc gat ggt tat ttg tgc gtg tcg ggc ctt ccc cac cgc 2927 Glu Thr Ile Gly Asp Gly Tyr Leu Cys Val Ser Gly Leu Pro His Arg 960 965 970 975 aat ggg cat gaa cac gcg caa cac atc gcc aaa atg tcg ttg gca ttc 2975 Asn Gly His Glu His Ala Gln His Ile Ala Lys Met Ser Leu Ala Phe 980 985 990 atg cgc aac ttg ggc agc ttc acc att ccc cac ttg ccc att gaa cgg 3023 Met Arg Asn Leu Gly Ser Phe Thr Ile Pro His Leu Pro Ile Glu Arg 995 1000 1005 ctt cgt ctc cgc att ggc att cac acc ggc tcc acc gtg gcg ggc 3068 Leu Arg Leu Arg Ile Gly Ile His Thr Gly Ser Thr Val Ala Gly 1010 1015 1020 gtt gtc ggt ctt tcc atg ccc cgt tat tgt ctg ttc ggc gac aca 3113 Val Val Gly Leu Ser Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr 1025 1030 1035 att aac aca gcg gca cgg ctg gaa agc agc tca aag ccg atg cga 3158 Ile Asn Thr Ala Ala Arg Leu Glu Ser Ser Ser Lys Pro Met Arg 1040 1045 1050 att cac att tcc acg acg acg aat cac ttt ttg gtc aat gtt ctc 3203 Ile His Ile Ser Thr Thr Thr Asn His Phe Leu Val Asn Val Leu 1055 1060 1065 gga ggt ttt gtc acc caa gcg cgt gga gaa att tta gtg aag gga 3248 Gly Gly Phe Val Thr Gln Ala Arg Gly Glu Ile Leu Val Lys Gly 1070 1075 1080 aag ggc gtt ctc gaa acc ttt tgg ctg ctt ggc ctc gaa ggc gac 3293 Lys Gly Val Leu Glu Thr Phe Trp Leu Leu Gly Leu Glu Gly Asp 1085 1090 1095 ccg gcg gtg atg cga atg ttg cac agt tcg gac ggt aat aat gcg 3338 Pro Ala Val Met Arg Met Leu His Ser Ser Asp Gly Asn Asn Ala 1100 1105 1110 act acg gaa tgaacaaaaa caaattgagg aagaaattga acacaaagga 3387 Thr Thr Glu 1115 aacagaaaaa ccaaaagaat gaatgaatga atgatttgtc atttgtaaaa attaaaatgt 3447 cggacaacaa aaaaaatcga aaggaacgaa aaaaaaaaaa aaaaaaaaaa aa 3499 5 1115 PRT Heterodera glycines 5 Phe Val Pro Met Phe Phe Gly Thr Ser Val Ala Val Val Leu Cys Trp 1 5 10 15 Leu Phe Cys Thr Phe Pro Thr Thr Phe Gly Gln Gln Gln Asn Gly Thr 20 25 30 Ala Pro Leu Ile Lys Val Gly Leu Met Met Pro His Asn Gln Ser Ser 35 40 45 Asp Leu Ser Phe Ala Arg Ser Ala Gly Ala Ile Ser Val Ala Leu Lys 50 55 60 His Ile Phe Asn Asp Asn Leu Leu Pro Pro Gly Thr Asn Phe Ser Phe 65 70 75 80 Ile Val Arg Phe Glu Glu Cys Leu Met Ser Val Ala Ala Gly Tyr Ala 85 90 95 Phe Asp Leu Leu Asp Gly Gln Gln Val Asp Leu Phe Ile Ala Pro Pro 100 105 110 Cys Thr Asp Ser Ala Gln Val Ala Leu Phe Val Ser Thr Phe Tyr Asn 115 120 125 Ile Pro Ser Ile Thr Trp Gly Gln Asn Ser Asp Ser Ser Phe Asn Ser 130 135 140 Gln Ser Asn Tyr Pro Thr Leu Leu Ser Ala Leu Pro Asn Tyr Ala Asp 145 150 155 160 Phe Gly Gln Ile Ile Ile Ser Leu Cys Ile Phe Phe Lys Trp Ser Val 165 170 175 Met Ala Leu Ile Tyr Gln Leu Ser Glu Thr Gly Gln Cys Ala Ser Phe 180 185 190 Gln Gln Asp Leu Gln Ile Ala Ile Asn Ser Asn Asp Lys Cys Asp Ile 195 200 205 Ser Tyr Arg Glu Glu Val Lys Ile Ser Ser Ala Gly Thr Ser Asp Ala 210 215 220 Gln Tyr Thr Ile Ser Gln Ile Gln Ser Arg Ala Arg Ile Val Ile Leu 225 230 235 240 Cys Phe Asp Glu Phe Val Gln Leu Arg Asn Phe Ala Ala Lys Leu Gln 245 250 255 Glu Gly Gly Leu Asp Ser Ala Asp Tyr Val Tyr Leu Ile Pro Gly Leu 260 265 270 