# Patent

## Title
Soybean FGAM synthase promoters useful in nematode control

## Abstract

The subject invention relates to nematode responsive domains obtained from soybean promoters for phosphoribosylformylglycinamidine ribonucleotide (FGAM) synthase paralogs. The nematode responsive domains can be used in promoters that are linked to heterologous DNA. Such constructs can be expressed in transfected or transformed soybean and used in the control of nematode infection of soybean.

## Background
GOVERNMENT INTEREST
The U.S. Government may have certain rights in this invention pursuant to United States Department of Agriculture Grant No. 96-35302-3385.


RELATEDNESS OF THE APPLICATION
The subject application claims the benefit of priority from U.S. Ser. No. 60/556,745, filed Mar. 26, 2004, which is incorporated herein in its entirety.
REFERENCE TO SEQUENCE LISTING
The present application incorporates by reference a file named:1231–221 Sequence Listing. ST25. txt including SEQ ID NO: 1 to SEQ ID NO: 20, provided in a computer readable form and paper copy. The sequence listing information recorded on the computer readable form is identical to the paper copy sequence listing.
FIELD OF THE INVENTION
The subject invention relates to nematode responsive domains from the soybean FGAM synthase gene, which can be useful in reducing parasite infection or infestation.
BACKGROUND OF THE INVENTION
The soybean cyst nematode (SCN) Heterodera glycines Ichinohe is considered the most economically debilitating disease-causing pathogen to affect soybean cultivation (Noel, G. R. (1992) in Riggs, R. D., Wrather, J. A. (eds) Biology and management of the soybean cyst nematode, APS Press, St. Paul, Minn., pp 8–10), causing losses of up to one billion dollars annually (Kim, D. G. et al. (1997) J. Nematol. 29:173–179). Several Hg types of SCN (Nieblack, T. L. et al. (2002) J. Nematol. 34:279–288) exist in the field (Riggs, R. D. et al. (1988) J. Nematol. 23:149–154) and several soybean genes that confer resistance have been identified. The most important of these genes have been mapped to linkage groups G and A2 of the soybean genetic map (Webb, D. M. et al (1995) Theor. Appl. Genet. 85:136–138; Concibido, V. C. et al. (1996) Theor. Appl. Genet. 93:234–241; and Meksem, K. et al. (2001) Theor. Appl. Genet. 103:710–718).
Several approaches have been undertaken to characterize nematode-responsive gene expression patterns within feeding sites of the soybean root. Changes in mRNA abundance were studied by in vitro translation to proteins (Hammond-Kossack, K. E. et al. (1989) Physiol. Mol. Plant Pathol. 37:339–354; Potenza, C. L. et al. (1996) J. Nematol. 28:475–484; and Oberschmidt, I. et al. (1996) Fourth annual meeting of the European union AIR-CAP on Mechanisms for resistance against plant parasitic nematodes, Toledo, Spain, p. 13). Subtractive hybridization of cDNA libraries prepared from nematode-infected and uninfected roots has yielded “infection-specific” clones. This approach has been utilized in tomato plants infected with root-knot nematodes (Van der Eycken, W. et al. (1996) Plant J. 9:45–54), and in potatoes infected with cyst nematodes (Niebel, A. et al. (1995) MPMI 8:371–378). Likewise, several PCR-based libraries have been constructed to permit the cloning of “giant cell-specific” transcripts (Wilson, M. A. et al. (1994) Phytopathol. 84:299–303; and Bird, D. M. et al. (1994) MPMI 7:419–424). Use of the differential display technique has yielded several interesting candidate genes in the Arabidopsis-Meloidogyne interaction (Vercauteren, I. et al. (2001) MPMI 14:288–299) and the soybean-SCN interaction (Hermsmeier, D. et al. (1998) MPMI 11:1258–1263). Promoter-GUS fusion (Opperman, C. H. et al. (1994) Science 263:221–223) and promoter trap (Barthels, N. et al. (1997) The Plant Cell 9:2119–2134; and Puzio, P. S. et al. (1998) Physiol. Mol. Plant Pathol. 53:177–193) approaches have also been implemented to identify nematode-responsive loci.
In a previous report (Vaghchhipawala, Z. E. et al. (2001) MPMI 14:42–54), we showed that several genes were up-regulated within the syncytium during colonization of the root by SCN. We determined the map locations of some of the soybean genes responsive to nematode infection by locating them on the public soybean map (Shoemaker, R. C. et al. (1996) in D. P. S. Verma and R. C. Shoemaker (eds) Biotechnology in Agriculture, No. 14, Soybean: genetics, Molecular Biology and Biotechnology, CAB International, Wallingford, Oxon, UK, pp. 37–56). A particularly interesting candidate was phosphoribosylformylglycinamidine ribonucleotide (FGAM) synthase. This gene mapped to the same 3.0-cM interval of Linkage Group G where the major soybean SCN resistance locus Rhg1 maps (Mudge, J. et al. (1997) Crop Sci. 37:1611–1615).
FGAM synthase was of interest because of its coincident location within the genomic interval containing Rhg1 and its up-regulated expression within the nematode feeding site. The enzyme FGAM synthase catalyzes the fifth step of the de novo purine biosynthetic pathway, effecting the ATP-dependent transfer of the glutamine amido group to the C-4 carbonyl of FGAR (5′-phosphoribosyl-N-formylglycinamide). To investigate this soybean gene further, we isolated and characterized two FGAM synthase loci. The two loci were highly similar in sequence. Analysis of the two copies revealed distinct functions and/or expression profiles during development and syncytium formation. As is described herein, the promoters of both FGAM synthase copies were found to contain novel nematode responsive domains that are active during syncytium formation.
SUMMARY OF THE INVENTION
