WO2002042478A2 - Nematode resistant plant - Google Patents

Abstract

Methods are disclosed for conferring nematode resistance to plants comprising transforming a plant with a polynucleotide operatively linked to a promoter induced by nematode infection wherein expression of the polynucleotide results in suppression of FGAM synthase activity. Also disclosed are vectors, host cells and plants containing recombinant polynucleotides comprising a nematode infection inducible promoter operatively linked to a polynucleotide wherein expression of the polynucleotide results in suppression of FGAM synthase activity.

Description

NEMATODE RESISTANT PLANT

Background

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) infection of soybean roots presents an important agronomic problem to soybean growers of the mid- western and southern United States. The cyst nematode infection of susceptible soybean lines is accompanied by a series of cellular changes within the plant at the site of feeding initiation that leads to a structure known as a syncytium. Some of the defining characteristics of syncytium development include enlarged nucleoli, increased density of endoplasmic reticulum, increased number of mitochondria, changes in vacuolization, and cell wall dissolution of surrounding cells (Endo, 1991). The complexity of this process suggests that the expression of several plant genes is involved in this developmental change. In fact, plant genes encoding proteins like extensins and late embryonic abundant (LEA) protein (Nan der Eycken et al., 1996), catalase (Νiebel et al., 1995), cyclin (Νiebel et al., 1996), membrane channel protein (Opperman et al., 1994; Wilson et al., 1994; Yamamoto et al., 1990), the E2 enzyme of the ubiquitination pathway, Myb proteins and RΝA polymerase II (Bird and Wilson, 1994) have been identified to be nematode-responsive in various plant systems. In contrast, little direct investigation of soybean genes involved in the SCΝ-plant interaction has been pursued.

Current methods for control of nematodes involve resistant varieties, cultivation techniques and application of chemicals. These methods may be used individually or combined in an integrated system. The use of chemicals to control nematodes has come under increased scrutiny due to concerns with environmental pollution and possible effects on humans and animals that consume products treated with nematicides. Cultivation techniques such as crop rotation, while avoiding the use of chemicals, increase the cost of production and are less effective in preventing infection than are other methods. Genetic resistance is appealing due to its effectiveness while avoiding the use of chemicals and not requiring increased labor inputs. Although nematode resistance plant varieties are available, these varieties often have characteristics which decrease their suitability from a commercial standpoint. For example, available nematode resistant varieties of soybeans often suffer from problems such as black seed coats, poor standability, seed shattering and low yield. Because of the multigenic nature of nematode resistance, crossing of resistant varieties with elite varieties results in heterogenous populations.

Resistance to SCN has been reported in several genotypes of soybean, and quantitative genetic analysis has allowed the development of various genetic models, generally involving multiple resistance loci acting in concert (Concibido et al., 1996b; Rao AreUi et al., 1992; Nieriing et al., 1996; Webb et al., 1995). At the cytological level, early stages of the infection process in a resistant (zero to three cysts per root) line may appear quite similar to the compatible interaction (Acedo et al., 1984; Mahalingam and Skorupska, 1996; Kim and Riggs, 1992). However, typically these formations do not support the full life cycle of the female nematode, and degenerate a few days after initiation. To achieve better resolution of the resistance mechanism, the development of inbred nematode strains and their recurrent selection for zero-cyst phenotype on a resistant plant genotype has proven an effective strategy for the genetic dissection of resistance. This strategy was used in the development of an inbred nematode strain, first reported as Hgl (Luedders, 1987) and recently re-designated to NL1 (Bird and Riddle, 1994; T.L. Νiblack, personal communication), utilizing soybean resistant line PI 88287 and susceptible line PI 89008. Within this genetic system, resistance is conferred by a single recessive resistance gene (Luedders, 1987). This system was chosen to study the cellular changes and genes induced during the infection process because of the effectiveness of this system in facilitating unambiguous classification of the resistant phenotype in a segregating population. Despite extensive research, the location of the single resistance gene remained unknown until mapped by the present inventors. Complicating factors that have slowed progress in studies of soybean-nematode interactions are the genetic heterogeneity of soybean cyst nematode field populations, the environmental sensitivity of the infection process, and the difficulties inherent in locating actively developing infection sites internal to the root and invisible at the surface. The present application has utilized a genetically simplified system to facilitate more in-depth investigation of plant- nematode interaction. This system uses a homogeneous inbred cyst nematode population together with a highly resistant plant genotype. Previous gene expression studies have provided evidence that several host genes are recruited in the process of establishing a compatible interaction between the nematode and the plant. Several of these host genes are homologous to genes with known functions. These include plant genes encoding extensins and late embryonic abundant (LEA) proteins (NanderEycken et al., 1996), cyclin (Νiebel et al., 1996), catalase (Νiebel et al., 1995), membrane channel protein (Opperman et al., 1994; Wilson et al, 1994, Yamamoto et al., 1990), the E2 enzyme of the ubiquitination pathway, Myb proteins, and RΝA polymerase II (Bird and Wilson, 1994). Hydroxymethylglutaryl coenzyme A reductase (HMGR) has been proposed to be enhanced upon nematode infection due to the dependence of the nematode on the plant host for sterol biosynthesis (Cramer et al., 1993). Additional enzymes such as endo-1.3-glucanase (Yoshikawa et al., 1990) and cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Laxalt et al., 1996) may be involved in a general response to pathogen infection.

Quantitative trait loci controlling resistance to soybean cyst nematode have been genetically mapped by several laboratories (Concibido et al., 1994; Webb et al., 1995; Concibido et al., 1996a, b; Nieriing et al., 1996; Chang et al., 1997). Most report only a genomic region in which a molecular marker was significantly associated with resistance, which does not allow a precise positioning of the QTL on the genomic map. However, the use of PCR-based and publicly available markers by Concibido et al., (1996b) and the mapping of SCΝ resistance as a qualitative trait, allowed the localization of a major SCΝ resistance gene between markers B_053T and Bng_122D on Linkage Group G (Mudge et al., 1997). Several methods have been described for altering plants using recombinant DΝA technology to confer nematode resistance. U.S. Patent No. 5,824,876 discloses a method for controlling nematodes in which a plant is transformed with a construct containing a nematode induced promoter combined with a region encoding a product disruptive of nematode attack. In U.S. Patent 5,589,622, the same construct as in the '876 patent is disclosed, except that the promoter induces expression adjacent to a nematode feeding site. U.S. Patent No. 5,866,776, discloses a method for protecting plants against a pathogen by transforming a plant containing a resistance gene with a polynucleotide encoding an elicitor protein corresponding to the resistance gene wherein expression of at least one of the resistance gene and the elicitor protein is regulated by a promoter induced by the pathogen. U.S. Patent No. 5,866,777 discloses a two component system in which a first promoter drives a first sequence encoding a substance which disrupts the cell of the nematode feeding structure and a second promoter drives a second sequence encoding a molecule that represses the effect of the first molecule. In this system, the first promoter is active at least in the cell containing a nematode feeding structure, while the second promoter is active in all cells in which the first promoter is active, but is not effective in the cell of the nematode feeding structure.

Summary

Using a homogeneous inbred cyst nematode population together with a highly resistant plant genotype, the present inventors have discovered that nematode resistance can be mapped to the region encompassing the FGAM synthase gene and that the FGAM synthase gene is induced upon nematode infection at the infection site. These discoveries provide a means to confer resistance to nematode infection in plants by suppression of FGAM synthase expression during nematode infection.

Accordingly, one aspect of the present invention is a method for conferring nematode resistance to a plant comprising, transforming a plant with a polynucleotide linked to a promoter inducible by nematode infection wherein expression of said polynucleotide results in suppression of FGAM synthase activity.

Another aspect is a method for conferring nematode resistance to a plant comprising transforming the plant with an expression cassette comprising, operatively linked in the 5' to 3' order, a nematode infection inducible promoter, a polynucleotide, and a termination signal, wherein expression of said polynucleotide results in suppression of FGAM synthase activity.

Yet another aspect provides a vector comprising a polynucleotide operatively linked to a nematode infection inducible promoter wherein expression of the polynucleotide results in suppression of FGAM synthase activity. Still another aspect provides a vector comprising an expression cassette where the expression cassette comprises, operatively linked in 5' to 3' order, a nematode infection inducible promoter, a polynucleotide, and a termination signal wherein expression of the polynucleotide results suppression of FGAM synthase activity. In one preferred embodiment, either of the above mentioned vectors are expression vectors.

An additional aspect provides a host cell comprising any of the previously mentioned vectors. In a further aspect, the host cell is a plant cell. Another aspect provides a plant produced by transforming the plant with a polynucleotide operatively linked to a promoter inducible by nematode infection wherein expression of the polynucleotide results in suppression of FGAM synthase activity. Still another aspect provides a plant produced by transforming the plant with an expression cassette comprising, operatively linked in 5' to 3' order, a nematode infection inducible promoter, a polynucleotide, and a termination signal, wherein the expression of the polynucleotide results in suppression of FGAM synthase activity. Still a further aspect provides a plant comprising a polynucleotide operatively linked to a nematode infection inducible promoter, wherein expression of the polynucleotide results in suppression of FGAM synthase activity. Yet another aspect provides a plant comprising an expression cassette comprising, operatively linked in 5' to 3' order, a nematode infection inducible promoter, a polynucleotide, and a termination signal, wherein expression of the polynucleotide results in suppression of FGAM synthase activity.

Yet another aspect provides a seed produced by any of the aforementioned plants, progeny of any of the aforementioned plants or hybrids produced by crossing any of the aforementioned plants with wild type and other plants.

In any of the aforementioned aspects, suppression of FGAM synthase activity can be achieved by any method known in the art, including, but not limited to, antisense suppression, dominant negative suppression, ribozyme cleavage, or co-suppression. In any of the aforementioned aspects the promoter can be a tissue specific promoter.

Still another aspect provides a method for determining a pathogen resistance gene comprising obtaining an inbred strain of the pathogen; obtaining a pathogen resistant strain of the host organism; infecting the resistance host organism and a wild type, non- resistant host organism with the pathogen; obtaining RNA from the infected resistant and wild type organisms; and comparing the RNA from both organisms to determine differences in gene expression.