Thr Met Asp Asp Ser Ile Glu Ser Val Asn Cys Val Phe Tyr Lys Ile 275 280 285 Gln Ile Cys Val Cys Phe Phe Ser Val Phe Asn Leu Leu Phe Val Leu 290 295 300 Gly Gly Ser Lys Ala Thr Ala Trp Trp Val Asp Pro Asn Pro Thr Ile 305 310 315 320 Gln Ser Ala Ala Tyr Arg Ile Ala Gln Arg Ser Leu Tyr Leu Met Leu 325 330 335 Asp Ile Phe Asn Lys Val Ala Thr Ser Gly Gln Val Gly Asn Gly Thr 340 345 350 Ser Phe Asp Gln Glu Val Ile Arg Gln Val Thr Gln Trp Pro Phe Phe 355 360 365 Cys Thr Asp Cys Asp Gln Ser Leu Gln Ala Ser Ser Tyr Ala Pro Leu 370 375 380 Leu His Asp Ser Phe Tyr Leu Tyr Ala Met Ala Leu Ser Lys Ala Ala 385 390 395 400 Lys Ile Ala Gly Ala Leu Ser Pro Ser Val Tyr Arg Asn Gly Gln Leu 405 410 415 Ile Arg Ser Gln Thr Ala Asn Leu Ser Phe Glu Gly Met Thr Gly Ser 420 425 430 Asn Lys Phe Gly Ser Asp Gly Leu Arg Asn Phe Ile Tyr Leu Val Ser 435 440 445 Met Tyr Ser Ser Leu Asn Gly Asp Leu Thr Ser Tyr Val Trp Leu Gln 450 455 460 Met Asn Asp Ala Gly Val Asn Ser Ser Trp Ile Asn Ala Thr Ala Glu 465 470 475 480 Lys Leu Ile Trp Ser Ser Arg Asn Gly Val Lys Pro Leu Ala Val Pro 485 490 495 Leu Cys Gly Phe Asp Gly Asn Gly Cys His Met Asp Phe Phe Thr Glu 500 505 510 Tyr Arg Gly Tyr Val Ile Ala Ala Gly Cys Leu Leu Leu Leu Ile Leu 515 520 525 Gly Ser Phe Ala Phe Gly Ile Tyr Trp Leu Phe Gln Ser Lys Ala Arg 530 535 540 Glu Met Glu Arg Gln Asn Arg Leu Trp Gln Ile Ala Tyr Ser Thr Leu 545 550 555 560 Thr Pro Ala Gly Thr Lys Lys Lys Met Met Glu Ser Val Arg Ser Leu 565 570 575 Gln Ser Ser Thr Ser Ser Gln Phe Thr Arg Asp Ser Ser His Ser His 580 585 590 Val Ser Ile Lys His Asn Phe Asn Gly Ile Val Tyr Ile Met Asn Gly 595 600 605 Glu Arg Val Ile Gly Ile Gln His Ser Val Gly Ile Arg Leu Ser Pro 610 615 620 Gln Asp Met Ala Glu Leu Arg Thr Met Arg Leu Leu Asp Gly Asp Asn 625 630 635 640 Val Asn Arg Phe Ile Gly Leu Ser Ile Asp Gly Ala Ala Leu Leu Ser 645 650 655 Leu Trp Arg Tyr Cys Ser Arg Gly Pro Leu Ser Asp Val Ile Ser Gly 660 665 670 Ser Ser Ser Leu Thr Met Asp Gly Phe Phe Ile Tyr Ser Leu Val Arg 675 680 685 Asp Val Ala Glu Gly Leu Arg Phe Leu His Ala Ser Ser Ile Gly Trp 690 695 700 Tyr Gly Asn Leu Arg Ser Thr Asn Cys Leu Ile Asp Asp Arg Trp Gln 705 710 715 720 Ile Lys Leu Ser Glu Phe Gly Leu Arg Phe Phe Arg Ala His Glu Lys 725 730 735 Arg Glu Ala Lys Asp Leu Val Trp Thr Ala Pro Glu Leu Leu Arg Asp 740 745 750 Asn Asp Ile Val Gly Asn Lys Phe Gly Asp Val Tyr Ser Phe Ser Ile 755 760 765 Val Ser Ser Glu Ile Val Asn Met Lys Pro Ile Trp Glu Gln Asp Glu 770 775 780 Ala Lys Gly Asn Val Glu Arg Val Arg Thr Gly Gly Lys Arg Ala Phe 785 790 795 800 Arg Pro Lys Leu Glu Pro Ser Ser Gln Asp Leu Ser Pro Ala Leu Leu 805 810 815 His Leu Ile Lys Asp Cys Trp Asp Glu Ser Pro Ala Glu Arg Pro Lys 820 825 830 Met Glu Thr Val Thr Ala Leu Leu Gln Ser Met Asn Thr Gly Arg Ser 835 840 845 Thr Asn Leu Met Asp His Val Phe Asn Met Leu Glu Val Tyr Ala Gly 850 855 860 Ser Leu Glu Glu Glu Val Glu Glu Arg Thr Lys Glu Leu Val Glu Glu 865 870 875 880 Lys Lys Lys Thr Asp Ile Leu Leu