The subject invention concerns the identification of soybean gene promoter sequences that contain nematode responsive domains. The nematode responsive domain is active during nematode establishment of a feeding site on the soybean, resulting in altered expression of downstream coding sequences.
As discussed herein, they soybean cyst nematode (SCN) is an economically debilitating disease-causing pathogen in soybean cultivation. Several soybean genes that confer resistance have been identified. One of the most important nematode resistance genes, rhg1, has been mapped to a distal region of MLG-G in soybean. A simplified genetic system to identify soybean genes with modified expression in response to SCN led to the identification of several genes within the nematode feeding sites (Vaghchhipawala et al. (2001) supra). The genes were mapped to reveal their linkage relationship to known QTLs associated with soybean cyst nematode (SCN) resistance. One candidate, a phosphoribosylformylglycinamidine (FGAM) synthase (EC# 6.3.5.3) gene, mapped to the same genomic interval as the major SCN resistance gene rhg1 within Linkage Group G. As is detailed herein, isolation of FGAM synthase from a soybean bacterial artificial chromosome (BAC) library revealed two highly homologous paralogs. The genes appeared to be well conserved from bacteria to humans. Promoter analysis of the two soybean homologs was carried out with the Arabidopsis thaliana-Heterodera schachtii system to investigate gene response to nematode feeding. As reported herein, the two promoters and their derived deletion constructions effected green fluorescent protein expression within nematode feeding sites. It was found that the 1.0-kbp promoter sequence immediately adjacent to the translation start site was sufficient to direct expression of GFP within syncytia at the feeding site. The observed expression of GFP within the feeding sites indicates that plant gene expression is redirected within feeding sites to benefit the parasitic nematode.
Thus, in one embodiment, the subject invention is a molecule that comprises a soybean promoter sequence that comprises a nematode responsive domain, i.e., a domain that is responsive to nematode establishment of a feeding site in the plant.
As is set forth in the Examples, the promoter sequence can comprise a sequence selected from the group consisting of soybean FGAM synthase Pr1-1.0 (nucleotides 1790–2483 of SEQ. ID NO. 2), Pr2-1.0 (nucleotides 1551–2547 of SEQ. ID NO. 1), Pr1-1.5 (nucleotides 1271–2483 of SEQ. ID NO. 2), Pr2-1.5 (nucleotides 991–2547 of SEQ. ID NO. 1), Pr1-2.5 (nucleotides 124–2483 of SEQ. ID NO. 2) and Pr2-2.5 (nucleotides 19-2547 of SEQ. ID NO. 1).
Further, the promoter sequence can be a sequence that has at least 50% homology with that of Pr1-1.0 or Pr2-1.0 of soybean FGAM synthase. With increasing preference, the promoter sequence has at least 60%, 70%, 80%, 90% or 95% homology to Pr1-1.0 or Pr2-1.0. To be encompassed within the scope of the subject invention, these variant promoter sequences must remain functional as nematode responsive domains.
It will be apparent that minor additions, deletions or substitutions can be made to Pr1-1.0 or Pr2-1.0, while retaining or perhaps enhancing the nematode responsive function. All of these variants are encompassed within the scope of the subject invention.
In another embodiment, the subject invention includes a molecule that is a promoter comprising the nematode responsive domain and a heterologous DNA operatively linked to the promoter. The heterologous DNA encodes a product that is disruptive of nematode attack. The disruptive product may be toxic to the plant cell or to the nematode.
The subject invention also includes a transfected plant (e.g., soybean) cell comprising the above-described molecule comprising the nematode responsive domain. It also includes transgenic plants comprising the transfected plant cells.
In another embodiment, the invention includes a method of reducing nematode infection of a plant (e.g., soybean) comprising transfecting plant cells of said plant with a vector comprising a promoter containing the nematode responsive domain and a heterologous DNA operatively linked to the promoter.
All references cited herein are incorporated in their entirety by reference.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is the nucleotide sequence and amino acid sequence for FGAM1 (SEQ. ID NOS. 1 and 2).
FIG. 1B is the nucleotide sequence and amino acid sequence for FGAM2 (SEQ. ID NOS. 3 and 4).
FIG. 2A is the sequence alignments of the soybean FGAM synthase gene with other known FGAM sequences. Multiple alignment of amino acid sequences (Higgins, D.G. et al. (1988) Gene 73:237–244) for genes FGAM1 (SEQ ID NO: 2) , FGAM2 (SEQ ID NO: 4), Drosophila melanogaster (SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 14), Homo sapiens (SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO: 15) and E.coli (SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13 and SEQ ID NO: 16) using the ClustalW program are shown. Only conserved domains are shown. Identical amino acids are in bold. The ATP-binding domain and the three glutamine-binding domains are overlined.
FIG. 2B is the Bestfit analysis of the sequence homology of promoter regions of FGAM1 (SEQ ID NO: 1) and FGAM2 (SEQ ID NO: 3) genes with the wun1 promoter (SEQ ID NO: 17 and SEQ ID NO: 18) from potato (Hansen, E. et al. (1996) Physiol. Mol. Plant Pathol. 48:161–170).
FIG. 3 is a diagrammatic representation of promoter organization for genes FGAM1 (A) and FGAM2 (B). Promoter deletions were generated from the approximately 2500 bp end using PCR. Domains identified in these promoters via functional or sequence analysis are indicated. WR indicates a wound response element identified by sequence homology and shown functionally in Pr1-2.5 plants. STRE designates a stress response element.