Another aspect of the invention comprises SEQ ID NO: 1, SEQ ID NO: 2, or fragments thereof. Another aspect provides polypeptides encoded by SEQ ID NO: 1, SEQ ID NO: 2, or fragments thereof. Yet another aspect provides recombinant vectors transformed with SEQ ID NO: 1, SEQ ID NO: 2, or fragments thereof. Also included in the present invention are host cells transformed with recombinant vectors comprising SEQ ID NO: 1, SEQ ID NO: 2, or fragments thereof.

Still another aspect provides isolated an polynucleotide comprising SEQ ID NO: 3 or SEQ ID NO: 4. A further aspect provides a method for inducing expression of a polynucleotide in response to nematode infection, particularly in a plant, comprising operatively linking in 5' to 3' order SEQ ID NO: 3 and the polynucleotide to be expressed. Yet another aspect provides a method for inducing expression of a polynucleotide in response to nematode infection, particularly in a plant, comprising operatively linking in 5' to 3' order SEQ ID NO: 4 and the polynucleotide to be expressed. Yet another aspect provides an expression cassette comprising, operatively linked in 5' to 3' order SEQ ID NO: 3, a polynucleotide to be expressed, and a termination signal. Another aspect provides an expression cassette comprising, operatively linked in 5' to 3' order SEQ ID NO: 4, a polynucleotide to be expressed, and a termination signal.

BRIEF DESCRIPTION OF THE FIGURES These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims and accompanying figures where:

Figure 1 shows light microscopic examination of syncytium development in susceptible (PI 89008) soybean roots infected with nematode inbred line NL1 (Heterodera glycines Ichinohe). Panels (A) to (G) show the susceptible response of PI 89008 to SCΝ observed between approximately 4 and 30 days after infection. All specimens were embedded in Quetol resin, except for samples in panel (E) which were embedded in LR White resin. The sample in panel E was prepared from an infected root section grown in vitro on a medium according to Lauritis et al. (1983). The scale bar in the lower left corner of each panel corresponds to 10 μm. Labels in panels specify: CWS, cell wall stubs; Ν, nematode; ΝC, necrotic matter; S, syncytium-component cell; N, vacuole; X, xylem element.

(A) A cross section through a root (PI 89008) at low magnification showing three syncytium-component cells [S] at different stages of development due to staggered re- infection inside the central vascular cylinder of the root, and adjacent to the xylem elements. The syncytium cells have distorted the morphology of the vascular cylinder. The nematode [N] is situated next to the endodermis surrounding the central vascular cylinder. The cavity in which the nematode resides is filled with necrotic material [NC] that stains dark.

(B) A young syncytium [S] showing seven cells in this section with different degrees of vacuolization [V]. Cell wall breakdown has just started to develop. Irregular depositions of cell wall material become visible predominantly in areas of the syncytium cell walls adjacent to xylem elements (arrowhead). Approximately 4 days after infection.

(C) A higher magnification of one syncytium shown in panel (A). At this stage, vacuolization has further progressed and significant cell wall breakdown generates cell wall stubs [CWS] and establishes a cytoplasmic continuum between syncytium- component cells. Approximately 8 days after infection.

(D) In the mature syncytium the vacuoles tend to be smaller in size and become irregular in form. Extensive finger-like ingrowths are evident (arrowheads) at cell walls adjacent to xylem cells. Approximately 14 days after infection. (E) In an aging syncytium [S] the vacuoles have disappeared and irregular membranous material resides in the cytoplasm. The syncytium has reached its maximum extension. Approximately 19 days after infection.

(F) High magnification of an older, degenerated syncytium with cell wall ingrowths (arrowheads) shown in panel (A). Cell wall stubs [CWS] mark the position of once intact cell walls. Approximately 24 days after infection.

(G) A decayed syncytium is void of cytoplasm. Cell-wall ingrowths (arrowheads) and perforated cell walls and cell wall stubs [CWS] remain of the nematode feeding site. Approximately 30 days after infection.

Figure 2 shows RT-PCR analysis of soybean gene induction during infection of susceptible line PI 89008 by nematode inbred strain NL1.

(A) Titration of root mRΝA against known quantities of leaf mRΝA by RT-PCR using primers specific for soybean 18S rRΝA. The right half of the figure illustrates that the leaf mRΝA was quantified spectrophotometrically and the indicated amounts were subjected to RT-PCR with soybean 18S rRΝA-specific primers (see Table 1). This titration series was compared to a series of 1 :25 to 25,600 dilutions of a root mRΝA preparation. Control, no mRΝA. The size of the amplification product (in base pairs) is indicated to the left of the gel. (B) Shown is a composite picture of 1.2% agarose gels (processed with Photoshop 4.0, Adobe, USA) after electrophoresis of PCR amplification products obtained by reverse transcription of mRNA from leaf (L), non-infected root (-) and infected root (+) tissues (sets of three lanes each in that order) with specific primers corresponding to the genes indicated at the top of the gels. The gene designations used and the identity of the primers refer to the descriptions in Tables 1 and 2. Abbreviations: Gluc-1, β-l,3-endoglucanase (AC # U41323); Gluc-2, β-l,3-endoglucanase (AC # U08405); LEA, Late embryonic abundant protein type 14; Catalase; Cyclin (AC # D508681); EF-la, Elongation Factor lot; HSP-1, heat shock protein HSP70 (AC # F000379); HSP-2, heat shock protein HSP70 (AC # X62799); n rRNA, nematode 28S ribosomal RNA; Glucosidase, β-Glucosidase (F000378); 18S rRNA, 18S ribosomal RNA; HMGR, Hydroxymethylglutaryl CoA reductase. Primers for the soybean 18S ribosomal RNA and glyceraldehyde-3-phosphate dehydrogenase (data not shown) were used as internal standards to equalize the amount of mRNA from the three sources used in the assays. Nematode 28S rRNA primers served as a negative control giving a strong signal only in the mRNA sample from roots infected by nematodes. Whereas expression of the selected genes is readily detected in leaves (except for clones of nematode origin), some are expressed at comparable levels in infected root tissue, but at undetectable levels, using the assay described herein, in the non-infected control. With primers matching most divergent DNA regions, the assay also discriminated between individual genes from within families such as HSP70, endo-glucanases and cyclins; only particular genes from each class demonstrated enhanced expression in infected root. Molecular weights (in base pairs) are provided to the left.

Figure 3 A shows placement of eight cDNAs on the public genetic linkage map of soybean. Linkage groups beginning with "MS" are from the F2 population of A81 356022 x PI 468.916 (Shoemaker et al., 1996). Linkage groups beginning with "BSR" are from the recombinant inbred line population of BSR 101 x PI 437.654 (Baltazar and Mansur, 1992). If a cDNA locus was mapped to the same linkage group in both populations, the BSR linkage group was placed physically to the right of the corresponding MS linkage group. cDNA locus names are shown in bold font and boxed, and include the restriction enzymes used in genomic DNA digestion. Abbreviations are: CYCLIN, cyclin (AC # D508681); EF-la, elongation factor EF-lα; GLUC-2, β-l,3-endoglucanase (U08405); HMGR, Hydroxymethylglutaryl CoA reductase; HSP70, heat shock protein HSP70 (AC # X62799); LEA14, Late embryonic abundant protein type 14; FGAM, Formylglycinamidine ribonucleotide synthetase. Previously reported quantitative trait loci (QTL) regions associated with soybean resistance to SCN were indicated with a vertical bar and "SCN QTL" with the reference at the bottom of the linkage group. Figure 3B shows a detailed map of a portion of Linkage Group G showing approximate mapping location of FGAM. The 3.0 cM interval delimited by markers B_053T and Bng_122E is shown in association to the FGAM locus. The mapping data set used to position FGAM and SCN_rad to linkage group G is that used in Cregan et al. (1999). Figure 4 shows SEQ ID NO: 1. The start codon is at position 2484 and the stop codon is at position 2463.

Figure 5 shows SEQ ID NO: 2. The start codon is at position 2614 and the stop codon is at position 6554.

Figure 6 shows SEQ ID NO: 3. Figure 7 shows SEQ ID NO: 4.

DEFINITIONS AND ABBREVIATIONS

FGAM synthase = phosphoribosylformylgylcinamidine synthase

SCN = cyst forming nematode

QTL = quantitative trait loci cM = centi Morgan

RT = reverse transcription

PCR = polymerase chain reaction

RIL = recombinant inbred line

AC # = GenBank accession number The term "expression" in the context of a gene or polynucleotide involves the transcription of the gene or polynucleotide into RNA. The term can also, but not necessarily, involve the subsequent translation of the RNA into polypeptide chains and their assembly into proteins.

As used herein, "expression cassette" means a genetic module comprising a polynucleotide and the regulatory regions necessary for its expression, which may be incorporated into a vector. As used herein, a "recombinant nucleic acid" is defined either by its method of production or its structure. In reference to its method of production, e.g., a product made by a process, the process is use of recombinant nucleic acid techniques, e.g., involving human intervention in the nucleotide sequence, typically selection or production. Alternatively, it can be a nucleic acid made by generating a sequence comprising fusion of two fragments which are not naturally contiguous to each other. Thus, for example, products made by transforming cells with any unnaturally occurring vector is encompassed, as are nucleic acids comprising sequences derived using any synthetic oligonucleotide process. Such manipulation is often done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, genetic manipulation is performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of functions not found in the commonly available natural forms. Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.

As used herein, "polynucleotide" and "oligonucleotide" are used interchangeably and mean a polymer of at least 2 nucleotides joined together by phosphodiester bonds and may consist of either ribonucleotides or deoxyribonucleotides.

As used herein, "sequence" means the linear order in which monomers occur in a polymer, for example, the order of amino acids in a polypeptide or the order of nucleotides in a polynucleotide.

As used herein, "peptide" and "protein" are used interchangeably and mean a compound that consists of two or more amino acids that are linked by means of peptide bonds.

DETAILED DESCRIPTION

The following detailed description is provided to aid those skilled in the art in practicing the present invention. Even so, this detailed description should not be construed to unduly limit the present invention as modifications and variations in the embodiments discussed herein can be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.

All publications, patents, patent applications, databases, and other references cited in this application are herein incorporated by reference in their entirety as if each individual publication, patent, patent application, database, or other reference were specifically and individually indicated to be incorporated by reference.