Tyr Arg Met Leu Pro Lys Gln Val 885 890 895 Ala Asp Lys Leu Lys Leu Gly Gln Ser Val Glu Pro Glu Thr Phe Asp 900 905 910 Cys Val Thr Val Phe Phe Ser Asp Val Val Ser Phe Thr Thr Ile Ala 915 920 925 Ser Lys Cys Ser Pro Leu Gln Val Val Asn Leu Leu Asn Asn Leu Tyr 930 935 940 Thr Leu Leu Asp Ser Ile Ile Ala Glu Phe Asp Val Tyr Lys Val Glu 945 950 955 960 Thr Ile Gly Asp Gly Tyr Leu Cys Val Ser Gly Leu Pro His Arg Asn 965 970 975 Gly His Glu His Ala Gln His Ile Ala Lys Met Ser Leu Ala Phe Met 980 985 990 Arg Asn Leu Gly Ser Phe Thr Ile Pro His Leu Pro Ile Glu Arg Leu 995 1000 1005 Arg Leu Arg Ile Gly Ile His Thr Gly Ser Thr Val Ala Gly Val 1010 1015 1020 Val Gly Leu Ser Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr Ile 1025 1030 1035 Asn Thr Ala Ala Arg Leu Glu Ser Ser Ser Lys Pro Met Arg Ile 1040 1045 1050 His Ile Ser Thr Thr Thr Asn His Phe Leu Val Asn Val Leu Gly 1055 1060 1065 Gly Phe Val Thr Gln Ala Arg Gly Glu Ile Leu Val Lys Gly Lys 1070 1075 1080 Gly Val Leu Glu Thr Phe Trp Leu Leu Gly Leu Glu Gly Asp Pro 1085 1090 1095 Ala Val Met Arg Met Leu His Ser Ser Asp Gly Asn Asn Ala Thr 1100 1105 1110 Thr Glu 1115 6 3008 DNA Heterodera glycines CDS (3)..(2849) 6 cg aaa atc aca att aat tat aaa aca cgc att ttc aac att caa tca 47 Lys Ile Thr Ile Asn Tyr Lys Thr Arg Ile Phe Asn Ile Gln Ser 1 5 10 15 tcc gac aca agc aca att gtc aat gca att cga gaa cgg gcg agg atc 95 Ser Asp Thr Ser Thr Ile Val Asn Ala Ile Arg Glu Arg Ala Arg Ile 20 25 30 gtt ttg ctt tgc ttt gac gat ttg aag cag atg cga act ttc gca ctt 143 Val Leu Leu Cys Phe Asp Asp Leu Lys Gln Met Arg Thr Phe Ala Leu 35 40 45 caa ttg ttc gat gga gga cta aac aca aaa gat tat gtt tac ata atg 191 Gln Leu Phe Asp Gly Gly Leu Asn Thr Lys Asp Tyr Val Tyr Ile Met 50 55 60 gtg gat aat gac atg tat tta tct ttc aat ttg acg aga tta cct ttt 239 Val Asp Asn Asp Met Tyr Leu Ser Phe Asn Leu Thr Arg Leu Pro Phe 65 70 75 tgg gta caa tcg agt aac aat tca aat acg ctc gac gga aga aac gcg 287 Trp Val Gln Ser Ser Asn Asn Ser Asn Thr Leu Asp Gly Arg Asn Ala 80 85 90 95 gac gcc gaa gtg att ggc cga ttg gcc tta tgg tgg cac tac gac atc 335 Asp Ala Glu Val Ile Gly Arg Leu Ala Leu Trp Trp His Tyr Asp Ile 100 105 110 act ttg tcc gcg ttg tcc aat caa aat tac tac ggc ttt ttc aaa aga 383 Thr Leu Ser Ala Leu Ser Asn Gln Asn Tyr Tyr Gly Phe Phe Lys Arg 115 120 125 gtg atc gac aga acg ggc gat tgg ccc ttt tat tgc gat gaa tcc aat 431 Val Ile Asp Arg Thr Gly Asp Trp Pro Phe Tyr Cys Asp Glu Ser Asn 130 135 140 tgc agc aaa gtg atc aat gca tcc atc tat tcg ctt ctg ttg tac gac 479 Cys Ser Lys Val Ile Asn Ala Ser Ile Tyr Ser Leu Leu Leu Tyr Asp 145 150 155 gca att tac aat tac gga atg gca ctg aac gaa tct ttc cgc caa ttt 527 Ala Ile Tyr Asn Tyr Gly Met Ala Leu Asn Glu Ser Phe Arg Gln Phe 160 165 170 175 ggc att cgg ccc gaa gtg tac cga aac ggc act ctg ttg gca cgg aac 575 Gly Ile Arg Pro Glu Val Tyr Arg Asn Gly Thr Leu Leu Ala Arg Asn 180 185 190 aac aga aag cca ttc atg ggt ttg acc ggc tat gtg acg gtg