DETAILED DESCRIPTION
The subject invention concerns the identification of soybean gene promoter sequences that contain a nematode responsive domain, and the use of said domain in the control of nematode infection of soybeans.
A “nematode responsive domain” is a region of the plant (e.g., soybean) promoter that is active in a nematode established feeding site on the plant. Without wishing to be bound by a particular theory, it is believed that a nematode protein or other molecule may bind to the nematode responsive domain of the promoter to control expression of downstream coding sequences during establishment of a feeding site. A nematode responsive domain is “functional” if the mRNA expression of the downstream coding sequence is up-regulated in the feeding site syncytium by at least 10% as compared to plant cells of the same tissue type that are not nematode feeding sites. “Functional” nematode responsive domain can also mean, with increasing preference, increased expression of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more. Methods for determining increase in amount of mRNA expression are known to persons skilled in the art.
A “promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
Plant transformation involves the construction of an expression vector which will function in plant cells. Such a vector comprises a heterologous DNA under control of or operatively linked to a regulatory element (for example, a promoter). The expression vector may contain one or more such operatively linked gene/regulatory element combinations. The vector(s) may be in the form of a plasmid, and can be used alone or in combination with other plasmids, to provide transformed plants, using transformation methods as described below to incorporate heterologous sequences into the genetic material of the plant.
The heterologous DNA may encode any product that is disruptive of nematode attack when that DNA is transcribed (and if necessary, translated) in a plant cell. The product can include proteins, peptides, and non-protein products such as antisense RNAs, aptamers and the like. Atkinson, H. J. et al. (2003) Ann. Rev. Phytopathol. 41:615, review information on direct effectors that act against the nematode and effectors that disrupt the nematode feeding site.
The heterologous DNAs may encode a product that is toxic to the plant cells, as described in U.S. Pat. No. 5,750,386 to Conkling et al. A wide variety of protein or peptide products which are toxic to plant cells can be used, including (but not limited to) enzymes capable of degrading nucleic acids (DNA, RNA) such as nucleases, restriction endonucleases, micrococcal nuclease, Rnase A, and Barnase (Bacillus amyloliquefaciens RNAse); enzymes which attack proteins such as trypsin, pronase A, carboxypeptidase, endoproteinase Asp-N, endoproteinase Glu-C, and endoproteinase Lys-C; ribonucleases such as RNase CL-3 and RNase T1, toxins from plant pathogenic bacteria such as phaseolotoxin, tabtoxin, and syringotoxin; lipases such as produced from porcine pancrease and Candida cyclindracea, membrane channel proteins such as glp F and connexins (gap junction proteins), and antibodies which bind proteins in the cell so that the cell is thereby killed or debilitated. Genes which produce antibodies to plant cell proteins can be produced as described in Huse, W. et al. (1989) Science 246:1275–1281. Proteins to which such antibodies can be directed include, but are not limited to, RNA polymerase, respiratory enzymes, cytochrome oxidase, Krebs cycle enzymes, protein kinases, aminocyclopropane-1-carboxylic acid synthase, and enzymes involved in the shikimic acid pathway such as enolpyruvyl shikimic acid-5-phosphate synthase. In preferred embodiments, the heterologous DNA is an anti-apoptosis gene (Dickman, M. B. et al. (2001) Proc. Natl. Acad. Sci. 98:6957), a gene involved in the hypersensitive response, a gene involved in MAPK signal transduction, or a gene encoding an RNA interference construct that down-regulates a gene needed for feeding site establishment (Campbell, M. A. et al. (2002) Transgenic Res. 11 (6):599).
Note that the toxic product may either kill the plant cell in which it is expressed or simply disable the cell so that it is less capable of supporting the pathogen. It is preferred that the plant-toxic product be non-toxic to animals, and particularly be non-toxic to humans.
The heterologous DNA may encode any other product disruptive of nematode attack, including but not limited to those described in U.S. Pat. No. 5,589,622 to Gurr et al. (e.g., products toxic to the nematode). Thus the heterologous DNA may encode a Bacillus thuringiensis crystal protein toxic to insects. Strains of B. thuringiensis which produce polypeptide toxins active against nematodes are disclosed in U.S. Pat. Nos. 4,948,734 and 5,093,120 (Edwards et al.). Additionally, the heterologous DNA may encode other natural pesticides such as that found in cyanobacterium Nostoc strain ATCC 53789 (Biondi et al. (2004) Appl. Environ. Microbiol. 70(6):3313).
Again note that the toxic product may either kill the nematode attempting to feed on the plant cell in which it is expressed or simply disable the nematode so that it is less capable of feeding on the plant cell or establishing a feeding site. For example, the heterologous DNA may encode a peptide, antibody or the like that disrupts feeding by interacting with the ingestion or digestion of food such as one of the antibodies described for soybean cyst nematode including that against the dorsal pharyngeal gland (Atkinson et al., 1988 Annals of Applied Biology 112:459–469), using the procedures for transgenic expression of antibodies in plants described by Hiatt, A. et al. (1989) Nature 342:76–78).
Again it is preferred that the nematode-toxic product be non-toxic to other animals, and particularly be non-toxic to birds, reptiles, amphibians, mammals and humans.
Plant transformation is achieved via known methods of using expression vectors. Expression vectors generally include at least one genetic marker, operatively linked to a regulatory element (a promoter) that allows transformed cells containing the marker to be either recovered by negative selection, i.e., inhibiting growth of cells that do not contain the selectable marker gene, or by positive selection, i.e., screening for the product encoded by the genetic marker. Many commonly used selectable marker genes for plant transformation are well known in the transformation arts, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or an herbicide, or genes that encode an altered target which is insensitive to the inhibitor. A few positive selection methods are also known in the art.
One class of marker genes for plant transformation require screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a coding sequence in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include β-glucuronidase (GUS), β-galactosidase, luciferase, chloramphenicol and acetyltransferase (Jefferson, R. A. (1987) Plant Mol. Biol. Rep. 5:387; Teeri et al. (1989) EMBO J. 8:343; Koncz et al. (1987) Proc. Natl. Acad. Sci. USA 84:131; and DeBlock et al. (1984) EMBO J. 3:1681).
Also available are in vivo methods for visualizing GUS activity that do not require destruction of plant tissue (Molecular Probes publication 2908, Imagene GreenTM p. 1–4 (1993); and Naleway et al. (1991) J. Cell Biol. 115:151a).