The present inventors have surprisingly discovered that nematode resistance in soybeans can be genetically mapped to the region encompassing the FGAM synthase gene and that nematode infection is accompanied by an increase in FGAM synthase gene expression. These discoveries provide a means for conferring nematode resistance to plants in which plants are transformed with a nucleotide construct operatively linked to a nematode induced promoter, wherein expression of the construct results in suppression of FGAM synthase activity. FGAM synthase (EC 6.3.5.3) is an enzyme in the pathway for de novo purine biosynthesis that catalyses the formation 5' phosphoribosylformylglycinamidine from ATP, 5'-phosphoribosylformylglycinamide, L- glutamine, and H₂O with the release of ADP, orthophosphate, and L-glutamate.

The genetic mapping of nematode resistance to the region of the FGAM gene was accomplished by the use of a homogeneous inbred cyst nematode population together with a highly resistant plant genotype. Any inbred strain of nematode can be used in this localization method. In general, nematodes are subjected to recurrent selection for zero- cyst phenotype on a resistant plant genotype. Methods for the development of inbred lines of nematodes are known in the art (Luedders, 1987). In one embodiment, the inbred line of nematodes used was the Hgl line (Luedders, 1987) which has recently been re- designated to NL1 (Bird and Riddle, 1994). Identification of genes associated with syncytium development following nematode injection was accomplished by cDΝA subtraction, differential display and empirical selection. Methods for production of cDΝA subtraction libraries and differential display are well known in the art and can be found for example in Ausubel et al., Short Protocols in Molecular Biology, 2nd ed., Wiley & Sons, 1992, unit 5.8; Nedoy et al., Braz. J. Med. Biol. Res. 32:877-884, 1999; Ausubel et al., 1988; Wilson et al., 1994; Innis et al., PCR Applications, Academic Press, 1999, Chap. 18. In general the production of a subtraction library consists of obtaining cDΝA from mRΝA obtained from infected and uninfected plants. The cDΝAs from the target tissues are then hybridized using a vast molar excess of driver cDNA from control (uninfected) tissues followed by separation of the double-stranded DNA hybrids from the single stranded cDNAs which corresponded to the differentially expressed mRNAs. Separation of single stranded from double stranded nucleic acids can be accomplished by any suitable method, but is most commonly by hydroxyapaptite or streptavidin-biotin interaction, or by suppression PCR. The subtracted cDNA is then used either as a probe to screen existing cDNA libraries or can be used to construct a subtracted cDNA library.

In general, differential display uses the amplification of mRNA from two populations coupled with a comparison of amplicons by gel electrophoresis and isolation of cDNA fragments which are expressed at different levels between the two populations. More particularly, mRNA is isolated from two populations of tissues, for example, infected and uninfected. First strand cDNA synthesis is accomplished by reverse transcription using anchored primers. Anchored primers contain two base-specific nucleotides at the 3' end of the oligo(dT) stretch which determine the specificity of the RT reaction. The cDNA is then amplified by PCR using arbitrary primers and the amplification products compared by denaturing polyacrylamide electrophoresis. Differentially expressed cDNA fragments are then isolated from the gel and reamplified by PCR and sequenced. Further details can be found in U.S. Patent No. 5,262,311 ; Liang and Pardee, Science, 257:967, 1992; Liang et al, Nuc. Acids Res., 22:5763-5764, 1994. Data obtained on differential gene expression is then used to identify genes whose expression is influenced by nematode infection. Genes associated with nematode resistance are then determined using genetic mapping techniques as described below. The discovery that FGAM synthase is involved in susceptibility to nematode infection can be used to produce constructs conferring nematode resistance to plants by genetic engineering. The present invention therefore, encompasses a method for conferring nematode resistance to a plant comprising, transforming a plant with a polynucleotide operably linked to a promoter inducible by nematode infection wherein expression of the construct results in suppression of FGAM synthase activity. A nucleic acid sequence is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked sequences are contiguous and, in the case of a secretory leader, contiguous and in reading phase. Linking is achieved by ligation at restriction enzyme sites. If suitable restriction sites are not available, then synthetic oligonucleotide adapters or linkers can be used as is known to those skilled in the art. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, (1989) and Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons (1995). In accordance with the present invention, therefore, nematode resistance can be conferred upon a plant by transforming the plant with a suppressor construct the expression of which results in suppression of FGAM synthase activity and in particular suppression of the increase in FGAM synthase activity normally associated with nematode infection. By suppressor construct is meant, a polynucleotide expression of which causes a suppression of FGAM synthase activity. By suppression of FGAM synthase activity is meant that the activity of FGAM synthase as measured by any method known in the art is less under a given set of conditions for a plant or plant cell containing the suppressor construct when compared to plant or plant cell which is identical except for the presence of the suppressor construct. Many methods for the suppression of FGAM synthase activity are known in the art and are encompassed by the present invention, including, but not limited to, antisense suppression, dominant negative suppression, ribozyme cleavage and co-suppression. One embodiment of the present invention, therefore, involves suppression of FGAM synthase activity by the use of antisense sequences. Antisense sequences can be produced by reversing the orientation of the transcribed region of a gene or polynucleotide sequence whose suppression is desired. When operatively coupled to a suitable transcriptional promoter such as those discussed herein, a transcript of the antisense DNA strand is produced. The production and use of antisense DNA is well known in the art and can be found, for example, in Green et al., (1986) Annu. Rev. Biochem. 55:569. The transcript of the antisense DNA is antisense RNA. Without being bound by theory, it is believed that an individual antisense RNA molecule may hybridize with a complementary "sense" mRNA molecule to form an RNA-RNA duplex. Such a duplex may prevent the sense mRNA molecule from being translated, effectively suppressing production of the ultimate gene product, a protein. The presence of RNA-RNA duplexes may also initiate a sequence-specific RNA degradation pathway with the antisense molecules and/or the RNA-RNA duplexes playing a role in initiating the degradation pathway, and both sense and antisense molecules serving as specific targets for degradation. It will be apparent to one of ordinary skill in the art.Jhat the antisense transcript need not encompass the entire gene or polynucleotide sequence, but may be a fragment which hybridizes to only a portion of the sense RNA. The antisense transcript should be of sufficient length to allow specificity in binding to the target (sense) transcript. In general, the antisense transcript should be at least 10 bases long, although the presence of rare sequences may allow the use of shorter antisense transcripts. The use of this technology to suppress the expression of specific plant genes has been described, for example in European Patent Publication No 271988; U.S. Patent Nos. 5.073,676, 5,107,065 and 5,569,831; Smith et al, (1988) Nature, 334, 724-726;and Smith et al, (1990) Plant Mol Biol 14, 369-380). In another embodiment, FGAM synthase suppression may be achieved by transforming a plant or plant cell with a suppressor construct containing a dominant negative mutation. Expression of a suppressor construct containing a dominant mutant mutation generates a mutant protein that, when coexpressed with the wild type protein inhibits the activity of the wild type protein. Methods for the design and use of dominant negative constructs are well known in the art and can be found, for example, in

Herskowitz, Nature, 329:219-222 (1987) and Lagna and Hemmati-Brivanlou, Curr. Topics Devel Biol, 36:75-98 (1998).

Numerous strategies can be used in suppression by dominant negative constructs and can be used in the practice of the present invention. One strategy involves a dominant negative mutation within the FGAM synthase gene so that the protein produced competes with the wild type enzyme but has reduced enzymatic activity. Thus, in this embodiment, mutations affecting catalysis and/or substrate binding are preferred. In another embodiment, a dominant negative mutation is introduced into a protein which regulates expression of FGAM synthase. In one embodiment, the dominant negative mutant is a transcription regulator that competes with the wild type regulator, but with altered activity. Non-limiting examples of this embodiment would be a transcription enhancer that competes with the wild type enhance but does not result in transcription or in reduced transcription when compared to the wild type. Another example is a transcription repressor that competes with the wild type repressor but has substantially increased repressor activity when compared to the wild type.

In another embodiment, the suppressor construct encodes a ribozyme that cleaves the RNA transcript of the FGAM synthase gene. Ribozymes are catalytic RNA molecules that can promote specific biochemical reactions without the need for auxiliary proteins. Reactions catalyzed by ribozymes can be either intramolecular or intermolecular. Examples of intramolecular reactions are self-splicing or self-cleaving reactions while intermolecular reactions involve other RNA molecules as substrates and more closely approximate true enzymatic reactions where the enzyme is unchanged after each reaction. In the present invention, ribozymes catalyzing intermolecular reactions are preferred. In one embodiment, the ribozyme introduced can result in abnormal splicing of the wild type FGAM synthase RNA transcript so that the resulting protein has no or reduced enzymatic activity. The use of ribozymes to alter expression of genes is discussed in Cech, J. Am. Med. Assoc, 260:3030-3034 (1988).

In yet another embodiment, suppression of FGAM synthase activity can be accomplished by co-suppression. Co-suppression can occur when the suppressor construct contains a polynucleotide encoding FGAM synthase, or a fragment thereof, in the sense orientation between a strong promoter and an appropriate 3' terminal sequence. Cosuppression results in reduced expression of the transgene as well as the endogenous gene. Insertion of a related gene or promoter into a plant can induce rapid turnover of homologous endogenous transcripts, a process believed to have many similarities to the mechanism responsible for antisense RNA inhibition. The effect depends on sequence identity between transgene and endogenous gene. Some cases of co-suppression resembles RNA interference (the experimental silencing of genes by the introduction of double-stranded RNA), as RNA seems to be both an important initiator and a target in these processes. Various regulatory sequences of DNA can be altered (promoters, polyadenylation signals, post-transcriptional processing sites) or used to alter the expression levels (enhancers and silencers) of a specific mRNA. It is preferred that the suppressor construct encode an incomplete or defective FGAM synthase although a construct encoding a fully functional FGAM synthase mRNA may be used. The present invention encompasses recombinant suppressor constructs which can be used to confer nematode resistance to plants comprising, a polynucleotide, expression of which supresses FGAM synthase activity and a nematode infection inducible promoter. Any polynucleotide which when expressed results in reduced FGAM synthase activity can be used in the present invention. In one embodiment the polynucleotide sequence used is derived from SEQ ID NO: 1 or SEQ ID NO: 2 which encode the FGAM synthase of Glycine max. For example, in one embodiment the suppressor construct can contain SEQ ID NO: 1, SEQ ID NO: 2, or a fragment thereof in the antisense orientation. In another example, the amino acid sequence encoded by SEQ ED NO: 1 or SEQ ID NO: 2 can be altered to produce a dominant negative mutant which competes with the wild type enzyme but has reduced activity. It will be appreciated by those of ordinary skill in the art that due to the degeneracy of the genetic code, multiple polynucleotides can encode the same FGAM synthase amino acid sequence. The use of such alternative sequences as well as degenerate nucleotide sequences are included within the scope of the present invention. Those of ordinary skill in the art are aware that modifications in the amino acid sequence of a peptide, polypeptide, or protein can be used to create dominant negative mutants. The present invention accordingly encompasses such modified amino acid sequences and polynucleotide sequences encoding such modified amino acid sequences. Alterations can include amino acid insertions, deletions, substitutions, truncations, fusions, shuffling of subunit sequences, and the like, provided that such alterations result in a dominant negative mutant FGAM synthase which confers nematode resistance.

