AU6373300A

AU6373300A - Arabidopsis thaliana cyclic nucleotide-gated ion channel/dnd genes; regulators of plant disease resistance and cell death

Info

Publication number: AU6373300A
Application number: AU63733/00A
Authority: AU
Inventors: Andrew F Bent; Steven J. Clough; Kevin A. Fengler; Roger K. Smith Jr.; I-Ching Yu
Original assignee: Wisconsin Alumni Research Foundation
Current assignee: Wisconsin Alumni Research Foundation
Priority date: 1999-07-23
Filing date: 2000-07-24
Publication date: 2001-02-13
Also published as: MXPA02000781A; CA2378107A1; JP2003505076A; EP1196553A1; WO2001007596A1

Description

WO 01/07596 PCT/USOO/20216 ARABIDOPSIS THALIANA CYCLIC NUCLEOTIDE-GATED ION CHANNEL/DND GENES; REGULATORS OF PLANT DISEASE RESISTANCE AND CELL DEATH CROSS REFERENCE TO RELATED APPLICATIONS The present application claims priority from United States Provisional Patent Application No. 60/145,310, filed July 23, 1999. 5 ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT The U.S. Government has certain rights in the invention based upon research support provided by National Institutes of Health Grant No. 1-5-33349 and U.S. Department of 10 Agriculture Grant No. 1-5-28629. BACKGROUND OF THE INVENTION This invention relates to plant physiology, in particular, plant genes, termed cyclic 15 nucleotide-gated ion channel genes or DND (Defense, No Death) genes as regulators for plant diseases and methods for controlling plant diseases. Numerous plant diseases have plagued humankind from the dawn of time. They have caused major economic disruptions, substantial crop losses to individual growers, famines and 20 even disruption of entire cultures. Plant diseases are estimated to cause in excess of nine billion US dollars in pre-harvest loss of cultivated crop plants each year.

WO 01/07596 PCT/USOO/20216 Growers presently control plant diseases with a combination of germplasm choice (plant genetics), adaptive plant culture practices, and exogenous pesticidal treatments. All three strategies are widely in use. However, for a given crop and disease, control measures may only be partially effective, or not affordable, or may not be available at all. In some cases, 5 growers entirely avoid cultivation of a valued plant species and shift to cultivation of other species because of disease problems. Gene-for-gene resistance is a form of plant disease resistance that is exploited widely by plant breeders for crop plants [McIntosh, R.A., et al., (1995) Wheat Rusts: An Atlas of 10 Resistance Genes Commonwealth Scientific and Industrial Research Organization, Australia, and Kluwer, Dordrecht, The Netherlands; Crute, I.R. et al., (1996) Plant Cell 8:1747-1755; Agrios, G.N. (1997) Plant Pathology (Academic, San Diego); Bent, A.F. (1996) Plant Cell 8:1757-1771]. The name "gene-for-gene" denotes the dependence of this resistance on matched specificity between a plant disease resistance gene and a pathogen avirulence gene [Flor, H.H. 15 (1941) Phytopathology 32:653-669]. In a process that is reminiscent of mammalian antibody antigen interactions, these genes control receptor-ligand interactions that activate complex defense responses [Bent, (1996) supra; Alfano, J.R. et al. (1996) Plant Cell 8:1683-1698; Hammond-Kosack, K.E. et al. (1996) Plant Cell 8:1773-1791]. 20 There are thousands of resistance genes that mediate the recognition of specific fungal, bacterial, viral, or nematode pathogen strains. The strong defense response that is triggered after a gene-for-gene interaction includes synthesis of antimicrobial enzymes and metabolites, generation of signaling molecules that activate defense in neighboring cells and reinforcement of plant cell walls surrounding the site of infection [Bent, (1996) supra; Hammond-Kosack, 25 (1996) supra; Dangl, J.L. et al. (1996) Plant Cell 8:1793-1807]. One of the most prominent features of gene-for-gene defense is the death of infected plant cells within hours after initial contact with pathogen, a process known as the hypersensitive response (HR) [Stakman, E.C., (1915) J. Agric. Resd. 4:193-199; Goodman, R.N. et al. (1994) The Hypersensitive Reaction in Plants to Pathogens: A Resistance Phenomenon (Am. Phytopathol. Soc., St. Paul)]. HR cell 30 death is a programmed cell death response that bears features of the apoptotic cell death processes that occur in other metazoan organisms [Dangl, (1996) supra]. Although HR cell WO 01/07596 PCT/USOO/20216 death is a hallmark of gene-for-gene disease resistance, the relative importance of cell death in this form of disease resistance is not clear and may vary depending on the target pathogen species [Hammond-Kosack, (1996) supra; Dangl, (1996) supra; Stakman, (1915) supra; Goodman, (1994) supra]. 5 Multiple plant defense responses are activated in response to pathogen infection [Bent, A.F. et al., (1999) Advances in Agronomy 66:251-298; Dixon, R.A. et al. (1990) Adv. Genet. 