Additionally, a gene encoding Green Fluorescent Protein (GFP) has been utilized as a marker for gene expression in prokaryotic and eukaryotic cells (Chalfie et al. (1994) Science 263:802). GFP and mutants of GFP may be used as screenable markers.
Numerous methods for plant transformation have been developed, including biological and physical plant transformation protocols. See, for example, Miki et al., “Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc. Boca Raton, 1993) pages 67–88.
One method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. See, for example, Horsch et al. (1985) Science 227:122. A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, Kado, C. I. (1991) Crit. Rev. Plant Sci. 10:1. Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by Gruber Miki et al., supra. and Moloney et al. (1989) Plant Cell Reports 8:238. See also, U.S. Pat. No. 5,563,055, issued Oct. 8, 1996.
Several methods of plant transformation collectively referred to as direct gene transfer, have been developed as an alternative to Agrobacterium-mediated transformation. A generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles measuring 1 to 4 μm. The expression vector is introduced into plant tissues with a ballistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes (Sanford, J. C. (1990) Physiol. Plant 7:206; Klein et al. (1992) Biotechnology 10:268; U.S. Pat. No. 5,015,580, issued May 14, 1991; and U.S. Pat. No. 5,322,783, issued Jun. 21, 1994).
Another method for physical delivery of DNA to plants is sonication of target cells (Zhang et al. (1991) Bio/Technology 9:996). Alternatively, liposome or spheroplast fusions have been used to introduce expression vectors into plants (Deshayes et al. (1985) EMBO J. 4:2731; Christou et al. (1987) Proc. Natl. Acad. Sci. USA 84:3962). Direct uptake of DNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol or poly-L-ornithine has also been reported (Hain et al. (1985) Mol. Gen. Genet. 199:161; and Draper et al. (1982) Plant Cell Physiol. 23:451). Electroporation of protoplasts and whole cells and tissues have also been described (Donn et al., in Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin et al. (1992) Plant Cell 4:1495–1505; and Spencer et al. (1994) Plant Mol. Biol. 24:51–61).
A transformed soybean cell is one which has been transformed or transfected with DNA constructs as described herein. The transformed or transfected cell is then clonally propagated using known methods to generate a soybean plant. Tissue culture of various tissues of soybeans and regeneration of plants therefrom is well known and widely published. For example, reference may be had to Komatsuda, T. et al., Crop Sci. 31:333–337 (1991); Stephens, P. A., et al., Theor. Appl. Genet. (1991) 82:633–635; Komatsuda, T. et al., Plant Cell, Tissue and Organ Culture, 28:103–113 (1992); Dhir, S. et al., Plant Cell Reports (1992) 11:285–289; Pandey, P. et al., Japan J. Breed. 42:1–5 (1992); and Shetty, K., et al., Plant Science 81:245–251 (1992); as well as U.S. Pat. No. 5,024,944 issued Jun. 18, 1991 to Collins et al., and U.S. Pat. No. 5,008,200 issued Apr. 16, 1991 to Ranch et al.
As used herein, the term “tissue culture” indicates a composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant. Exemplary types of tissue cultures are protoplasts, calli, plant clumps, and plant cells that can generate tissue culture that are intact in plants or parts of plants, such as embryos, pollen, flowers, seeds, pods, leaves, stems, roots, root tips, anthers, and the like. Means for preparing and maintaining plant tissue culture are well known in the art. By way of example, a tissue culture comprising organs has been used to produce regenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and 5,977,445 describe certain techniques, the disclosures of which are incorporated herein by reference.
The Examples set forth herein describe in detail the isolation and characterization of duplicate copies of the FGAM synthase gene from soybean. This gene was identified by differential display analysis and confirmed by RT-PCR to be upregulated within the feeding sites of Heterodera glycines in soybean roots (Vaghchhipawala et al. (2001), supra). Isolation and characterization of the gene from the Williams 82 cultivar of soybean revealed the presence of three copies of the gene, two with high sequence homology and one distantly related. The presence of multiple gene copies was anticipated given the duplicated nature of the soybean genome (Shoemaker, R. C. et al. (1996) Genetics 144:329–338).
As is discussed in the Examples, the FGAM1 gene was encompassed within BAC 53M17, while FGAM2 resides within the BAC 42013/52C8 contig. The high sequence similarity between the genes suggests that the two loci have likely arisen by gene duplication. The degree of sequence identity between the two open reading frames (95.5%) and promoter regions (85%) implies that the duplication occurred fairly recently in evolutionary terms. Although the two gene copies show high protein sequence identity, an estimation of the coalescence time following the procedure of Lynch et al. (Science 290:1151–1155 (2000)) yields a date of approximately 11 Mya. The two loci apparently continue to carry out duplicate functions in differing spatial and temporal patterns or in response to varying stimuli.
Evidence for multi-gene copies in soybean is extensive. A recent study (Jin et al., 1999) reported at least 12 classes of β-1,3-glucanase genes displaying divergent gene expression patterns. Members of a BURP domain-containing protein family, from soybean were also shown to possess diverse expression patterns (Granger, C. et al. (2002) Genome 45:693–701). Mahalingam, R. et al. (1999) MPMI 12:490–498, identified two copies of a polygalacturonase gene, also from soybean, with expression up-regulated during syncytium establishment. Yamamoto, E. (2001) Mol. Biol. Evol. 18:1522–1531, identified three soybean orthologs of A. thaliana receptor-like protein kinases showing high sequence homology and predicted to have arisen from recent duplication events. The advantage of gene redundancy in soybean and other plant genomes is not known, but it has been suggested that members of a gene family generally retain a set of standard functions but acquire unique expression patterns and responses to environmental stimuli. It has been proposed that tissue specificity is an early step in functional divergence of a gene family, while divergence at the amino acid level occurs later (Pickett, F. B et al. (1995) Plant Cell 7:1347–1356). The differential expression of FGAM1 and FGAM2 and the observed divergence between their promoters are consistent with this hypothesis.
The essential function provided by FGAM synthase would predict its activity in areas of rapid cell proliferation. These tissues should include reproductive organs and apical and lateral meristems. This anticipated pattern of expression was evident in the GUS expression assays for FGAM1 full-length promoter (Pr1-2.5). A surprising exception was the pollen sacs, in which no FGAM1 expression was detected. Possibly, sequences for anther expression were present further upstream to the region tested and were omitted from the tested constructions, or a different FGAM synthase copy might be expressing within anther tissues. Lack of detectable GUS expression in the FGAM1 promoter deletions (Pr1-1.5 and Pr1-1.0) suggests that enhanced expression levels or tissue specificity of expression may reside within the interval 1.5 kbp upstream to the translation start site.