gaa act 623 Asn Arg Lys Pro Phe Met Gly Leu Thr Gly Tyr Val Thr Val Glu Thr 195 200 205 gat cag aac acg cgg gtg ttc gtt ttg tcc aat cgg aag tcg agc gag 671 Asp Gln Asn Thr Arg Val Phe Val Leu Ser Asn Arg Lys Ser Ser Glu 210 215 220 aaa gga aat gcg cta cgc att tta atg caa ttc gca tgg gtc gag ggg 719 Lys Gly Asn Ala Leu Arg Ile Leu Met Gln Phe Ala Trp Val Glu Gly 225 230 235 aaa ttg caa ata tcg ttg cga aat ggc agc ttg tcc atg tgg tcc tcc 767 Lys Leu Gln Ile Ser Leu Arg Asn Gly Ser Leu Ser Met Trp Ser Ser 240 245 250 255 cgc ggt gga agc att cct ccg gcg gtg cca atc tgc ggc ttc gac ggc 815 Arg Gly Gly Ser Ile Pro Pro Ala Val Pro Ile Cys Gly Phe Asp Gly 260 265 270 aaa ggg tgt gcc gcg tcg gtg ttc gaa atg tat aaa ggc tat tta ttg 863 Lys Gly Cys Ala Ala Ser Val Phe Glu Met Tyr Lys Gly Tyr Leu Leu 275 280 285 ctt gga att gct ctt ttt gta gtg aca ata agc ggt agc act ttt act 911 Leu Gly Ile Ala Leu Phe Val Val Thr Ile Ser Gly Ser Thr Phe Thr 290 295 300 gtc ggc ttt ttg ata cac gct aaa ttt gtg gaa ggt cgg aga agc aac 959 Val Gly Phe Leu Ile His Ala Lys Phe Val Glu Gly Arg Arg Ser Asn 305 310 315 atg agt tgg aaa ata cca ttt gct ttg ctg acg aaa tcg aaa cca aaa 1007 Met Ser Trp Lys Ile Pro Phe Ala Leu Leu Thr Lys Ser Lys Pro Lys 320 325 330 335 cgt gcc gac cgc aca gcc gcc aac cga agt cgg cac tcc gtc cgc tcc 1055 Arg Ala Asp Arg Thr Ala Ala Asn Arg Ser Arg His Ser Val Arg Ser 340 345 350 aac caa acg aac att tcc tcg ctg acc cat tcg acc att ggc agt ttg 1103 Asn Gln Thr Asn Ile Ser Ser Leu Thr His Ser Thr Ile Gly Ser Leu 355 360 365 gca cgg tcc cga atc ttc tcc ctg tac tca tac aat ggg gaa aag tgc 1151 Ala Arg Ser Arg Ile Phe Ser Leu Tyr Ser Tyr Asn Gly Glu Lys Cys 370 375 380 att gtg cgc agc ttt ggc tcc aca aca atg gca aag gca ttt aca gtg 1199 Ile Val Arg Ser Phe Gly Ser Thr Thr Met Ala Lys Ala Phe Thr Val 385 390 395 aca caa atg gcc gag tgc cga acg atg cgt ctg ttc gac cat gag aat 1247 Thr Gln Met Ala Glu Cys Arg Thr Met Arg Leu Phe Asp His Glu Asn 400 405 410 415 gtg aac cgg ttt ttg ggg ctg agt ttg gac ggg gcc aat gtg ttg gcc 1295 Val Asn Arg Phe Leu Gly Leu Ser Leu Asp Gly Ala Asn Val Leu Ala 420 425 430 gtg tgg aac ttt tgc atg cgc ggg tcc atc aga gac gtg att ttg tct 1343 Val Trp Asn Phe Cys Met Arg Gly Ser Ile Arg Asp Val Ile Leu Ser 435 440 445 gaa aat gcc atg gtc aaa gat gtg ata ttc atc cag tcg gcc atc aaa 1391 Glu Asn Ala Met Val Lys Asp Val Ile Phe Ile Gln Ser Ala Ile Lys 450 455 460 gag att tgt gaa ggc att cat ttc ctg cac aat tcg ccc ctc caa ttc 1439 Glu Ile Cys Glu Gly Ile His Phe Leu His Asn Ser Pro Leu Gln Phe 465 470 475 cat ggc cga ctg aaa tcc tcc gct tgt ttg atc aat gac cgg tgg caa 1487 His Gly Arg Leu Lys Ser Ser Ala Cys Leu Ile Asn Asp Arg Trp Gln 480 485 490 495 gtc aaa att tca tat ttt ggg ctt cga tgg cta aag tct tca caa aaa 1535 Val Lys Ile Ser Tyr Phe Gly Leu Arg Trp Leu Lys Ser Ser Gln Lys 500 505 510 aat cgg gcg aaa gat ctt tta tgg cta tcg cct gaa caa tta cgg aaa 1583 Asn Arg Ala Lys Asp Leu Leu Trp