One factor that can be considered in making such changes is the hydropathic index of amino acids. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein has been discussed by Kyte and Doolittle ( J. Mol. Biol, 157: 105-132, 1982). It is accepted that the relative hydropathic character of amino acids contributes to the secondary structure of the resultant protein. This, in turn, affects the interaction of the protein with molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

Based on its hydrophobicity and charge characteristics, each amino acid has been assigned a hydropathic index as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (- 0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate/glutamine/aspartate/asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

As is known in the art, certain amino acids in a peptide or protein can be substituted for other amino acids having a similar hydropathic index or score and produce a resultant peptide or protein having similar biological activity, i.e., which still retains biological functionality. In making such changes, it is preferable that amino acids having hydropathic indices within ±2 are substituted for one another. More preferred substitutions are those wherein the amino acids have hydropathic indices within ±1. Most preferred substitutions are those wherein the amino acids have hydropathic indices within ±0.5.

Like amino acids can also be substituted on the basis of hydrophilicity. U.S. Patent No. 4,554,101 discloses that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilicity values have been assigned to amino acids: arginine/lysine (+3.0); aspartate/glutamate (+3.0 ±1); serine (+0.3); asparagine/glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ±1); alanine/histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine/isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). Thus, one amino acid in a peptide, polypeptide, or protein can be substituted by another amino acid having a similar hydrophilicity score and still produce a resultant protein having similar biological activity, i.e., still retaining correct biological function. In making such changes, amino acids having hydropathic indices within ±2 are preferably substituted for one another, those within ±1 are more preferred, and those within ±0.5 are most preferred.

As outlined above, amino acid substitutions in the peptides of the present invention can be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take various of the foregoing characteristics into consideration in order to produce conservative amino acid changes resulting in silent changes within the present peptides, etc., can be selected from other members of the class to which the naturally occurring amino acid belongs. Amino acids can be divided into the following four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral non- polar amino acids. Representative amino acids within these various groups include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and (4) neutral non-polar amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. It should be noted that changes which are not expected to be advantageous can also be useful if these result in the production of functional sequences.

The invention also contemplates that the suppressor construct contain an inducible promoter, and that the promoter be induced by infection by a nematode. Any promoter which is inducible by nematode infection and which is functional in the selected host is suitable. The nematode inducible promoters useful in the present invention can be of two types: promoters that do not normally cause expression of a polynucleotide sequence, but do cause expression in response to nematode infection, and promoters that normally drive expression of a polynucleotide sequence, but whose activity is increased following nematode infection resulting in enhanced expression. Pathogen and nematode inducible promoters are known in the art and as can be found, for example, in U.S. Patent No. 5,750,386, U.S. Patent No. 5,955,646 and the references cited therein. In one embodiment the promoter is SEQ ID NO: 3. In another embodiment the promoter is SEQ ID NO: 4. Other examples of nematode inducible promoters include those disclosed in Opperman et al., Science, 263:221-223 (1994); Hansen et al., Physiol Molec. Plant

Pathol, 48:161-170 (1996); and Mahalingam et al., Molec. Plant-Microbe Interactions, 12:490-498 (1999). In addition to being induced by nematode infection, promoters can be tissue- or cell-specific. By use of a tissue- or cell-specific promoter, expression of the suppressor construct can be restricted to the site of nematode infection. Commonly, the recombinant polynucleotides of the present invention will be included in a vector which is in turn used to transform a suitable host. The vector can be either a cloning vector or an expression vector. A cloning vector is a self-replicating DNA molecule that serves to transfer a DNA segment into a host cell. The three most common types of cloning vectors are bacterial plasmids, phages, and other viruses. An expression vector is a cloning vector designed so that a coding sequence inserted at a particular site will be transcribed into mRNA and translated into a protein. Both cloning and expression vectors contain nucleotide sequences that allow the vectors to replicate in one or more suitable host cells. In cloning vectors, this sequence is generally one that enables the vector to replicate independently of the host cell chromosomes, and also includes either origins of replication or autonomously replicating sequences. Various bacterial and viral origins of replication are well known to those skilled in the art and include, but are not limited to, the pBR322 plasmid origin, the 2μ plasmid origin, and the SV40, polyoma, adenovirus, VSV and BPV viral origins. Ausubel et al., ed., Sbort Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

One commonly used type of cloning vector is derived from filamentous phages, and in particular, the φX174 and the Ml 3 phages. The advantage of filamentous phage vectors is that DNA inserted into them can be recovered in both the double-stranded and single stranded forms. As typically used, the nucleotide sequence to be cloned is inserted into double-stranded vector and the vector containing the sequence is introduced into cells by transformation. In the case of the Ml 3 phage, the foreign sequence is inserted into a polylinker located in a non-essential region of the Ml 3 genome. Cells containing vectors with filamentous phage origins, usually the fl origin, are also infected with helper phage. The helper phage provides the gene 2 protein that drives the vector into the fl mode of replication and the DNA packaging and export functions. Once inside the cells, the double-stranded DNA replicates and produces both new double-stranded circles and single-stranded circles. Single-stranded circles are packaged into phage coats and secreted into the medium without lysis of the host cell. Because only the (+) strand is packaged efficiently, only foreign DNA that is in the same 5' -> 3' orientation as the phage (+) strand origin will be packaged. Methods for the use of filamentous phage vectors are well known in the art and can be found, for example, in Ausubel et al., Sbσrt Protocols in Molecular Biology, 3rd ed., John Wiley & Sons, pp. 1-24-1-27, 1995 and Messing, New Ml 3 Vectors for Cloning in Methods in Enzymology, 101 :20-79, 1983. Vectors for plant transformation have been reviewed in Rodriguez et al. (1988) Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston; Glick et al. (1993) Methods in Plant Molecular Biology and Biotechnology CRC Press, Boca Raton, Fla; and Cray (1993) In Plant Molecular Biology Labfax, Hames and Rickwood, Eds., BIOS Scientific Publishers Limited, Oxford, UK. In addition, any other vector that is replicable and viable in the host may be used. The recombinant polynucleotide may be inserted into the vector by a variety of methods. In the most common method, the sequence is inserted into an appropriate restriction endonuclease site(s) using procedures commonly known to those skilled in the art and detailed in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, (1989) and Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons (1995).

Expression and cloning vectors can and usually do contain a selection gene or selection marker. Typically, this gene encodes a protein necessary for the survival or growth of the host cell transformed with the vector. Examples of suitable selection markers include dihydrofolate reductase (DHFR) or neomycin resistance for eukaryotic cells and tetracycline or ampicillin resistance for E. coli. Selection markers in plants include bleomycin, gentamycin, glyphosate, hygromycin, kanamycin, methotrexate, phleomycin, phosphinotricin, spectinomycin, dtreptomycin, sulfonamide and sulfonylureas. Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Laboratory Press, 1995, p. 39.

The selection marker can have its own promoter so that expression of the marker occurs independent of the suppressor construct. The marker promoter can be either a constitutitive or an inducible promoter. Common promoters used include, but are not limited to, LTR or SV40 promoter, the E. coli lac or tip promoters, and the phage lambda PL promoter. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, (1989) and Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons (1995). In plants, often-used constitutive promoters include the CaMV 35S promoter (Odell et al. (1985) Nature 313: 810), the enhanced CaMV 35S promoter, the Figwort Mosaic Virus (FMV) promoter (Richins et al. (1987) NAR 20: 8451), the mannopine synthase (mas) promoter, the nopaline synthase (nos) promoter, and the octopine synthase (ocs) promoter. Useful inducible plant promoters include heat-shock promoters (Ou-Lee et al. (1986) Proc. Natl Acad. Sci. USA 83: 6815; Ainley et al. (1990) Plant Mol. Biol. 14: 949), a nitrate-inducible promoter derived from the spinach nitrite reductase gene (Back et al. (1991) Plant Mol. Biol. 17: 9), hormone-inducible promoters (Yamaguchi-Shinozaki et al. (1990) Plant Mol Biol 15: 905; Kares et al. (1990) Plant Mol. Biol. 15: 905), and light-inducible promoters associated with the small subunit of RuBP carboxylase and LHCP gene families (Kuhlemeier et al. (1989) Plant Cell 1: 471; Feinbaum et al. (1991) Mol. Gen. Genet. 226: 449; Weisshaar et al. (1991) EMBOJ. 10: 1777; Lam and Chua (1990) Science 248: 471; Castresana et al. (1988) EMBOJ. 7: 1929; Schulze-Lefert et al. (1989) EMBO J. 8: 651). Alternatively, the recombinant polynucleotide of the present invention may be part of an expression cassette that comprises, operably linked in the 5' to 3' direction, a promoter inducible by nematode infection, a polynucleotide, expression of which suppresses FGAM synthase activity, and a transcriptional termination signal sequence functional in a host cell. In addition to being induced by nematode infection, the promoter can be a cell- or tissue-specific promoter. The expression cassette can further comprise a nucleotide sequence encoding a selectable marker. As discussed previously, the selection marker can have its own promoter, which can in turn can be either a constitutive or an induced promoter.

The recombinant polynucleotides, vectors or expression cassettes of the present invention can be used to transform host cells to produce plants having nematode resistance. The level of resistance need not be absolute, but will in general be sufficient to provide an agricultural or economical advantage to the plant. In one embodiment, the degree of resistance is such than plants contain 0 to 3 cysts per plant using the screening procedures described herein.