28:165-234; Ryals, J.L. et al. (1996) Plant Cell 8:1809-1819; Hammond-Kosack (1996) supra]. While gene-for-gene systems control early and strong activation of plant defenses 10 following recognition of the invading pathogen, many of the same plant defenses are activated more gradually or to a lesser extent in other forms of disease resistance. Even disease susceptible plants activate a wide variety of defenses, appreciably slowing disease progression [Delaney, T.P. et al. (1994) Science 266:1247-1250; Glazebrook, J. et al. (1996) Genetics 143:973-982]. Although some defense responses are particularly effective against specific 15 pathogens, many plant species induce multifactorial defenses that are somewhat generic. These general or "broad-Spectrum" defense responses are often effective against a wide variety of viral, bacterial and fungal pathogens [Bent, (1999) supra; Dixon, (1999) supra; Ryals, (1996) supra]. Salicylic acid has been shown to be a key endogenous mediator that promotes expression of a diverse set of plant defenses [Delaney, (1996) supra; Gaffney, T. et al. (1993) 20 Science 261:754-756]. Salicylic acid can be required for effective gene-for-gene resistance, for other localized defense responses, and for systemic acquired resistance (SAR) [Ryals, (1996) supra]. Other defense pathways have been identified that are apparently independent of salicylic acid, such as many jasmonic acid-dependant defense responses [Penninckx, I.A. et al. (1996) Plant Cell 8:1809-1819]. Multigenically controlled defense pathways form 25 important barriers to infection, and plant breeding efforts are often devoted to improvement of these "quantitative" types of resistance [Agrios, (1997) supra]. Yu et al. [Yu et al. (1998) Proc. Natl. Acad. Sci. USA 95:7819-7824; Yu et al. (2000) Mol. Plant-Microbe Interactions 13:277-286] identified two Arabidopsis mutants, dndl and 30 dnd2, that do not develop the HR response to avirulent P. syringae pathogens. These dnd mutants exhibited gene-for-gene restriction of pathogen growth in the absence of extensive HR 3 WO 01/07596 PCTIUSOO/20216 cell death and also exhibited a constitutive systemic acquired resistance phenotype. This constitutive induction of systemic acquired resistance may substitute for HR cell death in potentiating the stronger gene-for-gene defense response. 5 Advances in molecular biology and genetic engineering now make it feasible to tailor important crops to better cope with pathogens and reduced losses to plant diseases. Many of the major crop species can routinely be transformed and regenerated [Christou, (1996) Trends Plant Sci. 1:423-431]. However, this type of genetic engineering requires a knowledge of the molecular processes involved. 10 In order to provide a basis for developing more efficient means to control diseases in plants, the present invention describes a class of plant genes exemplified by two Arabidopsis genes termed DND (Defense, No Death) 1 and 2 which were discovered herein to encode proteins formerly identified in the literature as putative cyclic nucleotide-gated ion channels 15 (cDNAs of AtCNGC2 and AtCNGC1, respectively) [Kohler, C. et al (1999) Plant J. 18:97-104; Kohler, C. et al. (1998); Plant Physiol. 116:1604; Leng et al. (1999) Plant Physiol 121:753 761]. Therefore, the terms "DND1" and "DND2" as used herein are intended to be synonymous with AtCNGC2 and AtCNGC1, respectively. Note, however, that in this previous work by others on AtCNGC2 and AtCNGC1, no association was made with plant disease 20 resistance, cell death or related whole-plant phenotypes. The AtCNGC/DND genes regulate disease resistance, i.e. modified forms of these genes cause enhanced resistance against a broad range of viral, bacterial and fungal pathogens in the absence of cell death. Therefore, manipulation of these genes or other related genes allows the generation of plants with improved disease resistance. 25 SUMMARY OF THE INVENTION The object of the invention is to provide methods for improving disease resistance in plants. A second object of the invention is to provide methods for control of cell death in 30 plants. The plant genes DND (Defense, No Death) 1 and 2 of Arabidopsis thaliana described herein regulate broad-spectrum disease resistance and cell death in plants. The nucleotide 4 WO 01/07596 PCT/USOO/20216 coding sequences of the DND 1 and 2 genes of the present invention are identical to previously known cDNA molecules that encode proteins that function as cyclic nucleotide-gated ion channels 2 (AtCNGC2) and 1 (AtCNGC1), respectively. Such cyclic nucleotide-gated ion channels are ubiquitous in plants, generally. Plants that do not express AtCNGC/DND genes 5 due to a mutation exhibit elevated resistance against a broad range of viral, bacterial, and fungal pathogens, and also exhibit a decrease in the HR cell death response to avirulent pathogens and a decrease in cell death induced by Fumonisin B1 toxin. Therefore, this invention discloses various methods for improving disease resistance by modifying the AtCNGC2/DNDJ or AtCNGC1/DND2 gene or gene product, or genes or gene products in other 10 plants that share substantial structural or functional similarity to the AtCNGC2/DND1 or AtCNGC1/DND2 gene or gene product. The modifying means include, but are not limited to transcriptional or translational down-regulation, mutations including nucleotide substitution, deletion or insertion, inactivation of the gene or gene product, and chemical inhibition of the gene product. These genes may also be down-regulated or inactivated by antisense technology, 15 sense-strand suppression, virus-induced gene silencing, double strand RNA and other inactivation methods known in the art. The invention further includes a transformed or genetically modified plant, plant tissue or seed made by the described method. 