Leu Ser Pro Glu Gln Leu Arg Lys 515 520 525 atg gga gac agc gaa att gtg gag ggg tca aaa cat tct gac att tac 1631 Met Gly Asp Ser Glu Ile Val Glu Gly Ser Lys His Ser Asp Ile Tyr 530 535 540 acg atg gca tta atc ttc acc gaa atg gtt aat atg tct ccg tgt tgg 1679 Thr Met Ala Leu Ile Phe Thr Glu Met Val Asn Met Ser Pro Cys Trp 545 550 555 gac agc agc gaa gcg gac gga gca gag gct gac cgg gcc gag gat gga 1727 Asp Ser Ser Glu Ala Asp Gly Ala Glu Ala Asp Arg Ala Glu Asp Gly 560 565 570 575 gaa gag caa aac gga acg gaa atg tcg cga aga aag caa acg gcg gaa 1775 Glu Glu Gln Asn Gly Thr Glu Met Ser Arg Arg Lys Gln Thr Ala Glu 580 585 590 acg gag gga gaa acg gca cag cgg cgc ccg ggg cga cgc gcg agg gga 1823 Thr Glu Gly Glu Thr Ala Gln Arg Arg Pro Gly Arg Arg Ala Arg Gly 595 600 605 cgc aac gcg gag gaa atc gct tat ttg gtg aag cgg ggc gga atc gtt 1871 Arg Asn Ala Glu Glu Ile Ala Tyr Leu Val Lys Arg Gly Gly Ile Val 610 615 620 ccg ctg cgg ccg atc att cgg ccg gca ttt gac cat ctg aac acg gaa 1919 Pro Leu Arg Pro Ile Ile Arg Pro Ala Phe Asp His Leu Asn Thr Glu 625 630 635 gtg att cat ctg atc cgc gac tgt tgg gtc gaa acg ccg agc gaa cgg 1967 Val Ile His Leu Ile Arg Asp Cys Trp Val Glu Thr Pro Ser Glu Arg 640 645 650 655 ccg acc att gaa aaa gtg cga cag aaa ttg cgg caa atg ggt gcc caa 2015 Pro Thr Ile Glu Lys Val Arg Gln Lys Leu Arg Gln Met Gly Ala Gln 660 665 670 cgg agg gtc aat ttg atg gac cat gtg ttc gac atg ttg gag cag tac 2063 Arg Arg Val Asn Leu Met Asp His Val Phe Asp Met Leu Glu Gln Tyr 675 680 685 gcc aac aaa ttg gag gag gaa gtg cag gag cgg acc aaa gag ttg gag 2111 Ala Asn Lys Leu Glu Glu Glu Val Gln Glu Arg Thr Lys Glu Leu Glu 690 695 700 ggg gag aag cga aag tcg gac att ctt ctc tat cgg atg atg cca cgc 2159 Gly Glu Lys Arg Lys Ser Asp Ile Leu Leu Tyr Arg Met Met Pro Arg 705 710 715 caa gtg gcg gac cga cta aag ctc ggc caa tcc gtg gag ccc gag cag 2207 Gln Val Ala Asp Arg Leu Lys Leu Gly Gln Ser Val Glu Pro Glu Gln 720 725 730 735 ttc gac tgt gtg acg gtg ttc ttc tcg gac att gtc caa ttc gcg gca 2255 Phe Asp Cys Val Thr Val Phe Phe Ser Asp Ile Val Gln Phe Ala Ala 740 745 750 ctg tcc aac caa atg cgg ccg ctg cag gtg gtc aat ctg atg aac gaa 2303 Leu Ser Asn Gln Met Arg Pro Leu Gln Val Val Asn Leu Met Asn Glu 755 760 765 ctg tac acc atc ttc gac gca atc att gac gag cac gac gtg tac aag 2351 Leu Tyr Thr Ile Phe Asp Ala Ile Ile Asp Glu His Asp Val Tyr Lys 770 775 780 ggc gat ggt tat ttg tgc gtg tct ggc ctt ccc aat cgg aat ggc act 2399 Gly Asp Gly Tyr Leu Cys Val Ser Gly Leu Pro Asn Arg Asn Gly Thr 785 790 795 ttg cat gcc aaa cac tgt gct gat atg gcg atc aaa ttt atg caa gcg 2447 Leu His Ala Lys His Cys Ala Asp Met Ala Ile Lys Phe Met Gln Ala 800 805 810 815 ctg ctc aat ttc cga att ccc gac ctt cca aat gag cgc gtc cgt ctc 2495 Leu Leu Asn Phe Arg Ile Pro Asp Leu Pro Asn Glu Arg Val Arg Leu 820 825 830 cga att ggg ctg cac agc ggc cca tgc gtc gcg gga gtc gtc ggg ttg 2543 Arg Ile Gly Leu His Ser Gly Pro Cys Val Ala Gly Val Val Gly Leu 835 