Plants encompassed by the present invention can take many forms. The plants may be chimeras or mosaics of transformed cells and non-transformed cells; the plants may be clonal transformants such that all cells in the plant contain the recombinant polynucleotide sequence of the present invention. The plants may also comprise graphs of transformed and untransformed tissues. Also encompassed by the present invention are uniform populations of plants containing the recombinant polynucleotides of the present invention. The transformed plants may be propagated by a number of methods, including, but not limited to, clonal propagation or classical breeding techniques. For example, a first generation of heterozygous transformed plants may be selfed to produce a homozygous second generation which is further propagated by classical breeding techniques. To assist in breeding, the polynucleotide of the present invention can also comprise a dominant selectable marker, many of which are known in the art.

A variety of different methods can be employed to introduce recombinant polynucleotides into plant protoplasts, cells, callus tissue, leaf discs, meristems, etc., to generate transgenic plants. These methods include, for example, Agrobacterium-meάiateά transformation, particle gun delivery, microinjection, electroporation, polyethylene glycol-mediated protoplast transformation, liposome-mediated transformation, etc. (Potrykus Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 205, 1991; Maliga et al., Methods in Plant Molecular Biology, Cold Spring Laboratory Press, 1995).

In general, transgenic plants comprising cells containing and expressing recombinant polynucleotides conferring nematode resistance described herein can be produced by transforming plant cells with a DNA construct as described above via any of the foregoing methods; selecting plant cells that have been transformed on a selective medium; regenerating plant cells that have been transformed to produce differentiated plants; and selecting a transformed plant that possesses nematode resistance by the methods described herein. Specific methods for transforming a wide variety of dicots and obtaining transgenic plants are well documented in the literature (Gasser and Fraley (1989) Science 244: 1293; Fisk and Dandekar (1993) Scientia Horticulturae 55: 5; Christou (1994) Agro Food Industry Hi Tech, p. 17; and the references cited therein).

Examples of successful transformation and plant regeneration in monocots are as follows: asparagus (Asparagus officinalis; Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84: 5345); barley (Hordeum vulgarae; Wan and Lemaux (1994) Plant Physiol. 104: 37); maize (Zea mays; Rhodes et al. (1988) Science 240: 204; Gordon-Kamm et al. (1990) Plant Cell 2: 603; Fromm et al. (1990) Bio/Technology 8: 833; Koziel et al. (1993)

Bio/Technology 11: 194); oats (Avena sativa; Somers et al. (1992) Bio/Technology 10: 1589); orchardgrass (Dactylis glomerata; Horn et al. (1988) Plant Cell Rep. 7: 469); rice (Oryza sativa, including indica and japonica varieties; Toriyama et al. (1988) Bio/Technology 6: 10; Zhang et al. (1988) Plant Cell Rep. 7: 379; Luo and Wu (1988) Plant Mol Biol. Rep. 6: 165; Zhang and Wu (1988) Theor. Appl. Genet. 76: 835; Christou et al. (1991) Bio/Technology 9: 957); rye (Secale cereale; De la Pena et al. (1987) Nαtwre 325: 274); sorghum (Sorghum bicolor; Cassas et al. (1993) Proc. Natl. Acad. Sci. USA 90: 11212); sugar cane (Saccharum spp.; Bower and Birch (1992) Plant J. 2: 409); tall fescue (Festuca arundinacea; Wang et al. (1992) Bio/Technology 10: 691); turfgrass (Agrostis palustris; Zhong et al. (1993) Plant Cell Rep. 13: 1); and wheat (Triticum aestivum; Vasil et al. (1992) Bio/Technology 10: 667; Weeks et al. (1993) Plant Physiol. 102: 1077; Becker et al. (1994) Plant J. 5: 299). Of course, gametes, seeds, embryos, progeny and hybrids of plants containing the recombinant polynucleotides of the present invention produced by traditional breeding methods are also included within the scope of the present invention.

Also included are plants containing recombinant polynucleotides of the present invention which are apomictic. Apomixis is a genetically controlled method of reproduction in plants where the embryo is formed without union of an egg and a sperm. There are three basic types of apomictic reproduction: 1) apospory where the embryo develops from a chromosomally unreduced egg in an embryo sac derived from the nucellus, 2) diplospory where the embryo develops from an unreduced egg in an embryo sac derived from the megaspore mother cell, and 3) adventitious embryony where the embryo develops directly from a somatic cell. In most forms of apomixis, psuedogamy or fertilization of the polar nuclei to produce endosperm is necessary for seed viability. In apospory, a "nurse" cultivar can be used as a pollen source for endosperm formation in seeds. The nurse cultivar does not affect the genetics of the aposporous apomictic cultivar since the unreduced egg of the cultivar develops parthenogenetically, but makes possible endosperm production. Apomixis is economically important, especially in transgenic plants, because it causes any genotype, no matter how heterozygous, to breed true. Thus, with apomictic reproduction, heterozygous transgenic plants can maintain their genetic fidelity throughout repeated life cycles. Methods for the production of apomictic plants are known in the art. See, U.S. Patent No. 5,811,636 and references cited therein. The present invention is useful for conferring resistance against numerous nematodes and in particular cyst forming nematodes. These include such cyst forming nematodes as Globodera pallida and Globodera rostochiensis (potato cyst nematodes); Heterodera glycines (soybean cyst nematode); Heterodera shachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode); Heterodera carotae (carrot cyst nematode),

Heterodera oryzae (rice cyst nematode); and Gobodera tabacum (tobacco cyst nematode). Plants which may be protected by the practice of the present invention include, but are not limited to, tomatoes, potatoes, soybeans, sugar beets, rape, wheat, oats, barely, rice, carrots, brassicas and tobacco. EXAMPLES

Example 1 Biological Materials and Inoculation Procedures for Cytological Examinations The nematode inbred strain VL1 was provided by Dr. T. Niblack (University of Missouri), and seeds of soybean lines PI 88287 and PI 89008 were provided by Dr. R. Nelson (University of Illinois). Plants were grown in growth chambers at 25 °C under fluorescent light illumination with a 14-hr/10-hr (day /night) schedule. Seven day-old seedlings (four per planting) were transferred to 500 cm³ of a mixture of sterilized soil and sand (1:1) and inoculated with 10 cracked cysts of nematode strain VL1. An infection cycle was completed approximately four to five weeks after inoculation (Table 1). The infection site was localized by identifying sites on the root's surface at which the infecting female protrudes. For mRNA and protein preparations, and for fixation and resin embedding of infected root material, roots were inspected under the stereo microscope and the protruding female was carefully removed from the infection site. The infection sites were then carefully dissected from the root, and either frozen in liquid nitrogen or submerged into fixative, respectively. Although this was an effective means of delimiting infection sites, it limited dissection to only middle to late stages of syncytium development. For controls, sections of uninfected roots of the same age were harvested in parallel.

Example 2

Specimen Preparation for Cytology To confirm that observations made using susceptible host PI 89008 infected with inbred VL1 would be representative of a normal cyst nematode infection process, a cytological study during infection was conducted. Plant tissues were cut into small blocks of about 1 mm length and immediately fixed in phosphate buffer, pH 7.3, containing ImM 3-maleidobenzoic acid N-hydroxysuccinimide ester, 0.5% Triton X-100, and 50mM EGTA for one hour, followed by fixation in phosphate buffer containing 3% glutaraldehyde. After dehydration in an ethanol series, embedding was performed in LR White or Quetol resins (Electron Microscopy Sciences, Fort Washington, PA) according to the manufacturer's instructions. Thick sections (1 μm) were cut with a glass knife on a Reichert-Jung Ultracut-E microtome (Vienna, Austria). The sections were mounted on glass slides, dried on a hot plate and stained with 0.5% of each methylene blue and azure II in 0.5% sodium borate. The sections were cover slipped and viewed under a Microphot- FX light microscope system (Nikon, Japan). Figure 1 (panels A to G) demonstrates a normal development of the feeding site.

In a compatible interaction between susceptible line PI 89008 and VL1, the process involved the proliferation of primary syncytium initials, radiating out with wall dissolution of adjacent cells, the formation of a highly vacuolar cytoplasm (panels B to D) and the development of cell wall ingrowths typical of transfer cells (Gunning and Steer, 1996) alongside xylem elements (panel D). Upon completion of the nematode life cycle the cytoplasm of the syncytium-component cells deteriorates (panels E, F), eventually leaving nearly empty cells with cell wall stubs and ingrowths (panel G). In contrast, nematode penetration of the root of the resistant line, PI 88287, only occasionally resulted in the formation of initial syncytium structures, and in the development of a syncytium that degenerated before completion of a nematode life cycle as indicated by the detachment of the cytoplasm from the cell wall (data not shown). Characteristically, no development of cell wall ingrowths was observed. The cytological observations resemble earlier descriptions of the resistant reaction in different genetic systems (Acedo et al., 1984; Mahalingam and Skorupska, 1996; Kim and Riggs, 1992).

Example 3

Soybean Clone Generation and Selection Infected and uninfected lateral root tissues were collected from inoculated and non- inoculated seedlings, respectively. For infected roots, tissue was sampled from roots at 6 to 14 days after inoculation and pooled for mRNA preparation (Sambrook et al., Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, 1989). The procedures for cDNA subtraction and differential display, using tissues from infected versus uninfected (driver) susceptible line PI 89008, were as follows.

The protocol for differential display analysis was adapted from that of Liang et al (1995) and Liang and Pardee (1992). Total RNA was extracted from uninfected and VL1- infected roots, as outlined in Gelvin et al. (1994). Differential display protocol bands of interest were excised and eluted from the gel. These cDNAs were reamplified with the same primer combinations (primers contained restriction endonuclease designation [EcoRI] sites) used in the differential display which allowed for cloning into the pGΕM- 3Zf vector (Promega, Madison, WI).

The cDNA subtraction protocol followed procedures described in Ausubel et al., (1988) with specifications according to Wilson et al., (1994). Briefly, RNA prepared from VL1 -infected (tracer) and uninfected (driver) tissues was reverse transcribed to cDNA and ligated to EcoRI adapters. After subtraction procedures, the enriched cDNAs were ligated into the EcoRI site of the pGΕM-3Zf vector.