To investigate the divergent expression that has arisen between the two loci, we focused on promoter sequence differences. Alignment of promoter sequences revealed a FGAM1 stress response element close to the translation start site. Moreover, FGAM2 promoter constructions showed no GUS expression, suggesting that expression of this locus is much lower or responsive to particular stimuli.
Sequences responsible for feeding site GFP expression were located within an upstream 1.0-kbp interval present in both promoters. Observation of enhanced GFP expression in feeding sites from all constructions, and the considerable sequence homology within the upstream 1.0-kbp interval that confers nematode-responsive expression, suggest that nematode-inducible activity was acquired prior to the gene duplication event.
It is conceivable that nematode responsiveness in the expression of FGAM synthase has facilitated co-evolution of the host-nematode interaction. Purine biosynthesis gene expression in the root has already been shown to be inducible by Rhizobium (Schnorr, K. M. et al. (1996) Plant Molec. Biol. 32:751–757). In fact, several examples of reprogrammed plant gene expression have been found in response to nematode infection (Gheysen, G. et al. (2002) Ann. Rev. Phytopathol. 40:191–219). Juergensen, K. et al. (2003) Plant Physiol 131:61–69, demonstrated activated expression of AtSuc2, which mediates the transmembrane transfer of sucrose into syncytia that acts as nutrient sinks for the nematode. Down regulation of a novel Glycine max ethylene-responsive element-binding protein 1 (GmEREBP1) has also been reported. This protein binds to GCC motifs located within PR gene promoters in H. glycines-infected soybean roots during a susceptible interaction (Mazarei, M. et al. (2002) MPMI 15:577–586) to undermine host defenses. Vercauteren, I. et al. (2002) MPMI 15:404–407, report the up-regulation of a pectin acetylesterase gene in feeding sites of root and cyst-knot nematodes. This gene encodes a pectin-degrading enzyme that may be involved in softening and loosening the primary cell wall in nematode-infected plant roots, leading to expansion of the syncytium. These reports reflect the very broad spectrum of genes thought to be redirected in expression by the nematode for feeding site establishment. The feasibility of disrupting gene expression patterns essential to feeding site establishment as a method of plant protection has not been fully assessed.
Sijmons, P. C. et al. (1991) Plant J. 1: 245–254, were first to document in detail the requirements for successful infection of Arabidopsis by economically important nematodes. In Golinowski, W. et al. (1996) Protoplasma 194:103–116; and Golinowski, W. et al. (1997) in Cellular and molecular aspects of plant-nematode interactions (C. Fenoll et al. (eds.), pp. 80–97, ultrastructural studies were undertaken on root cellular architecture to follow the course of development of H. schachtii in Arabidopsis roots. The nematode developmental life-cycle ( ̃6 weeks) of H. schachtii is similar to that of Heterodera glycines. Likewise, the sequence of changes in Arabidopsis root cell morphology appears to follow a similar course to that in soybean roots. For these reasons, it appears that the observations made in Arabidopsis are likely to parallel events in the infected soybean root.
Interestingly, the expression profiles observed in the full length and deletion constructions for the FGAM1 promoter were similar to the pattern reported for the promoter of gene pyk20, isolated from Arabidopsis thaliana by a promoter tagging strategy (Puzio, P. S. et al. (2000) Plant Sci. 157:245–255). This approach was used to identify genes that were active in nematode feeding sites. The investigators detected expression within the feeding sites as well as floral organs, and a wound response within leaves. Likewise, they reported a region of 963 bp upstream to the first ATG of pyk20 that was sufficient to direct expression within the nematode feeding site in Arabidopsis roots. The lack of expression within feeding sites by vector control constructions (35S::GFP) in our study agrees with previous published data (Urwin et al., 1997, Plant J. 12(2):455–61 and van Poucke et al., 2001, Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet. 66(2b):591–8).
Opperman, C. H. et al. (1994) Science 263:221–223, reported a requirement of 300 bp of upstream sequence to the TobRB7 gene of tobacco for localized expression in Meloidogyne-induced giant cells. Moreover, Escobar, C. et al. (1999) MPMI 12:440–449, identified a sequence 111 bp upstream of the TATA box where nuclear proteins from nematode-induced galls formed DNA protein complexes. These reports indicate that putative nematode responsive domains are generally present in regions of the promoter very close to the transcription initiation sites. It is conceivable that an array of common nematode responsive promoter domains serve as the primary means of coordinating plant gene expression during syncytium establishment.
Based on observations described herein, it appears that the FGAM1 locus likely serves housekeeping functions, while FGAM2 may respond to specific environmental stimuli. Yamamoto, E. et al. (2000) MPMI 12: 440–449, reported the cloning of two identical CLAVATA 1-like genes from soybean which show differential expression patterns and suggest that the function of the two genes is slightly different in different organs. In contrast, both FGAM full-length promoters (and deletion fragments thereof) were found to be nematode inducible, indicating that the nematode inducible domain is located in the 1.0 kbp domain immediately 5′ to the translation start site.
The present invention is explained in greater detail in the following non-limiting Examples.
EXAMPLES
Example 1—Materials and Methods
Vectors and Strains
The genomic copies of FGAM synthase were isolated from a bacterial artificial chromosome (BAC) library prepared from the partial Hind Ill digestion of genomic DNA of the soybean line ‘Williams 82’(Marek, L. F. (1997) Genome 40:420–427). Gene promoter constructions utilized the vector pCAMBIA 1303. Transgene constructions were introduced into ELECTROMAX DH10B cells (Life Technologies, USA) of Escherichia coil via electroporation.
DNA Gel Blot Analysis, PCR and DNA Sequencing Procedures
DNA gel blot analysis was carried out using standard procedures (Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). DNA sequencing was accomplished using the fluorescently-labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Intl., Buckinghamshire, England) in an ALFexpress automated sequencer (Pharmacia, Biotech AB, Ume{dot over (a)}, Sweden). The polymerase chain reaction (PCR) was carried out using genomic DNA from transgenic Arabidopsis leaves prepared according to published protocol (Li, J. et al. (1998) in Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) as template. Primers were designed from the uidA sequence to amplify a product of approximately 1189 bp.