20 The invention discloses methods for identifying other AtCNGC2/DND1 or AtCNGC1/DND2 related disease resistance genes or structural or functional homologs thereof. The AtCNGC2/DND1 or AtCNGCJ/DND2 related genes or homologs or gene products thereof thus identified can be modified as described herein to improve disease resistance. These genes 25 and gene products can be used in a screen to identify inhibitors for enhancing disease resistance in plants. The invention also provides use of genetic markers for improving plant disease resistance via prevailing plant breeding practices. The genetic markers are identified because 30 of their similarity to or close genetic proximity to AtCNGC2/DND1 or AtCNGC1/DND2 or their proximity to homologs of AtCNGC2/DND1 or AtCNGC1/DND2. 5 WO 01/07596 PCT/USOO/20216 The identification of the CNGC/DND genes as disease resistance regulators provides additional means to identify other molecules which interact with them in exhibiting disease resistance. These include effector genes or proteins or chemicals which interact with a CNGC/DND gene or gene product or homolog. 5 The methods disclosed in the invention to improve disease resistance in plants may also be used to control disease-induced cell death. Accordingly, the invention includes: A method for controlling cell death in a plant by down-regulating, mutating or inactivating a CNGC/DND gene or gene product of the plant. Specifically, such method can include inactivating a 10 CNGC/DND gene using an appropriate CNGC/DND antisense or sense DNA. Accordingly, the invention includes antisense DNA molecules of DND1 or DND 2 genes or similar genes from other plant species. The invention further includes a transformed or genetically modified plant, plant tissue or seed made by the described method, or transformed to express the described antisense or sense DNA. 15 In another aspect, the invention provides a method for improving pathogen resistance of a plant by down-regulating, mutating or inactivating a cyclic nucleotide-gated ion channel gene or gene product or a homolog thereof of the plant. Similarly, the invention provides transformed or genetically modified plant, plant tissue or seed made by the described method. 20 The invention further provides a method for identifying a disease-resistance gene by screening for a cyclic nucleotide-gated ion channel gene, including AtCNGC2/DND1 and AtCNGC1/DND2 genes. 25 BRIEF DESCRIPTION OF THE DRAWINGS Figs. lA-1D shows HR cell death defect in dndl mutant. Leaves of wild-type parent (Col) and dndl mutant (dndl) plants were inoculated with a high dose (2 x 108 cfu/ml) of avirulent, HR-stimulating P. syringae pv. glycinia Rct 4 pV288 (Psg avrRp2+) or the isogenic, 30 nonavirulent control strain P. syringae pv. glycinia Race 4 pVSP61(Psg). At 24 h postinoculation, leaves were harvested, fixed, and examined for autofluorescent dead cells by 6 WO 01/07596 PCT/USOO/20216 using a fluorescence microscope. Fig. 1C shows the edge of an inoculated zone, revealing confluent cell death in response to bacteria only on the left (inoculated) side. Figs. 2A-2B illustrate the growth of bacteria within plant leaves. Fig. 2A shows 5 Arabidopsis lines Col (Col-0 wild-type, RPS2/RPS2; DND1/DND1), rps2 (Col-0 rps2 201/rps2-201; DNDJ/DND1), and dndl (Col-0 and RPS2/RPS2; dndl/dndl) inoculated with P. syringae pv. tomato DC3000 pV288 (avrRpt2*). Fig. 2B shows Arabidopsis lines Col-O and dndl inoculated with isogenic P. syringae pv. tomato DC3000 differing by the presence (pAvrRpml, filled symbols) or absence (pVSP61, open symbols) of avirulence gene avrRpml 10 carried on plasmid pVSP61. Both plant lines are RRM1/RPM1 genotype. All data points are mean + SD. Figs. 3A-3C show pathogenesis-related gene expression monitored by RNA blot analysis of Col-O wild-type (Col) and Col-O dndl/dndl mutant (dndl) plants. Fig. 3A 15 illustrates P-glucanase expression 72 h after treatment of leaves with 10 mM MgCl 2 containing no pathogen (0), the nonavirulent control strain P. syringae pv. tomato DC3000 pVSP61 (vir), or the isogenic avrRp2-expressing strain P. syringae pv. tomato DC3000 pV288 (avr). Fig. 3B illustrates PR-1 expression 24 h after treatment as in Fig. 3A. Fig. 3C shows Phosphorimager quantification of PR-1 expression from blot shown in Fig. 3B, normalized to 20 level of constitutive P-ATPast mRNA. Similar results were obtained in multiple experiments. Figs. 4A-4B show the levels of salicylic acid and glucoside-conjugated salicylic acid compounds in Col-0 and mutant Col-O dndl-1dndl-1 or Col-O dnd2-1/dnd2-1 plants. 25 Figs. 5A and 5B illustrate that the dndl and dnd2 mutant plants show more resistance to cell death induced by Fumonisin B1 (an inhibitor of ceramide synthase) compared to the wild type Arabidopsis. Fig. 5A is a dose-response curve of Fumonisin B1 generated using control (Col) and dndl mutant plants. Fig. 5B shows the delayed response of the dnd2 mutant plants after Fumonisin treatment compared to the wild type Arabidopsis. The Y axis in both 30 graphs indicates the severity of necrosis rated on a 0-5 scale (O=no lesions, 5=complete necrosis). 7 WO 01/07596 PCT/USOO/20216 Fig. 6 shows nucleotide sequences of the genomic region containing AtCNGC2/DND1 gene, 5,897 nucleotides in length. The notable features are as follows: nt 1632; 5' end of the AtCNGC2/DND1 cDNA, nt 1663; ATG putative start codon, nt 1716; end of exon 1 of AtCNGC2/DND1 gene, nt 2088-2763; exon 2, nt 2928-3143;exon 3, nt 3333-3652; exon 4, nt 5 3747-3863; exon 5, nt3953-4192; exon 6, nt 4275-4363; exon 7, nt 4478-5153; 3' end of AtCNGC2/DND1 cDNA, nt 4987 and TAA putative stop codon. The dndl mutant described in the invention contains G to A point mutation creating a stop codon at position 3101 nt as underlined. The sequences shown herein are identical to those of SEQ ID NO:1 10 Fig. 7 shows the amino acid sequence (SEQ ID NO:3) of the protein encoded by the DND1 gene. Fig. 8 shows the nucleotide sequence of AtCNGC2/DND1 cDNA (SEQ ID NO:2). 