Plasmids from the clones retrieved through the aforementioned procedures were isolated, and the inserts were retrieved for hybridization to soybean genomic DNA by digestion with EcoRI, according to standard procedures (Sambrook et al., Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, 1989). Clones were subjected to sequence analysis by the fluorescent labeled primer cycle sequencing kit with 7-deaza- dGTP (Amersham International, Little Chalfont, Buckinghamshire, U.K.) in an ALFexpress automated sequencer (Pharmacia Biotech, Uppsala, Sweden). Clone identities were determined by blasting sequences against the databases at GenBank (http://www.ncbi.nlm.nih.gov/) and SWISS-PROT (http://www.ebi.ac.uk/). Vector sequence contamination was detected with VecScreen.

Example 4 RNA Preparation and Quantification for RT-PCR

For isolating RNA from infected tissues, areas of PI 89008 roots with heavy SCN infestations were chosen. Total RNA was then extracted from these roots by the RNeasy Plant Mini Kit (Qiagen, Chatsworth, CA) and subjected to DNase treatment using RNase free DNase (Stratagene, La Jolla , CA). The resulting RNA was subjected to RNA-PCR with specific gene primers to ensure the absence of DNA template and then quantitated in a spectrophotometer (Beckman Coulter, Fullerton , CA). For the RT-PCR assay, lOOng of RNA prepared from uninfected and infected tissues was taken in RNase-free microtubes and dried in a vaccum concentrater (Labconco, Kansas City, MO), after which the RNA was resuspended in equal volumes of RNase-free water to achieve equal concentrations of RNA per micro liter. These RNA templates were then used in the RT-PCR reaction described below. Example 5 mRNA Preparation and Quantification for RT-PCR Total leaf RNA was isolated by the RNeasy Plant Mini Kit (Qiagen, Chatsworth, CA) followed by mRNA isolation using the PolyATtract® System 1000 (Promega, Madison, WT). Root mRNA was isolated directly from uninfected and infected tissues (5 mg starting material) of soybean susceptible line PI 89008 with the PolyATtract® System 1000 (Promega, Madison, WI). Direct isolation of mRNA from root tissue was carried out in order to minimize the loss of template. Spectrophotometric quantification was not possible with root mRNA samples due to the minute quantities of mRNA isolated. Hence, quantification was carried out by a comparative dilution series (25 to 25,000 fold) using unknown starting amounts of root mRNA and known amounts of leaf mRNA subjected to RT-PCR. Specific primers for soybean 18S rRNA (see Table 1) were used in the PCR reaction, taking advantage of rRNA as a regular contaminant in mRNA samples purified with Oligo(dT) to measure mRNA concentrations. This approach was adopted from the Relative RT-PCR protocol by Ambion (Austin, TX). After RT-PCR (for conditions, see below) the entire reaction was run out on a 1.2% agarose gel, stained with ethidium bromide, photographed, and the image saved digitally (Gel Print 2000i, BioPhotonics Corp., USA). The image was then analyzed by the computer program GPTools (BioPhotonics Corp., USA) which quantifies pixel intensities of each band. According to quantification results, mRNA volumes were adjusted in the RT reaction until equal intensity of the bands produced by PCR for the samples being compared was achieved. This titration allowed nearly equal amounts of template to be subjected to the first strand cDNA reaction. This approach is similar in its experimental basis to the Relative RT-PCR protocol of Ambion, Inc. (Austin, TX) wherein the signal of 18S rRNA is attenuated by adjusting the ratio of non-extendable 18S primers to normal 18S rRNA primers to achieve equal signal intensities. The dilution approach utilized applies the same principle in allowing equal amplification of the control 18S rRNA sequences from the samples under consideration.

Example 6 Oligonucleotide Primer Design for RT-PCR

Based on sequence information, primers were designed to produce amplification products in the range of 250 to 450 base pairs by RT-PCR. Primers had an overall G+C content of 60 to 65%, and ended with a mandatory G or C on both ends. The calculated T_m (at 50mM salt concentration and 50nM primer concentration) was approximately 62 °C (Breslauer et al., 1986). For genes selected based on reports of enhanced expression in other plant-pathogen interactions (Cramer et al., 1993; Laxalt et al., 1996; Niebel et al., 1995 and 1996; VanderEycken et al, 1996; Wang et al., 1995; Yoshikawa et al., 1990), sequence information was retrieved from GenBank and corresponding primers were designed. Primers discriminating between members of the same gene family were designed according to the most divergent gene sections, chosen on the basis of a prior multiple amino acid sequence alignment using the program CLUSTAL (Higgins and Sharp, 1988). For cytosolic GAPDH and HMG CoA reductase, no soybean sequence data were available. Therefore, conservative regions of homologous plant genes were first identified by using the program CLUSTAL (Higgins and Sharp, 1988). Degenerate primers were then designed for PCR displaying the structure: 5'> GCHAARGCHGTKGGWAARGT <3' SEQ ID NO: 5 and 5'> TAWCCCCAYTCRTTRTCRTACCA <3' SEQ ID NO: 6 for NAPDH, and 5'> CCWGCHGCHGTKAAYTGGAT <3' SEQ ID NO: 7 and 5'> GCWCCYTTMACWCC <3' SEQ ID NO: 8 for HMG CoA reductase. Upon sequence verification of the cloned genomic DNA, non-degenerate, matching primers were synthesized to be used in RT-PCR experiments.

Example 7

Reverse Transcription of RNA/mRNA and Polvmerase Chain Reaction (RT-PCR For first strand cDNA synthesis, the following components were included in a 25 μl reaction: 400μM of each dNTP, reverse transcription buffer (50mM Tris-HCl pH 8.3, 50mM KC1, lOmM MgCl₂, 0.5mM spermidine and lOmM DTT), 40 μM random primer (Boehringer Mannheim, Indianapolis, IN), template RNA or mRNA and DEPC-treated water. 2.5 units of AMV reverse transcriptase (Promega, Madison, WI, or Boehringer Mannheim, Indianapolis, IN) were added to the reaction last and the mixture was incubated at 55°C for 30 minutes in a thermal cycler with heated lid (MJ Research). This first strand cDNA template was then used in a normal PCR reaction. A 25-μl PCR reaction contained the following components: 125 μM dNTPs, Taq

DNA polymerase reaction buffer (50mM KC1, lOmM Tris-HCl pH 9.0, 0.1% Triton X- 100), lOOnM of specific primers (for primer design, see above), 2.5 units of Taq DNA polymerase and 2.5μl of first strand cDNA template from the above reaction. Reactions were performed in thin-walled tubes in an MJ Research PTC 100 programmable thermal cycler (Watertown, MA). The PCR cycling profile was 94°C for 2 minutes for initial denaturation followed by 35(RNA template)-40(mRNA template) cycles of 94°C for 30s, 55°C for 30s, 72°C for 1 minute, ending with a final extension at 72°C for 7 minutes. The entire reaction was subjected to electrophoresis in 1.2% agarose, stained with ethidium bromide and photographed. The positive control 18S rRNA template reaction was run each time with all primer sets used. Each assay was run with negative controls (water in place of mRNA), and the obtained product size was evaluated to match the expected size according to available nucleotide sequence data. A single RT reaction was used to generate template for all the PCR reactions required to test candidate genes together with control sequences for a single experiment; this eliminated variation inherent in multiple RT reactions. All PCR amplification results were confirmed a minimum of four times from templates obtained independently. The possibility of amplification from genomic DNA contamination was ruled out by treatment of RNA-mRNA templates with DNase (RNase-free). As an added precaution, primers were designed for particular genes (EF-lα and HMGR) to span intron sequences and allow amplification of different sized products from cDNA versus genomic DNA templates (data not shown). For preparation of gene mapping probes, the RT-PCR products were eluted from the gel using GenElute columns (Supelco, Bellefonte, PA), re-amplified with the same pair of primers used in its initial amplification, and run in a preparative agarose gel. The amplified fragment was retrieved from the gel in the same way, further purified with the Wizard DNA Purification Kit (Promega, Madison, WI) and quantified spectrophotometrically.

Example 8 Plant Populations for Genetic Analysis Reciprocal crosses were made between PI 89008 and PI 88287. Each cross was harvested individually, and crosses that produced two-seeded and three-seeded pods were analyzed genetically. One seed from each of 10 pods was grown in 18-cm pots containing organic soil mix to produce the F₂ generation. The plants were grown in the greenhouse. DNA samples from each F, plant were evaluated by RFLP analysis to ensure that they were hybrids. A second seed from these pods was used in nematode screening experiments.

Example 9 Gene Mapping

Two soybean populations were used to map the selected cDNAs. The first was a population of 57 F₂ individuals derived from the cross of a G. max parent, A81 356022, and a G. soja parent, PI 468.916 (Shoemaker et al., 1996). The second, a population of 100 recombinant inbred lines (RIL) in the F_6;7 generation, was derived from a cross of BSR 101 and PI 437.654 (Baltazar and Mansur, 1992). PI 437.654 is resistant to all known races of soybean cyst nematode in the United States (Myers and Anand, 1991). Resistance to SCN in this population was mapped to Linkage Groups A (near the / locus), G and M (Webb et al., 1995).

Radioactively labeled RT-PCR products were used as probes against parental DNA blots of the two populations. Parental DNA of both populations was digested with Dral, EcoRI, EcoRV, Haelϊl, H dIII, and Taql. Parental DNAs from the F₂ population were, in addition, digested with Accl, Alul, BamHl, Bell, Hhal, Hnfl, Rsal, and Sspl. Once polymorphisms were detected, the radioactively labeled probes were hybridized against the population DNAs. In the RIL population, DNA was extracted from at least 30 seedlings per line. The DNA extraction, blotting, hybridization and autoradiography methods used followed Keim et al. (1990).