Genomic, Plasmid and BAC DNA Preparations and Sequence Homology Searches
Genomic DNA was prepared by the method of Vallejos, C. E. et al. (1992) Genetics 131:733–740. Plasmid DNA preparations were carried out using the CONCERTTM plasmid miniprep kit (Life Technologies, USA), while BAC DNA was prepared using a modified alkaline lysis protocol (Felicielo, I. et al. (1993) Anal. Biochem. 212:394–401). GCG package software “SEQWEB” function “Bestfit” was used to identify sequence homologies, and the “motifs” function was used to locate protein motifs of interest.
Preparation of Promoter Constructions
The subcloning of the promoter region was carried out in the vector pCAMBIA1303, which incorporates the reporter genes β-glucuronidase (GUS) and enhanced green fluorescent protein (GFP) under the control of the CaMV 35S promoter. Cloning was accomplished by excising the 35S promoter from the vector by digestion with enzymes BamHI and NcoI, and introducing putative promoter fragments from the two identified FGAM synthase genes, FGAM1 and FGAM2. Promoter inserts (2.48 kbp) and their derived truncations were generated by PCR amplification with primers designed to contain BamHI and NcoI restriction sites.
Generation of Arabidopsis Transformants
The transformation of Arabidopsis thaliana, grown in a 16-hr day, 8-hr night light regime, was carried out using the floral dip method (Clough, S. J. (1998) Plant J. 16:735–743). The Agrobacterium tumefaciens strain C58C1 (provided by Dr. Thomas Clemente, University of Nebraska-Lincoln) was used to transform Arabidopsis ecotype Columbia. Transgene constructions were mobilized into the Agrobacterium strain via electroporation. Upon transformation, selection of transgenic plants was carried out by plating surface-sterilized seeds on 0.5×MS-B medium with 2% (w/v) sucrose, vitamins and 20 mg/L Hygromycin. Selected plants were subjected to GUS staining (Jefferson, R. A. et al. (1987) EMBO J. 6:3901–3907), PCR analysis, and DNA gel blot analysis before inclusion in the nematode assay.
GUS Staining and Microscopy Procedures
Plant tissues were immersed in X-Gluc (0.8 mg/ml) solution and kept overnight at 37° C. for color development. After staining, 70% (v/v) ethanol was added for clearing of pigments, following the procedure of Jefferson et al. (1987), supra. The infection of transgenic Arabidopsis roots by Heterodera schachtii was examined for GFP fluorescence with a Confocal Laser Scanning Microscope (CLSM) (Bio-Rad, USA).
Heterodera schachtii Infection Assays of Arabidopsis Transformants
Seeds from confirmed transgenic Arabidopsis plants were germinated on selective media as described above, then transferred to individual wells of a 12-well petri plate containing 1.5 ml of a modified Knop's medium (Sijmons et al. (1991), supra) minus antibiotics. Infection was carried out on 11 to 13-day-old seedlings whose roots had penetrated into the medium. Each 12-well plate contained 10 individual T1 transgenic seedlings derived from one independent transformant; the last two plants in the plate served as uninoculated controls. This system, following the procedure of Baum, T. J. et al. (2000) J. Nematol. 32:166–173, provided ample experimental replications without undue contamination. The plants were inoculated near the roots with 50–100 surface-sterilized J2 juveniles of Heterodera schachtii suspended in 1.5% (w/v) low melting agarose. After 6–8 days incubation in a growth chamber at 25° C. and 16 hr daylength, allowing for feeding site establishment on the roots, plants were examined for GFP expression at root feeding sites by confocal laser scanning microscopy. Subsequently, GUS expression was assayed by filling the entire well with X-Gluc staining solution and incubating at 37° C. overnight. Clearing of tissues involved adding 70% (v/v) ethanol, and cleared roots were observed under the dissecting microscope.
Surface Sterilization of Heterodera schachtii J2 Juveniles
Worms freshly hatched after a 2–3-day incubation in a hatch chamber in 3.14 mM ZnSO4 were used for inoculation. Juveniles were counted in a haemocytometer and approximately 100,000 individuals were placed in a sterile 50-ml centrifuge tube. The samples were washed once in sterile distilled water by pelleting at 1500–2000 rpm for 3 minutes in a centrifuge using a swinging bucket rotor and no brake. The nematodes were resuspended in 50 ml of 0.001% Hibitane (Chlorhexidine, diacetate salt, Sigma #C6143) for 30 min mixing continuously. The sample was centrifuged at 1500 rpm for 3 minutes and resuspended in 50 ml of 0.01% (w/v) HgCl2. This suspension was incubated for 7 minutes, including the time to pellet the worms and remove supernatant. The sample was centrifuged to remove the HgCl2, followed by 3 washes with sterile distilled water. After the last wash, enough 1.5% (w/v) LMP agarose was added to achieve the desired final concentration of nematodes, and the sample was maintained at 37° C. The slurry was pipeted over roots in each well. J2 motility was observed after the LMP agarose had solidified.
Example 2—Assembly of Soybean FGAM Synthase Gene Contigs
The sequence of FGAM synthase cDNA (AF000377) was used to generate two primers for use in RT-PGR. Primer 113: 5′-GGT AU GAT GGA GGG AAA GAC AG-3′ (SEQ ID NO: 19) and Primer 114: 5′- GCC ATC TCT AAG GCA GAA ACT AG-3′ (SEQ ID NO: 20) were used to screen soybean genomic BAG library DNA pools by PCR. The search yielded four putative hits and the corresponding BAG clones 81J4, 42013, 53M17, and 52C8 were selected. The four BAG clones were digested with NotI enzyme and subjected to pulsed field gel electrophoresis to estimate insert sizes ranging from 110 kb to 160 kb. To assemble the BAG clones into contigs, multi-enzyme DNA digestions were separated by agarose gel electrophoresis. The BAG clones 42O13 and 52C8 were found to share several bands in common, while the fingerprint of BAG 53M17 shared fewer bands. BAG 81J4 had a distinct banding pattern. Overlaps were confirmed by DNA gel blot hybridization. When probed with the FGAM synthase cDNA clone (890 bp), BACs 42O13 and 52C8 produced identical hybridization patterns, while the pattern produced by BAG 53M17 differed. A very faint hybridization signal was detected in BAG 81J4, suggesting that the FGAM homology contained within this locus was weak. The two distinct forms of FGAM synthase represented in BACs 53M17 and 42O13/52C8 were henceforth referred to as FGAM1 and FGAM2, respectively. Digestion of Williams 82 genomic DNA with HhaI also revealed 2 prominent and one faint band, consistent with presence in the genome of two homologous loci and one divergent sequence.
Genetic mapping of the original FGAM cDNA in the soybean genome indicated that at least one copy of the FGAM loci is derived from Linkage Group G at the same map location as the major SCN resistance gene, Rhg1. Mapping data were derived from a mapping population of 57 F2 individuals and a RIL mapping population of 100 individuals (Vaghchhipawala et al. (2001) supra). BAC analyses confirmed that the FGAM locus is duplicated. However, the location of the duplicate FGAM locus was not determined. Overlapping fragment analysis was used to determine full-length genomic sequence of genes FGAM1 and FGAM2 using the FGAM synthase cDNA clone to generate end probes. At the 5′ end of each gene, approximately 2.5 kb of promoter sequence was also determined.