15 Fig. 9 illustrates the results of the complementation studies. The three complementing cosmids derived from BAC3H2 (1A8, 1H2 and 1H3) are depicted by solid bars. Striped bars represent cosmids that failed to complement the dndl dwarf phenotype. Numerical data are the number of size-complemented plants out of the total number of T2 plants examined for each cosmid. 20 Fig. 10 shows response of T2 Col-0 dndl/dndl plants transformed with cosmids 1A8 and 1H2. T2 plants segregated 3:1 (wild type:dwarf) for size. Plants of both types were inoculated with Psg R4 avrRpt2 or with Psg R4 (no avr). HR was scored 24 hours post inoculation. The degree of HR was scaled from 0 (no HR) to 5 (severe HR) respectively. For 25 plants of dwarf stature, 7, 3, and 4 plants were tested for dndl, 1A8, and 1H2 respectively. The number indicates the average of three leaves per plant (avr) and one leaf per plant (no avr) for wild-type size plant. Dwarf plant scores represent the average of at least six inoculated leaves (avr) or three leaves (no avr). 30 Fig. 11 shows growth of virulent P. syringae pv. tomato (pst) DC3000 in Col-0 dndl/dndl plants transformed with cosmid 1H3. Six-week old T2 plants segregating 3:1 (wild 8 WO 01/07596 PCTUSOO/20216 type:dwarf) size were inoculated with Pst DC3000 with no avr gene. Bacterial growth was sampled 0, 2, and 4 days post inoculation for T2 plants of wild-type size, as well as the Col-0 and dndl controls. For T2 plants of dwarf stature, bacterial growth was sampled only at 3 days post inoculation (depicted by the X). 5 Fig. 12 shows complementing cosmids and subclones. Complementing cosmids are represented by solid bars. Complementing subclones are represented by spotted bars and are depicted immediately above their parent cosmid. Cosmids and subclones that failed to complement are represented by white bars. Subclones were generated using EcoRI and/or 10 XbaI, except where noted. Fig 13 shows the nucleotide sequence of the genomic region containing the Arabidopsis DND2 (AtCNGC1) gene (SEQ ID NO:4). 15 Fig. 14 shows the nucleotide sequence of DND2 (AtCNGC1) cDNA (SEQ ID NO:5). Fig. 15 shows the amino acid sequence of the protein encoded by the DND2(AtCNGC1) gene (SEQ ID NO: 6). 20 Fig. 16 shows the results of the complementation studies of dnd2 small rosette size phenotype by transformation with the Arabidopsis genomic DNA fragment shown in Fig. 13 (SEQ ID NO:4), encoding AtCNGC1/DND2. "Col + vector" represents the wild type plants transformed with vector only, "dnd2+vector" represents the dnd2 mutant plants transformed with vector only, and "dnd2 +AtCNGC1" represents the dnd mutant plants transformed with 25 a vector containing the Arabidopsis genomic DNA frgment shown in Fig. 13 (SEQ ID NO:4), encodidng AtCNGC1/DND2. 9 WO 01/07596 PCT/USOO/20216 DETAILED DESCRIPTION OF THE INVENTION As used in the present invention, the following terms are defined as follows: 5 The term "down-regulation", as used herein, refers to a general method of reducing the level of gene products (RNA or protein). Thus, down-regulation of a gene may be achieved either transcriptionally or translationally. For example, an antisense molecule may be introduced into a cell or tissue to down-regulate the gene from which the antisense molecule is derived. 10 The term "mutation" as used herein refers to a modification of the natural nucleotide sequence of a nucleic acid molecule made by deleting, substituting, or adding a nucleotide(s) in such a way that the protein encoded by the modified nucleic acid is altered. The resulting proteins often exhibit altered functionality. 15 The term "antisense molecule" as used herein is intended to mean a single stranded nucleic acid molecule consisting of the complementary nucleotides of a sense molecule. The sense molecule in general refers to the strand of DNA or RNA which has the sequence of mRNA encoding the protein. 20 The term "disease resistance" or "pathogen resistance" as used herein refers to any process by which a plant response to pathogen attack functions to enhance the plant's ability to survive and/or maintain productivity despite that attack. 25 "Improved resistance" in a plant variety means that the damage associated with pathogen attack in that variety is reduced when compared to a control variety, as measured by an art-recognized criterion. The ultimate goal of improved resistance is to provide a higher crop yield, on average, from the variety having improved resistance, compared to the control. Since crop yields require time-consuming field trials, various laboratory tests have been 30 devised to measure resistance in individual plants, such tests being art-recognized as predictive of improved yield in the presence of the pathogen. These tests include, but are not limited to, 10 WO 01/07596 PCT/USOO/20216 measurement of pathogen growth in the infected plant, measurements of extent of necrosis, plant cell death and hypersensitivity response. Such measurements are generally preferred because they can be conducted under controlled conditions, controlled pathogen level, timing of pathogen introduction, temperature, humidity and the like. In a specific example described 5 herein, disease resistance is measured as restriction of pathogen growth, i.e., growth of an inoculated pathogen (i.e. P. syringae pv. tomato) was much less in the dnd mutant compared to the wild type. Thus, when a plant is modified