The computer program MAPMAKΕR 3.0 (Lander et al., 1987) was used to map the cDNAs in the two populations. The defaults of a LOD score of 3.0, Ηaldane estimation (Ηaldane, 1919), and a maximum recombination of 30 percent were used. Linkage groups were identified according to the USDA-ARS public map (Shoemaker et al., 1996), and gene orders were assigned. The "compare", "try" and "ripple" commands were used to include a locus on a pre-existing linkage group. Example 10 Nematode Pathotvpe Testing To characterize the pattern of VLl infection, a replicated standard race test was performed (Golden et al., 1970). For each test, three plants of differential host lines Lee, Picket, Peking, PI 88788, and PI 90763 were inoculated. Each differential host was scored as susceptible (+) or resistant (-) if the number of mature females was above or below 10%, respectively, the number of mature females recovered from the susceptible control Lee. Soybean cyst nematode differential host lines were provided by the University of Illinois germplasm bank and Dr. J. Wilcox, Purdue University. Based on testing, it was concluded that VLl behaves as a pathotype of "race" 5.

Example 11 Assay for Soybean Reaction to VLl Inoculation in Segregating Populations Nematode cysts were collected from the root and soil using the procedure described by Faghihi et al., (1986). Collected and rinsed cysts were crushed using a rubber stopper and passed through a 100-mesh sieve nested in a 400-mesh sieve. Intact cysts were collected on the 100-mesh sieve and the crushing repeated. The inoculum was diluted to 4500 eggs per ml.

Seven F_[ plants (second seed from two-seeded or three-seeded pods) and 165 F₂ seeds were screened with VLl . Seeds were germinated in sand, and roots of seven-day old seedlings were inoculated with 4500 VLl eggs/plant. Plants were grown in 7 cm x 10 cm pots containing a 3:1 sand: soil mixture. Pots were maintained in a growth chamber with a 14 hour photoperiod and were fertilized (Masterblend 20: 10:20) weekly. After 28 days, cysts were collected as previously described and counted under a dissecting microscope.

Example 12 Modulation of Root Gene Expression in Susceptible Host PI 89008 in

Response to Infection by Nematode Inbred Strain VLl The dramatic cytological changes induced in the soybean root upon infection by the cyst nematode imply that several plant genes are recruited by the nematode to act in concert to facilitate syncytium development. To test this hypothesis, cDNA subtraction, differential display, and empirical selection of several gene candidates produced a collection of plant gene clones that were further examined for response to VLl infection in the susceptible host PI 89008. Several gene candidates were selected based on previous reports (Cramer et al., 1993; Laxalt et al., 1996; Niebel et al., 1995 and 1996; VanderEycken et al., 1996; Wang et al., 1995; Yoshikawa et al., 1990). Gene expression was assayed by reverse transcription of RNA/mRNA followed by polymerase chain reaction (RT-PCR). The responsive genes selected are listed by their identity and origin in Tables 1 and 2.

For preparation of mRNA used to construct the cDNA subtraction library and carry out the differential display procedures, it was necessary to identify those root tissues most densely populated with feeding sites, and to estimate the timing of syncytium development. Inoculations were made with prepared eggs rather than stage J2 nematode juveniles, so infections were not well synchronized. As shown in Table 3, however, infection results were highly reproducible. Since interest focused on the changes of the soybean root gene expression pattern defining the sedentary accommodation of females in the root (around day 10 after infection, Table 3) plant material of PI 89008 from days 3 to 21 after infection was pooled for RNA/mRNA isolation. For control mRNA preparation, noninfected PI 89008 root material of the same developmental stages was collected and pooled.

To confirm the enhanced expression by RT-PCR, careful dissection of the regions encompassing the feeding sites was carried out, producing extremely small tissue samples (5mg per collection). Subsequent preparation of poly A+ RNA resulted in a very low mRNA yield. Thus, a quantitative assay for template concentration was developed with RT-PCR based on known concentrations of leaf mRNA, as demonstrated in Figure 2, panel A. RT-PCR assays were carried out using mRNA templates from infected and uninfected root of the susceptible line PI 89008, and leaf tissues. Results of the RT-PCR assay, with regard to enhanced amplification upon infection, are summarized in Tables 1 and 2, with sample RT-PCR results demonstrated in Figure 2, panel B. The results shown in Figure 2, panel B were independently replicated in subsequent RT-PCR assays using spectrophotometrically quantified RNA templates (data not shown) prepared as mentioned in methods and materials. Clones which were regarded as enhanced in their expression had to show differential expression using both templates. RT-PCR assay results indicated that several clones represent genes with enhanced expression in the root samples upon infection with nematode strain VLl . Although many of these were unidentifiable based on sequence analysis and database searches (data not shown), other clones selected for RT-PCR could be identified based on DNA sequence homologies (Tables 1 and 2).

Example 13 Genetics and Mapping of Susceptibility in PI 88287 to Nematode Inbred VLl Gene expression data, supported by cytological observations, suggested that several plant genes are influenced in their expression during nematode infection. Therefore, it was appropriate to investigate further the genetics of plant-nematode interaction in this system, which was done in two ways. First, the genetics of soybean susceptibility to nematode inbred strain VLl was examined providing a tentative location of a gene conferring susceptibility on the public soybean map, and second, the map locations of several of the soybean genes responsive to nematode infection were determined.

Genetic characterization of the inbred nematode strain VLl was limited to examining its ability to establish a compatible interaction on several host differentials commonly used for the assignment of infection pathotype (Golden et al., 1970). Table 4 shows the results of infection with VLl inbred strain on host differentials, resulting in the conclusion that VLl behaves as a pathotype of "race" 5.

Using VLl, a single gene in PI 88287 was identified that confers susceptibility to cyst nematode. In the VLl screening procedure, susceptible and resistant classes were distinct as evidenced in Table 3 and Table 5, with resistance classified as a zero to three- cyst phenotype. An F, progeny from a cross between PI 89008 x PI 88287 produced a wider range of cysts per plant than did the parents under controlled growth conditions (Table 5). Despite this variation, the F, population was susceptible to VLl.

The number of cysts per plant varied among the susceptible class in the F₂ population, although the data best fit a 1:3 model (resistant : susceptible) of a single dominant gene for cyst nematode susceptibility (Table 5). These results parallel Luedder's (1987) conclusions. A PI 89008 x PI 88287 population of 55 F₂ plants was used to map the susceptibility locus based on linkage associations with known markers on the public soybean map. The population segregated 40:15 (susceptible : resistant) (χ _3:1=0.148). Restriction endonuclease digestion of genomic DNAs with EαmHl enzyme allowed the detection of a dominant (45:10, χ² _3:1=1.36) DNA polymorphism using, as probe, marker B 053 from linkage group G (Shoemaker et al., 1996). The polymorphic fragment demonstrated linkage (χ² _9:3:3:ι⁼28.3, P<0.01) with the susceptible phenotype in segregating F₂ progeny. Of the 55 plants tested, five appeared recombinant for the marker. It was not feasible to further resolve the map position of the susceptibility locus in this mapping population because of lack of DNA polymorphisms for other markers in the vicinity

(Bngl22, C006V; ten enzymes tested). However, linkage of the SCN resistance gene to this map location was further confirmed by the identification of a R-APD marker linked to the susceptibility locus. This marker (SCN_rad) also mapped to the region encompassing marker B_053 (Figure 3B). Linkage detected with marker B_053 and SCN_rad allows the map position of the susceptibility locus to be tentatively assigned to linkage group G (see Figure 3). This is the vicinity in which a major cyst nematode resistance quantitative trait locus (QTL) has been identified in two other studies using multiple sources of resistance (Concibido et al., 1996b; Webb et al., 1995).

Example 14 Map Locations of 8 SCN-responsive Loci in Soybean

Several QTLs have been identified in association with resistance to cyst nematode in soybean (Concibido et al., 1994; 1996a; 1996b; 1997; Chang et al., 1997; Vierling et al., 1996; Webb et al., 1995). Therefore, it was appropriate to determine whether any of the genes identified to be responsive to nematode infection reside within a map location associated with nematode infection. In this study, eight of the SCN-responsive loci were placed on the public soybean map (Shoemaker et al., 1996).

Restriction enzymes used to detect polymorphisms in the F₂ and recombinant inbred (RIL) population were Acc , BamTTl, Dral, EcoRI, EcoRV, Hαelll, Hhd, Hmdlll, Sspl and Taql. In the F₂ population derived from A81-356022 and PI 468.916 (Shoemaker et al., 1996), cDNAs were mapped to ten linkage groups as shown in Figure 3 A. In the RIL population, derived from BSR 101 and PI 437.654 (Baltazar and Mansur 1992), cDNAs mapped to five linkage groups (Figure 3). Members of the gene family elongation factor lα (EF-lα) and HMG CoA reductase mapped to three and five linkage groups (Al, L, Q, and Cl, Dl, I, K, P), respectively, in the F₂ population. No polymorphisms were detected with these cDNAs in the RIL population. However, two of the cDNAs, glucanase (U08405) and catalase, were mapped only in the RIL population (linkage groups J and B2, respectively). Three of the cDNAs (LEA14, cyclin and heat shock protein [HSP70]) were mapped in both populations (linkage groups G, C2, Dl, respectively). FGAM synthase mapped to linkage group G in both populations. EF-lα elongation factor was polymorphic in the RIL population with the restriction enzyme EcoRV but did not link to any known linkage group.

Four cDNAs mapped within the vicinity of previously reported SCN QTL markers. ΕF-lcc elongation factor positioned 5.8 cM from SCN QTL marker A 023 on Linkage Group L (Concibido et al., 1996a) in the F₂ population. LΕA14 was 30.0 cM from SCN QTL marker A 378 on Linkage Group G (Concibido et al., 1997) in both the F₂ and RIL populations. Glucanase (U08405) was 39.6 cM away from SCN QTL marker locus B 032 on Linkage Group J (Concibido et al., 1994, 1996a, 1997) in the RIL population. FGAM synthase was mapped to a <3.0 cM interval flanked by markers Bng_122E and B_053T at the top of linkage group G (Figure 3B). Interestingly, the major soybean SCN resistance QTL (rhgl locus) also maps to the same interval on linkage group G (Mudge et al., 1997). Four of the cDNAs mapped in regions known to contain resistance-gene analogs

(RGAs, Kanazin et al., 1996). EF-lα co-segregated with RGA7 on Linkage Group L in the F₂ population. One locus of HMG CoA reductase on Linkage Group P in the F₂ population was located 5.7 cM from RGAδ. Glucanase (U08405) was 6.7 cM from RGA3 on Linkage Group J in the RIL population, and HSP70 was 10.7 cM from RGAlf on Linkage Group Dl in the F₂ population (data not shown).