840 845 gcc atg ccc cgt tac tgt ttg ttt ggg gat acg gta aac acc gcc tcg 2591 Ala Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr Val Asn Thr Ala Ser 850 855 860 cgc atg gaa agt tct tca agc cca aac aaa att cac atg tcc agt gaa 2639 Arg Met Glu Ser Ser Ser Ser Pro Asn Lys Ile His Met Ser Ser Glu 865 870 875 acg ctc gaa ttg ctg cac aaa aat ttc aac ggc tct tat cac acg gag 2687 Thr Leu Glu Leu Leu His Lys Asn Phe Asn Gly Ser Tyr His Thr Glu 880 885 890 895 agc aga ggc gaa gtg atc ata aag ggc aaa ggc gtc atg gag acc ttt 2735 Ser Arg Gly Glu Val Ile Ile Lys Gly Lys Gly Val Met Glu Thr Phe 900 905 910 tgg ctg ttg ggc caa gtc gaa aat gga aca aca att aac gcc gat tat 2783 Trp Leu Leu Gly Gln Val Glu Asn Gly Thr Thr Ile Asn Ala Asp Tyr 915 920 925 gcg cat aga atg cat ctg ccg gtg atc aaa ttt ggg gag gag gga aat 2831 Ala His Arg Met His Leu Pro Val Ile Lys Phe Gly Glu Glu Gly Asn 930 935 940 gaa acc gga aaa aat gcg taaagaaatg gtgatgaagc caccacattt 2879 Glu Thr Gly Lys Asn Ala 945 agactgaaat gcataatgaa agagcaaaga tcaagacttt caaaatgcct gaaattaatg 2939 taactttacc caaaatttag caaaaaattt caatttatta cgaaaaaaaa aaaaaaaaaa 2999 aaaaaaaaa 3008 7 949 PRT Heterodera glycines 7 Lys Ile Thr Ile Asn Tyr Lys Thr Arg Ile Phe Asn Ile Gln Ser Ser 1 5 10 15 Asp Thr Ser Thr Ile Val Asn Ala Ile Arg Glu Arg Ala Arg Ile Val 20 25 30 Leu Leu Cys Phe Asp Asp Leu Lys Gln Met Arg Thr Phe Ala Leu Gln 35 40 45 Leu Phe Asp Gly Gly Leu Asn Thr Lys Asp Tyr Val Tyr Ile Met Val 50 55 60 Asp Asn Asp Met Tyr Leu Ser Phe Asn Leu Thr Arg Leu Pro Phe Trp 65 70 75 80 Val Gln Ser Ser Asn Asn Ser Asn Thr Leu Asp Gly Arg Asn Ala Asp 85 90 95 Ala Glu Val Ile Gly Arg Leu Ala Leu Trp Trp His Tyr Asp Ile Thr 100 105 110 Leu Ser Ala Leu Ser Asn Gln Asn Tyr Tyr Gly Phe Phe Lys Arg Val 115 120 125 Ile Asp Arg Thr Gly Asp Trp Pro Phe Tyr Cys Asp Glu Ser Asn Cys 130 135 140 Ser Lys Val Ile Asn Ala Ser Ile Tyr Ser Leu Leu Leu Tyr Asp Ala 145 150 155 160 Ile Tyr Asn Tyr Gly Met Ala Leu Asn Glu Ser Phe Arg Gln Phe Gly 165 170 175 Ile Arg Pro Glu Val Tyr Arg Asn Gly Thr Leu Leu Ala Arg Asn Asn 180 185 190 Arg Lys Pro Phe Met Gly Leu Thr Gly Tyr Val Thr Val Glu Thr Asp 195 200 205 Gln Asn Thr Arg Val Phe Val Leu Ser Asn Arg Lys Ser Ser Glu Lys 210 215 220 Gly Asn Ala Leu Arg Ile Leu Met Gln Phe Ala Trp Val Glu Gly Lys 225 230 235 240 Leu Gln Ile Ser Leu Arg Asn Gly Ser Leu Ser Met Trp Ser Ser Arg 245 250 255 Gly Gly Ser Ile Pro Pro Ala Val Pro Ile Cys Gly Phe Asp Gly Lys 260 265 270 Gly Cys Ala Ala Ser Val Phe Glu Met Tyr Lys Gly Tyr Leu Leu Leu 275 280 285 Gly Ile Ala Leu Phe Val Val Thr Ile Ser Gly Ser Thr Phe Thr Val 290 295 300 Gly Phe Leu Ile His Ala Lys Phe Val Glu Gly Arg Arg Ser Asn Met 305 310 315 320 Ser Trp Lys Ile Pro Phe Ala Leu Leu Thr Lys Ser Lys Pro Lys Arg 325 330 335 Ala Asp Arg Thr Ala Ala Asn Arg Ser Arg His Ser Val Arg Ser Asn 340 345 350 Gln Thr Asn Ile Ser Ser Leu Thr His Ser Thr Ile Gly Ser Leu Ala 355 360 365 Arg Ser Arg Ile Phe Ser Leu Tyr