to exhibit improved disease resistance, or improved pathogen resistance according to the methods described herein, it is understood that similar growth restriction to a given pathogen ultimately would result in reduced damage to the 10 plant and higher crop yield. The term "gene" refers to a deoxynucleic acid molecule that encodes a protein or peptide upon transcription and translation. Thus, "gene product" as used herein refers to either an RNA molecule or protein which is generated by expression of a given gene. 15 "DND gene" as used herein is intended to mean any gene that has structural homology to DND1, DND2 or other genes whose product would closely resemble that of an intact or mutated cyclic nucleotide-gated ion channel gene and that, when down-regulated, mutated, or inactivated, causes improved resistance or improved cell death traits as described in the present 20 invention. Accordingly, it includes not only the AtCNGC2/DND1 or AtCNGC1/DND2 gene of Arabidopsis but also those corresponding or related genes of other plant species which have structural or functional homology with the AtCNGC2/DNDJ or AtCNGC1/DND2 gene disclosed herein. It will be understood in the art that variant structures of the AtCNGC2/DND1 or AtCNGC1/DND2 can exist in other plants, and that such variants can be identified, as herein 25 described, by structural homology, by functional homology, or by similarity of phenotype in genetic analyses, or by any combination of the foregoing. The meaning of a "homolog" as used in the present invention is intended to include any gene or gene product which has a structural or functional similarity to the gene or gene 30 product in point. Accordingly, a structural homolog of the CNGC/DND gene is defined as one hybridizing with the Arabidopsis AtCNGC2/DND1 (SEQ ID NO: 2) or CNGC1/DND2 (SEQ 11 WO 01/07596 PCTIUSOO/20216 ID No: 5) genomic DNA or cDNA at a herein defined level of stringency of the conditions of hybridization, at low stringency, preferably at medium stringency or more preferably at high stringency. A second and equally valid definition of "homolog" is a gene for which the derived amino acid sequence of a translation product bears significant similarity to previously 5 characterized cyclic nucleotide-gated ion channels, including the hallmark six transmembrane domains, a pore domain between the fifth and six transmembrane domain, and a cytoplasmic cyclic nucleotide interaction domain [Zagotta and Siegelbaum (1996) Ann. Rev. Neurosci. 19:235-263; Kohler, et al. (1999) supra]. As a third and equally valid definition of "homolog," a functional homolog of a DND gene product is a cyclic nucleotide-gated ion 10 channel protein that can potentially function or can be caused by mutation, down-regulation or chemical inhibition to function as a disease resistance regulator or a regulator of cell death. A functional homolog of the CNGC/DND gene product is one which potentially functions upon modification as a regulator of disease resistance and/or cell death. 15 The present invention discloses two plant genes, DND1 and DND2 of Arabidopsis thaliana as regulators of disease resistance and cell death. Plants homozygous for mutated DND1 or DND2 gene exhibit enhanced disease resistance in the absence of cell death. Therefore, the manipulation of the DND1 or DND2 gene offers new possibilities of controlling various plant diseases. 20 To address the relationship between HR cell death, resistance gene-mediated defense signal transduction, and the actual restriction of pathogen growth, mutants of Arabidopsis thaliana that were deficient in the HR were isolated and characterized. A mutagenized M 2 population of Arabidopsis line Col-O, which expresses the RPS2 resistance gene, was screened 25 by inoculating plants with a strain of the bacterial plant pathogen P. syringae pv. glycinia expressing the RPS2-complementary avirulence gene avrRP2 [Kunkel, B.N., et al. (1993) Plant Cell 5:865-875]. An extremely high titer of pathogen, 2 x 108 cfu/ml, was used so that plants undergoing a wild-type HR would exhibit visible collapse of leaf tissue. 30 Two of the mutants isolated from this screen were called dndl and dnd2. These mutants exhibited several similar phenotypes; both are recessive to wild type, homozygous 12 WO 01/07596 PCTIUSOO/20216 mutant plants show an extreme reduction in the extent of cell death in response to avirulent P. syringae, and dwarfism. Because dndl and dnd2 mutants were analyzed in a similar manner and found to exhibit similar mutant phenotypes, the following description is taken largely from the dndl mutant analysis. However, it is easily understood by a person skilled in the art that 5 these methods are readily applicable to the dnd2 mutant analysis. The dndl mutant was recovered from this screen as a line displaying reduced rosette size and a clear HR- phenotype. Progeny lines derived from the dndl mutant failed to produce an HR not only when inoculated with pathogens expressing avrRpt2 but also in response to P. 10 syringae that express avirulence genes avrRpml or avrB (Kunkel, (1993) supra; Bisgrove, S.R. et al. (1994) Plant Cell 6:927-933]. Two separate resistance genes (RPS2 and RPM1) control responsiveness to these three separate avirulence genes. Accordingly, it is predicted that the dnd1 line is disrupted in a common component of the plant defense response that is shared by initially distinct gene-for-gene signal transduction pathways. 