Conclusion

In light of the detailed description of the invention and the examples presented above, it can be appreciated that the several aspects of the invention are achieved.

It is to be understood that the present invention has been described in detail by way of illustration and example in order to acquaint others skilled in the art with the invention, its principles, and its practical application. Particular formulations and processes of the present invention are not limited to the descriptions of the specific embodiments presented, but rather the descriptions and examples should be viewed in terms of the claims that follow and their equivalents. While some of the examples and descriptions above include some conclusions about the way the invention may function, the inventors do not intend to be bound by those conclusions and functions, but put them forth only as possible explanations.

It is to be further understood that the specific embodiments of the present invention as set forth are not intended as being exhaustive or limiting of the invention, and that many alternatives, modifications, and variations will be apparent to those of ordinary skill in the art in light of the foregoing examples and detailed description. Accordingly, this invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and scope of the following claims.

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Table 1. Identified clones obtained by differential display and cDNA subtractive hybridization cDNA Identity Clone designation³ Database AC. #^b Oligonucleotide primers for

* Clones obtained by differential display (clone designation ending in D) and obtained from subtractive hybridization (clone designation ending in S). Among the nine clone one was clearly of nematode origin. Whereas _- tubulin showed an identical sequence to the available database sequence, both EF-1_ and HSP70 clones were divergent fro the described soybean sequences within the database. ^b GenBank/EMBL/DDBJ accession numbers. ^c RT-PCR results demonstrating enhanced expression (+) upon nematode infection of the susceptible host, no enhanced expression (-) or serving as internal control fo reaction and template concentrations (control). ^d Mapped as detailed in Figure 4.

Table 2. Selected soybean genes examined for enhanced expression upon nematode infection. cDNA Identity Clone designation³ Database#^b Oligonucleotide primers for RT-PCR RT-PCR Mapped*¹

results⁰

^{*, b} Clone designations and GenBank EMBL/DDBJ accession numbers, respectively. ^c RT-PCR results demonstrating enhanced expression (+) upon nematode infection of the susceptible host, no enhanced expression (-) or serving as internal control fo reaction and template concentrations (control). ^d + indicates mapped as detailed in Figure 4.

' Cytosolic GAPDH was equally expressed in mRNA samples from leaf, infected and uninfected root, in line with reports on its housekeeping function (Wang et al., 1995).

Claims

What is claimed is:

1. A method for conferring nematode resistance to a plant comprising, transforming a plant with a polynucleotide operatively linked to a promoter inducible by nematode infection wherein expression of said polynucleotide results in suppression of FGAM synthase activity.

2. The method of claim 1, wherein said nematode is a cyst forming nematode.

3. The method of claim 2, wherein said cyst forming nematode is selected from the group consisting of Globodera pallida, Globodera rostochiensis, Heterodera glycines, Heterodera shachtii, Heterodera avenae, Heterodera carotae, Heterodera oryzae, and Gobodera tabacum.

4. The method of claim 1, wherein said plant is selected from the group consisting of tomatoes, potatoes, soybeans, sugar beets, rape, wheat, oats, barely, rice, carrots, brassicas and tobacco.

5. The method of claim 1, wherein said plant is a soybean plant.

6. The method of claim 1 , wherein said promoter is a tissue specific promoter.

7. The method of claim 6, wherein said tissue specific promoter is a root specific promoter.

8. The method of claim 1, wherein said promoter is SEQ ID NO: 3 or SEQ ID NO: 4.

9. The method of claim 1, wherein said polynucleotide is SEQ ID NO: 1.

10. A method for conferring nematode resistance to a plant comprising transforming said plant with an expression cassette comprising, operatively linked in a 5' to 3' order a nematode infection inducible promoter, a polynucleotide, and a termination signal wherein expression of said polynucleotide results in suppression of FGAM synthase activity.

11. The method of claim 10, wherein said nematode is a cyst forming nematode.

12. The method of claim 11, wherein said cyst forming nematode is selected from the group consisting of Globodera pallida, Globodera rostochiensis, Heterodera glycines, Heterodera shachtii, Heterodera avenae, Heterodera carotae, Heterodera oryzae, and Gobodera tabacum.

13. The method of claim 10, wherein said plant is selected from the group consisting of tomatoes, potatoes, soybeans, sugar beets, rape, wheat, oats, barely, rice, carrots, brassicas and tobacco.

14. The method of claim 10, wherein said plant is a soybean plant.

15. The method of claim 10, wherein said promoter is a tissue specific promoter.

16. The method of claim 15, wherein said tissue specific promoter is a root specific promoter

17. The method of claim 10, wherein said promoter is SEQ ID NO: 3 or SEQ ED NO:

4.

18. The method of claim 10, wherein said polynucleotide is SEQ ID NO: 1 or a fragment thereof in an antisense orientation.

19. A vector comprising a polynucleotide operatively linked to a nematode infection inducible promoter wherein expression of said polynucleotide results in suppression of FGAM synthase activity.

20. A vector comprising an expression cassette said expression cassette comprising operatively linked in 5' to 3' order, a nematode infection inducible promoter, a polynucleotide expression of which results in suppression of FGAM synthase activity, and a termination signal.

21. The vector of claim 19 or 20, wherein said vector is an expression vector.

22. The vector of claim 19 or 20, wherein said polynucleotide is SEQ ID NO: 1 or a fragment thereof in an antisense orientation.

23. The vector of claim 19 or 20 wherein said promoter is SEQ J-D NO: 3 or SEQ ID NO: 4.

24. A host cell comprising the vector of claim 19 or 20.

25. The host of cell of claim 24, wherein said host cell is a plant cell.

26. A plant produced by the method of claim 1 or 10.

27. A plant comprising a polynucleotide operatively linked to a nematode infection inducible promoter wherein expression of said polynucleotide results in suppression of FGAM synthase activity.

28. The plant of claim 27, wherein said polynucleotide is SEQ ID NO: 1 or a fragment thereof in an antisense orientation.

29. The plant of claim 27, wherein said promoter is SEQ ID NO: 3 or SEQ ID NO: 4.

30. The plant of claim 27, wherein said plant has increased resistance to nematode infection when compared to the wild type.

31. The plant of claim 30, wherein said increased resistance is to a cyst forming nematode.

32. The plant of claim 31, wherein said cyst forming nematode is selected from the group consisting of Globodera pallida, Globodera rostochiensis, Heterodera glycines, Heterodera shachtii, Heterodera avenae, Heterodera carotae, Heterodera oryzae, and Gobodera tabacum.

33. The plant of claim 27, wherein said plant is chosen from the group consisting of tomatoes, potatoes, soybeans, sugar beets, rape, wheat, oats, barely, rice, carrots, brassicas and tobacco.

34. The plant of claim 27, wherein said plant is a soybean plant.

35. A seed produced by the plant of claim 27.

36. A progeny of the plant of claim 27.

37. A hybrid plant produced by crossing the plant of claim 27 with another plant.

38. A plant comprising an expression cassette said expression cassette comprising operatively linked in 5' to 3' order, a nematode infection inducible promoter, a polynucleotide, and a termination signal wherein expression of said polynucleotide results in suppression of FGAM synthase activity.

39. The plant of claim 38, wherein said plant has increased resistance to nematode infection when compared to the wild type.

40. The plant of claim 39, wherein said increased resistance is to a cyst forming nematode.

41. The plant of claim 40, wherein said cyst forming nematode is selected from the group consisting of Globodera pallida, Globodera rostochiensis, Heterodera glycines, Heterodera shachtii, Heterodera avenae, Heterodera carotae, Heterodera oryzae, and Gobodera tabacum.

42. The plant of claim 38, wherein said polynucleotide is SEQ ID NO: 1 or a fragment thereof in an antisense orientation.

43. The plant of claim 38, wherein said promoter is SEQ J-D NO: 3 or SEQ ID NO: 4.

44. The plant of claim 38, wherein said plant is selected from the group consisting of tomatoes, potatoes, soybeans, sugar beets, rape, wheat, oats, barely, rice, carrots, brassicas and tobacco.

45. The plant of claim 38, wherein said plant is a soybean plant.

46. A seed produced by the plant of claim 38.

47. A progeny of the plant of claim 38.

48. A hybrid plant produced by crossing the plant of claim 38 with another plant.

49. A method for determining a pathogen resistance gene comprising, obtaining an inbred strain of the pathogen; obtaining a pathogen resistant strain of the host organism; infecting the resistance host organism and a wild type, non-resistant host organism with the pathogen; obtaining RNA from the resistant and wild type organisms; and comparing the RNA from both organisms to determine differences in gene expression.

50. The method of claim 50, wherein said pathogen is a nematode.

51. The method of claim 50, wherein said host organism is a plant.

52. The method of claim 50, wherein said determination of differences in gene expression is by cDNA subtraction or differential display.

53. An isolated polynucleotide comprising SEQ ID NO: 1 or a fragment thereof.

54. An isolated polypeptide comprising the amino acid sequence encoded by SEQ ID NO: 1 or a fragment thereof.

55. An isolated polynucleotide comprising SEQ ID NO: 2 or a fragment thereof.

56. An isolated polypeptide comprising the amino acid sequence encoded by SEQ ID NO: 2 or a fragment thereof.

57. A vector comprising SEQ J-D NO: 1 or SEQ ID NO: 2.

58. A host cell transformed with the vector of claim 57.

59. An isolated nucleic acid sequence comprising SEQ ID NO: 3.

60. An isolated nucleic acid sequence comprising SEQ LD NO: 4.

61. A method for inducing expression of a polynucleotide in response to nematode infection comprising operatively linking in the 5' to 3' order, SEQ ID NO: 3 and said polynucleotide.

62. A method for inducing expression of a polynucleotide in response to nematode infection comprising operatively linking in 5' to 3' order, SEQ ID NO: 4 and said polynucleotide.

63. The method of claim 61 or 62 further comprising a termination signal located 3' to said polynucleotide.

64. The method of claim 61 or 62, wherein said expression is in a plant.

65. An expression cassette comprising operatively linked in 5' to 3* order, SEQ ID NO:3, a polynucleotide to be expressed, and a termination signal.

66. An expression cassette comprising operatively linked in 5' to 3' order, SEQ ID NO:4, a polynucleotide to be expressed, and a termination signal.