Ser Tyr Asn Gly Glu Lys Cys Ile 370 375 380 Val Arg Ser Phe Gly Ser Thr Thr Met Ala Lys Ala Phe Thr Val Thr 385 390 395 400 Gln Met Ala Glu Cys Arg Thr Met Arg Leu Phe Asp His Glu Asn Val 405 410 415 Asn Arg Phe Leu Gly Leu Ser Leu Asp Gly Ala Asn Val Leu Ala Val 420 425 430 Trp Asn Phe Cys Met Arg Gly Ser Ile Arg Asp Val Ile Leu Ser Glu 435 440 445 Asn Ala Met Val Lys Asp Val Ile Phe Ile Gln Ser Ala Ile Lys Glu 450 455 460 Ile Cys Glu Gly Ile His Phe Leu His Asn Ser Pro Leu Gln Phe His 465 470 475 480 Gly Arg Leu Lys Ser Ser Ala Cys Leu Ile Asn Asp Arg Trp Gln Val 485 490 495 Lys Ile Ser Tyr Phe Gly Leu Arg Trp Leu Lys Ser Ser Gln Lys Asn 500 505 510 Arg Ala Lys Asp Leu Leu Trp Leu Ser Pro Glu Gln Leu Arg Lys Met 515 520 525 Gly Asp Ser Glu Ile Val Glu Gly Ser Lys His Ser Asp Ile Tyr Thr 530 535 540 Met Ala Leu Ile Phe Thr Glu Met Val Asn Met Ser Pro Cys Trp Asp 545 550 555 560 Ser Ser Glu Ala Asp Gly Ala Glu Ala Asp Arg Ala Glu Asp Gly Glu 565 570 575 Glu Gln Asn Gly Thr Glu Met Ser Arg Arg Lys Gln Thr Ala Glu Thr 580 585 590 Glu Gly Glu Thr Ala Gln Arg Arg Pro Gly Arg Arg Ala Arg Gly Arg 595 600 605 Asn Ala Glu Glu Ile Ala Tyr Leu Val Lys Arg Gly Gly Ile Val Pro 610 615 620 Leu Arg Pro Ile Ile Arg Pro Ala Phe Asp His Leu Asn Thr Glu Val 625 630 635 640 Ile His Leu Ile Arg Asp Cys Trp Val Glu Thr Pro Ser Glu Arg Pro 645 650 655 Thr Ile Glu Lys Val Arg Gln Lys Leu Arg Gln Met Gly Ala Gln Arg 660 665 670 Arg Val Asn Leu Met Asp His Val Phe Asp Met Leu Glu Gln Tyr Ala 675 680 685 Asn Lys Leu Glu Glu Glu Val Gln Glu Arg Thr Lys Glu Leu Glu Gly 690 695 700 Glu Lys Arg Lys Ser Asp Ile Leu Leu Tyr Arg Met Met Pro Arg Gln 705 710 715 720 Val Ala Asp Arg Leu Lys Leu Gly Gln Ser Val Glu Pro Glu Gln Phe 725 730 735 Asp Cys Val Thr Val Phe Phe Ser Asp Ile Val Gln Phe Ala Ala Leu 740 745 750 Ser Asn Gln Met Arg Pro Leu Gln Val Val Asn Leu Met Asn Glu Leu 755 760 765 Tyr Thr Ile Phe Asp Ala Ile Ile Asp Glu His Asp Val Tyr Lys Gly 770 775 780 Asp Gly Tyr Leu Cys Val Ser Gly Leu Pro Asn Arg Asn Gly Thr Leu 785 790 795 800 His Ala Lys His Cys Ala Asp Met Ala Ile Lys Phe Met Gln Ala Leu 805 810 815 Leu Asn Phe Arg Ile Pro Asp Leu Pro Asn Glu Arg Val Arg Leu Arg 820 825 830 Ile Gly Leu His Ser Gly Pro Cys Val Ala Gly Val Val Gly Leu Ala 835 840 845 Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr Val Asn Thr Ala Ser Arg 850 855 860 Met Glu Ser Ser Ser Ser Pro Asn Lys Ile His Met Ser Ser Glu Thr 865 870 875 880 Leu Glu Leu Leu His Lys Asn Phe Asn Gly Ser Tyr His Thr Glu Ser 885 890 895 Arg Gly Glu Val Ile Ile Lys Gly Lys Gly Val Met Glu Thr Phe Trp 900 905 910 Leu Leu Gly Gln Val Glu Asn Gly Thr Thr Ile Asn Ala Asp Tyr Ala 915 920 925 His Arg Met His Leu Pro Val Ile Lys Phe Gly Glu Glu Gly Asn Glu 930 935 940 Thr Gly Lys Asn Ala 945 8 30 DNA Artificial sequence Oligonucleotide probe 8 agcggatccc gtccgcgcat ggacattgtg 30 9 30 DNA Artificial sequence Oligonucleotide probe 9 ccgctcgagc gttgcggcac tcgcatttct 30