Example 3—Characterization of the Duplicate FGAM Synthase Loci
DNA sequence analysis of FGAM1 (Genbank AY178840) and FGAM2 (Genbank AY178839) revealed an open reading frame of 3132 bp and 3940 bp respectively (see FIGS. 1A and 1B). The two DNA sequences were 95.5% identical. Cluster analysis to assess amino acid sequence conservation among homologous FGAM synthase sequences available for soybean, Drosophila, Human and E. coli revealed highest sequence conservation among these genes within the ATP-binding domain and three glutamine binding domains as shown in FIG. 2A. Dendrogram analysis of 12 FGAM sequences from Genbank revealed a separate clustering of microbial and higher eukaryotic sequences. Among the higher eukaryotic genes identified, plant and animal sequences form distinct groups. Sequence analysis of the 2.5-kbp promoter region of the FGAM1 and FGAM2 genes revealed 85% identity. Scanning of the promoter sequences for various motifs revealed the presence of a stress response element (STRE) (Schuller, C. et al. (1994) EMBO J. 13:4382–4389) within the promoter of gene FGAM1 (nt 2361–2369 from 5′ end) with 97% conservation of the consensus. This element is shown to activate transcription of a yeast gene in response to a variety of stress stimuli (Schuller et al. (1994), supra). Alignment of the two promoter sequences to the wun1 wound-inducible promoter from potato, inducible during cyst nematode infection (Hansen, E. et al. (1996) Physiol. Mol. Plant Pathol. 48:161–170), revealed a 39-bp interval with 95% sequence identity within the FGAM1 promoter but only 68% identity within the FGAM2 promoter (FIG. 2B).
Example 4—Promoter Analysis in the Arabidopsis thaliana-Heterodera schachtii System
To determine which FGAM synthase gene was responsive to nematode infection, we conducted transgenic promoter analysis in the established A. thaliana-H. schachtii system (Sijmons et al. (1991), supra). This system has been reported to parallel cellular events of the soybean-SCN infection process (Golinowski, W. et al. (1996) Protoplasma 194:103–116). To determine which promoter intervals were serving to modify gene expression within syncytia, we developed two deletion constructions from each full-length promoter. The deletions were made at the 5′ end of each original 2.48-kbp promoter, leaving 1.5-kbp and 1.0-kbp sequences immediately 5′ to the translation start site in association with GUS (uidA) and gfp reporter genes as diagrammed in FIG. 3. The most divergent interval between the two promoters was located between nucleotides −1483 and −1983 (in relation to the +1 translation start site) in the FGAM2 promoter and nucleotides −1314 and −1014 (in relation to +1 start site) in the FGAM1 sequence. Within this region exists a stretch of sequence of 70 nucleotides in the FGAM2 promoter that is absent from the FGAM1 promoter. To test whether the divergent sequences might account for nematode responsiveness, two deletion constructions containing this region, Pr1-1.5 (FGAM1) and Pr2-1.5 (FGAM2), were derived. The effect of deleting these divergent regions was assessed with constructions Pr1-1.0 and Pr2-1.0 (FIG. 3).
Example 5—FGAM1 and FGAM2 Promoter Expression
Transformants for the six promoter constructions of FGAM1 and FGAM2, as well as the vector control, were stained with X-Gluc solution. Two independent vector-transformed control lines, harboring the 35S promoter fused to GUS-GFP, produced GUS staining in leaves, inflorescence, stem and roots. Five independent transformants containing the full length (2.48-kbp) FGAM synthase promoter from gene FGAM2 (Pr2-2.5) were evaluated for GUS expression, and none produced detectable GUS staining in any part of the seedling including inflorescence. The same results were obtained for the four independent transformants of deletion construction Pr2-1.5 and for seven transformants of construction Pr2-1.0.
Experiments with the 2.48-kbp full-length FGAM1 promoter (Pr1-2.5) produced four independent transformants. With some minor plant variation, Pr1-2.5 transformants showed GUS staining in leaf margins and veins, the root tip and lateral root meristems and inflorescence with the exception of anthers. The FGAM1 deletion constructions, Pr1-1.5 (two events) and Pr1-1.0 (two events) showed no visible GUS staining anywhere in the seedling including flowers. Non-transformed seedlings produced no GUS staining. These results imply that the two promoters differ markedly in strength as a consequence of sequences located more than 1.5 kbp from the translation start site in FGAM1. They also indicate that sequences located more than 1.5 kbp from the translation start site are important in housekeeping growth functions unrelated to nematode responsive expression in established feeding sites.
Example 6—Promoter Expression Analysis in H. schachtii-inoculated Arabidopsis roots
Twelve individual T3 progeny per gene construction were used in the H. schachtii infection assay carried out in twelve-well plates. Two plants served as uninoculated controls. Each plant was infected with 50–100 J2 juveniles, maintained in the growth chamber for 6 days, and then observed under a confocal laser-scanning microscope for GFP expression within feeding sites.
Roots of the vector control showed a uniform green fluorescence, and did not show significant elevation of GFP fluorescence at the sites of infection. Localized at the region of the root where a nematode had established a syncytium, a significant elevation of GFP expression above background was observed in all FGAM1 and FGAM2 promoter constructions. This observation was documented at least five times in each inoculated well (50 replicates for each independent transformant) for all promoter constructions. No localized elevation of GFP expression was seen in the uninoculated controls. Instances in which the nematode had penetrated the root tissue but had not yet established a feeding site showed no localized elevation of GFP. This observation suggests that the establishment of a feeding site was necessary for the enhancement of local GFP expression levels, and indicates that the elevated expression was not simply a localized wound response.
Example 7—Wound Response
Sequence homology data indicated that the FGAM1 gene promoter contains a 39-bp sequence with 95% sequence identity to the wun1 wound inducible promoter from potato. The FGAM2 gene promoter displayed only 68% sequence identity to the wun1 promoter. A leaf from each transformant was excised from the seedling and assayed for GUS expression. Of all transformants tested, one containing the full-length FGAM1 promoter construction (Pr1-2.5) showed what appeared to be a wound response. The excised leaf produced a visible staining pattern in the area around the wounded edge, while the remainder of the leaf remained unstained. This observation suggests that the FGAM1 promoter effects a weak wound response. None of the transformants containing the FGAM2 full length or deletion promoter constructions showed evidence of wound response. These results, again, imply that the nematode responsive expression observed in all transformants did not represent a general wound response.