15 To confirm the absence of hypersensitive cell death in response to avirulent pathogens in the dndl mutant, fluorescence microscopy was used to monitor cells within inoculated leaf tissue [Klement, Z. et al. (1990) in Methods in Phytobacteriology, eds. Klement, Z., Rudolph, K. & Sands, D.C. (H. Stillman, Budapest), pp. 469-473]. Plant cells that undergo the HR 20 display a marked increase in fluorescence due primarily to the production and release of phenolic compounds upon cell death. In "low titer" experiments, P. syringae pv. glycinia expressing avrRP2 were introduced into leaf mesophyll tissue at a concentration of ~5 x 10' cfu/ml, a dose at which a majority of the plant cells are not initially in contact with pathogen. As expected, leaves from the wild-type parental line infected at this dose with P. syringae 25 expressing avrRP2 contained numerous isolated autofluorescent cells. In contrast, very few autofluorescent foci were present in dndl leaves inoculated with the same avirulent strain. The dndl leaves instead resembled uninoculated leaves or leaves inoculated with the nonavirulent P. syringae control. 30 When leaves of the parental Col-O line were inoculated with an extremely high titer of avirulent P. syringae (2 x 108 cfu/ml), the expected confluent collapse of host cells was 13 WO 01/07596 PCT/US00/20216 observed (Fig. 1) [Kunkel, (1993) supra; Yu, G.-L. et al. (1993) Mol. Plant-Microbe Interact 6:434-443]. However, even at this high pathogen dose, very little cell death above that seen in negative controls was detected in dndl plants (Fig. 1). Separate experiments that used Evans Blue to stain dead or dying cells gave similar results. The autofluorescence assay 5 method was preferred because of greater clarity and less laborious tissue preparation. With the autofluorescence assay, absence of HR cell death in dnd1 plants was observed in multiple experiments, including experiments that used initial bacterial titers as high as 2 x 10 9 cfu/ml. A slight increase in cell death was observed in z5-8% of the dndl leaves inoculated with 2 x 108 cfu/ml of avirulent P. syringae but only in isolated areas that represented a fraction of the 10 inoculated tissue. Cell death in these small areas was patchy rather than confluent, and similar small patches of cell death could be observed at a lower frequency in control Col-0 plants inoculated with the nonavirulent P. syringae strain. No stimulation of cell death by avirulent P. syringae could be detected in the vast majority of the inoculated dndl leaves. 15 To determine whether the absence of the HR in the Arabidopsis dndl mutant is associated with compromised disease resistance, growth of P. syringae pv. tomato within plants was monitored quantitatively over time [Whalen, M. et al. (1991) Plant Cell 3:49-59]. Pathogenic strains that express an avirulence gene are virulent on plants that do not express the corresponding resistance gene, but their growth is reduced severely on plants which possess 20 the appropriate resistance gene. Fig. 2A shows the growth of P. syringae pv. tomato expressing avrRpt2 in wild-type Arabidopsis Col-O (RPS2/RPS2), in a Col-O line lacking functional RPS2 (rps2-201/rps2-201), and in the Col-0 dnd1 mutant. Despite the absence of the HR, dndl was very similar to wild type in successfully restricting the growth of P. syringae expressing avrRP2. Strong avirulence and resistance gene-dependent restriction of 25 pathogen growth also was observed in quantitative experiments with P. syringae expressing avrRpml, avrRps4, or avrB (Fig. 2B). These results demonstrate that extensive HR cell death is not always required for resistance gene/avirulence gene-dependent plant disease resistance. Having established that dndl plants are resistant to avirulent to P. syringae despite the 30 absence of the HR, the response of the dndl mutant to virulent P. syringae was examined. Fig. 2B shows the growth of the virulent P. syringae pv. tomato strain DC3000 (pVSP61) in 14 WO 01/07596 PCT/USOO/20216 wild-type Col-0 and in Col-0 dndl/dndl plants (open symbols). This strain does not trigger gene-for-gene resistance in plants of the Col-0 gentoype [Kunkel, (1993) supra; Whalen, (1991) supra], yet leaf populations of this pathogen strain were reduced 10- to 100-fold in experiments with the dndl mutant. Similar results were obtained in multiple experiments and 5 in studies with the virulent P. syringae pv. maculicola strain 4326. The dndl plants express a level of resistance to virulent P. syringae that is typical of plants exhibiting systemic acquired resistance, induced systemic resistance, or other forms of resistance gene-independent disease resistance [Ryals, J.L. et al. (1996) Plant Cell 8:1809-1819; Pieterse, C.M. et al. (1996) Mol. Plant-Microbe Interact 8:1225-1237]. This broad spectrum resistance phenotype co-segregated 10 with the other dndl mutant phenotypes in all cases tested. Important to note, Fig. 2B also shows that growth of populations of P. syringae that do express avrRpml (closed symbols) was restricted to a much greater extent than was growth of the virulent pathogen strain. A 1,000- to 10,000-fold reduction of pathogen growth was 15 observed if the otherwise virulent P. syringae strains DC300 or 4326 expressed avirulence genes avrRpm1 or avrRpt2 (Fig. 2B). These experiments demonstrated that gene-for-gene resistance can be induced over and above the weaker resistance gene-independent resistance in dndl plants. 20 To examine the extent of the lower level resistance to virulent pathogens in the dndl mutant, plants were inoculated with virulent strains of other pathogen species (Lee, J.