Claims

that which is claimed is:

1. An isolated DNA encoding a nematode guanylyl cyclase chemoreceptor selected from the group consisting of:

(a) isolated DNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 6;

(b) isolated DNA that hybridizes to DNA of (a) above under stringent conditions and encodes a nematode guanylyl cyclase chemoreceptor; and

(c) isolated DNA that differs from the DNA of (a) or (b) above due to the degeneracy of the genetic code, and encodes a nematode guanylyl cyclase chemoreceptor encoded by (a) or (b) above.

2. An isolated DNA according to claim 1 selected from the group consisting of:

(a) isolated DNA having a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6;

(b) isolated DNA that hybridizes to DNA of (a) above under stringent conditions in which said isolated DNA does not hybridize to DNA having a nucleotide sequence of SEQ ID NO: 1, and encodes a nematode guanylyl cyclase chemoreceptor; and

3. An isolated DNA according to claim 1, which nematode guanylyl cyclase chemoreceptor is selected from the group consisting of order Tylenchida and order Aphelenchida chemoreceptors.

4. An isolated DNA according to claim 1, which nematode guanylyl cyclase chemoreceptor is selected from the group consisting of cyst nematode, root knot nematode, lesion nematode, and reniform nematode chemoreceptors.

5. An isolated DNA according to claim 1 having a nucleotide sequence according to SEQ ID NO: 1.

6. An isolated DNA according to claim 1, having a nucleotide sequence according to SEQ ID NO: 3.

7. An isolated DNA according to claim 1 having a nucleotide sequence according to SEQ ID NO: 4.

8. An isolated DNA according to claim 1 having a nucleotide sequence according to SEQ ID NO: 6.

9. An oligonucleotide that specifically binds to an isolated DNA of claim 1.

10. An oligonucleotide according to claim 8, which oligonucleotide comprises DNA or RNA.

11. An antisense oligonucleotide that specifically binds to an mRNA transcript of a DNA according to claim 1.

12. A DNA that encodes an antisense oligonucleotide according to claim 11.

13. A double-stranded RNA that is complementary to a DNA according to claim 1 and interferes with the expression thereof in a cell that expresses the encoded protein.

14. An expression cassette comprising a DNA according to claim 1 and a heterologous promoter operatively associated therewith.

15. A cell that contains an expression cassette of claim 14 and expresses said nematode guanylyl cyclase chemoreceptor.

16. A cell according to claim 15, which cell is a yeast cell.

17. A cell according to claim 15, which cell is a plant cell.

18. A cell according to claim 15, which cell is an insect cell.

19. An isolated nematode guanylyl cyclase chemoreceptor protein encoded by a DNA of claim 1.

20. A protein or peptide that specifically binds to a protein of claim 19.

21. A protein or peptide according to claim 20, wherein said protein or peptide is an antibody.

22. A protein or peptide according to claim 21, wherein said antibody is a monoclonal antibody.

23. A method of screening a compound for the ability to disrupt plant parasitic nematode feeding or chemotaxis, said method comprising:

determining whether or not said compound selectively binds to a nematode guanylyl cyclase chemoreceptor protein encoded by a DNA of claim 1;

the presence of such binding indicating said compound is useful in disrupting plant parasitic nematode feeding or chemotaxis.

24. A method according to claim 23, wherein said determining step is carried out in vitro.

25. A method according to claim 23, wherein said determining step is carried out in vivo in a cell culture comprising cells that expresses said guanylyl cyclase chemoreceptor protein.

26. A method according to claim 26, wherein said compound is a member of a combinatorial library.