## Claims

1. An isolated nucleic acid molecule comprising a soybean promoter sequence that comprises a nematode responsive domain, wherein the promoter sequence comprises a sequence selected from the group consisting of: Pr1-1.0 (the sequence from nucleotides 1790 to 2483 of SEQ ID NO: 2), Pr2-1.0 (the sequence from nucleotides 1551 to 2547 of SEQ ID NO: 1), Pr1-1.5 (the sequence from nucleotides 1271 to 2483 of SEQ ID NO: 2), Pr2-1.5 (the sequence from nucleotides 991 to 2547 of SEQ ID NO: 1), Pr1-2.5 (the sequence from nucleotides 124 to 2483 of SEQ ID NO: 2) and Pr2-2.5 (the sequence from nucleotides 19 to 2547 of SEQ ID NO: 1).

2. The nucleic acid molecule of claim 1, further comprising a heterologous DNA operatively linked to the promoter.

3. The nucleic acid molecule of claim 2, wherein the heterologous DNA encodes a product that is disruptive of nematode attack.

4. The nucleic acid molecule of claim 3, wherein the heterologous DNA encodes a product that is toxic to a plant cell.

5. The nucleic acid molecule of claim 4, wherein the heterologous DNA comprises a gene selected from the group consisting of anti-apoptosis genes, genes involved in the hypersensitive response, genes involved in MAPK signal transduction, and genes for RNA interference that down-regulate a gene needed for nematode feeding site establishment.

6. The nucleic acid molecule of claim 3, wherein the heterologous DNA encodes a product possessing anti-helminthic properties.

7. The nucleic acid molecule of claim 6, wherein the heterologous DNA encodes a Bacillus thuringiensis crystal protein toxic to nematodes.

8. A transfected soybean cell comprising the nucleic acid molecule of claim 1.

9. A transgenic soybean plant comprising the plant cell of claim 8.

10. A method of reducing nematode infection of a soybean plant comprising transfecting cells of the soybean plant with the nucleic acid molecule of claim 3.