-M. et al. (1996) Mol. Plant-Microbe Interact. 9:729-735; Bent, A., et al. (1992) Mol. Plant-Microbe Interact 5:372-378; Parker, J.E. et al. (1993) Mol. Plant-Microbe Interact 6:216-224; Parker, J.E. et al. (1997) Plant Cell 9:879-894]. Tobacco ringspot virus spread systemically in only 25 9% of dndl plants as opposed to 71% for wild-type Col-0. Xanthomonas campestris pv. campestris and X. c. pv. raphani (bacteria) only produced mild yellowing on dndl rather than the necrotic lesions produced on Col-0. Peronospora parasitica (oomycete) produced three fold fewer spores on dndl as opposed to Col-0 [3.0 +2.2 vs. 10.7 + 3,1 mean + SE if (spores x 103) per leaf]. Microscopy of leaves infected with virulent P. parasitica confirmed 30 that restriction of mycelial growth was not associated with HR-like host cell necrosis or autofluorescence. At 3 days postinoculation, mycelia of virulent P. parasitica strain Noco2 15 WO 01/07596 PCTUSOO/20216 typically had formed haustoria on 2-10 host cells in dndl plants, whereas in wild-type Col-O plants a typical mycelium ramified extensively and formed haustoria on 15-30 host cells. Significantly reduced growth of Erysiphe orontii (fungus) in dndl plants also has been observed. 5 Constitutively elevated broad spectrum resistance has been observed previously in a number of contexts, such as in Arabidopsis cpr, cim, lsd, and acd mutants [Dangl, (1996) supra], in hybrid tobacco lines derived from crosses between disparate Nicotiana species [Ahl Goy, et al. (1992) Physiol. Mol. Plant Pathol. 41:11-21], and in plants expressing systemic 10 acquired resistance in response to prior pathogen infection or treatment with salicylic acid or synthetic salicylic acid mimics [Ryals, (1996) supra]. Elevated resistance often is associated with increased expression of pathogenesis-related (PR) genes [Ryals, (1996) supra], and examination of uninoculated dndl plants revealed constitutively increased expression of the PR genes P-glucanase and PR-1 (Figs. 3A and 3B) [Cao, H. et al. (1994) Plant Cell 6:1583-1592; 15 Ausubel, F.M. et al. (1997) Current Protocols In Molecular Biology (Wiley, New York)]. Although plants infected by virulent P. syringae pv. tomato displayed elevated levels of P glucanase or PR-1 mRNA, inoculation of dndl or wild-type Col-O with avirulent P. syringae expressing avrRp2 caused an even greater elevation in PR-1 mRNA (Fig. 3C) (25, 33). Similar or more pronounced results were obtained with four distinct RNA sets prepared, 20 blotted, and probed in entirely separate experiments. These results demonstrate, at the level of gene expression, that gene-for-gene signal transduction and defense response activation are functional in dndl plants and are inducible over and above constitutive broad spectrum resistance. 25 Enhanced PR gene expression and broad spectrum resistance can be induced by elevated levels of endogenous or applied salicylic acid compounds [Ryals, 1996) supra]. We observed constitutively elevated levels of both free salicylic acid and glucoside-conjugated salicylates in dndl plants (Fig. 4). Although salicyclate is likely to be a primary mediator of heightened resistance in dndl plants, the mechanism by which the dndl mutation causes salicylate 30 elevation remains to be discovered. 16 WO 01/07596 PCT/USOO/20216 Plant mutants that display gene-for-gene disease resistance with no HR cell death are not common. However, other Arabidopsis mutants that exhibit constitutively elevated resistance have been isolated, such as the cpr, cim, Isd, and acd mutants [Dangl, (1996) supra; Bowling, S.A. et al. (1997) Plant Cell 9:1573-1584; Bowling, S.A. et al. (1994) Plant Cell 5 6:1845-1857; Lawton, K. et al. (1993) in Mechanisms of Defence Responses in Plants. eds. Fritig, B. & Legrand, M. (Kluwer, Dordrecht, The Netherlands), pp. 422-432]. Accordingly, dndl plants were compared with a number of these lines. In contrast to the acd and lsd mutants, no lesion-mimic phenotype was observed in dndl mutants when leaf tissue from uninoculated plants was inspected by naked eye, by autofluorescence microscopy as described 10 in Yu, (1993) supra, or after trypan blue staining as described in Parker, J.E., et al. (1993) Plant J. 4:821-831. Genetic complementation tests demonstrated that dndl is a separate locus from the two published cpr loci, CPR1 and CPR5 (see Examples section). In addition, the dndl mutant apparently does not resemble many of the other unpublished cpr or cim mutants because the dndl mutant does not exhibit traits observed in preliminary analysis of those 15 mutants such as dominant or semi-dominant behavior, very low fertility, glabrousness, or distorted leaf shape. In particular, previously described cpr and cim mutants do not display the dnd phenotype of gene-for-gene defense with no HR cell death. The dndl mutant does exhibit a dwarf phenotype, as is observed in Arabidopsis cpr, cim, and other constitutive PR expression mutants, but dndl plants otherwise appear normal in their growth and development. 20 The dnd mutants were examined to determine whether they are also resistant to other inducers of cell death. As shown in Fig. 5A-5B and additional experiments, both dndl and dnd2 mutants exhibited delayed response and reduced sensitivity to Fumonisin B 1-induced cell death compared to the wild type Arabidopsis, indicating that the dnd mutants may have more 25 general suppression of programmed cell death. Fumonisin B 1 is a known inhibitor of ceramide synthase which induces apoptosis in diverse organisms. To determine the genetic basis of the dndl phenotype, segregation analysis and gene mapping studies were carried out. Crosses of dndl to wild-type Col-O and No-O ecotypes 30 yielded F1 individuals that display the wild-type HR' phenotype, demonstrating the recessive nature of the mutant phenotype. F2 of a Col-0 x dndl cross segregated 24:7 for HR*:HR-, F2 17 WO 01/07596 PCT/USOO/20216 of a No-O x dndl cross segregated 154:55, and F2 of a reciprocal dndl x No-O cross segregated 132:45. These data are consistent with a 3:1 ratio (for X 2 test, P = 0.59, 0.66, and 0.90, respectively), indicating that a single mutant locus controls the observed phenotypes. The reduced rosette size phenotype was also recessive, and absolutely co-segregated with the 5 HR phenotype in these and all other F2 plants analyzed. The gene symbol DND1 was chosen for this locus, reflecting the mutant phenotype of Defense with No HR cell Death. PCR-based microsatellite and cleared amplified polymorphic sequence genetic markers were used to map the mutated locus. No linkage was detected except to markers for the top arm of chromosome 5. Fine-structure mapping with 536 F2 individuals from No-0 dndl crosses yielded only six 10 recombinant chromosomes between dnd1 and CHS1. These experiments placed DND1 within the ~1.6-cM interval between CHS1, and nga 106 and a different 11 recombinant chromosomes between dnd1 and CHS1. These experiments placed DND1 within the z 1.6-Cm interval between CHS1 and nga 106 on the upper arm of Arabidopsis Chromosome 5. This location defines a map position that has not been associated previously with defense-related 15 genes. Genetic mapping data suggested that the DAD1 locus resides to the north and close to marker pCIT1243 on the top of Arabidopsis chromosome 5. In order to isolate the clone for the DAD] gene, four contiguous BACs (8M21, 3H2, 22L1 and 23B17) were generated which 20 subsequently used to generate a redundant cosmid library. The detailed techniques for creating BACs, cosmid library, and the use of RFLPs are well known in the art and can be found in Ausubel, (1997). Once a small number of cosmids were identified to span the DAD1 locus region, each clone was tested for the capacity to functionally complement the dndl mutation. This was accomplished by transforming mutant dnd1 plants with each of the cosmids via 25 Agrobacterium-mediated transformation and screening transformants for reversion to wild-type characteristics. To simplify this process, putative transformants were initially screened solely on the basis of size. Because all known phenotypes of dndl mutants appear to be tightly linked, complementation of dwarf size was considered to represent genetic complementation of the DAD] locus. In general dndl plants exhibited a significantly lower transformation rate, 30 relative to wild-type Col-0 ( - .001 % transformants per total number of seeds tested compared to ~-.2-.5 % for Col-0). The transformants were planted to soil and after 2-3 weeks analysed 18 WO 01/07596 PCTIUSO0/20216 for the size. TI plants from the three cosmids (1A8, 1H2 and 1H3) exhibited size similar to that of wild-type controls and overlapped to the same region of BAC 3H2 (see Fig. 9). In summary, complementation data delimited the location of the DND1 locus and demonstrated that the gene encoded in the region is responsible for the loss of function, i.e. dwarfism, in the 5 dndl plants. To confirm genetic complementation of the DND1 locus further, HR assays and bacterial growth curves were performed on T2 plants from the three size complementing cosmids to verify reversion to wild-type defense responses. Because plants transformed with 10 Agrobacterium are typically hemizygous for the transgene, it was not surprising to observe T2 plants from each of the cosmids segregating 3:1 (wild-type: dwarf) for size: 1A8 (23:6), 1H2(32:10), and 1H3 (29:13). Thus, these segregating T2 populations contain dndl plants complemented by the cosmid transgene, as well as noncomplemented mutant dndl plants. 15 As expected, T2 plants of wild-type size exhibited defense responses similar to that of Col-O, while T2 plants of dwarf stature displayed defense responses comparable to that of dndl plants. The trademark phenotype of dndl is the absense of a HR while challenged with avirulent Psg. However, dndl plants transformed with cosmid 1A8 or 1H2, that were of wild type size, displayed a strong HR response to Psg R4(avrRpt2*) (Fig. 10). Conversely, dwarf 20 T2 plants were defective in HR cell death indicating that these dndl plants did not contain a complementing cosmid transgene (Fig. 10). Another defense response characteristic of dndl mutation is elevated resistance to virulent pathogens. T2 plants transformed with cosmid 1H3 that were wild-type in size were 25 susceptible to Pst DC3000 (i.e. their response mirrored that of Col-0). As shown in Fig. 11, a day three growth analysis of dwarf T2 plants provided data to indicate that these plants retained elevated resistance characteristic of the dnd1 mutation. Thus these plants did not contain a complementing cosmid transgene. 30 A series of subcloning and subsequent functional testing as described above yielded the subclones and complementation data summarized in Figure 12. Note in particular that the 19 WO 01/07596 PCT/USOO/20216 generic region encoding DND1 was closely delineated by successful complementation with subclones 18B and 27.1, and by the failure to complement with 56.2 or 61.1. Subcloning also yielded a clone (17.1) of 5.2 kb in length. A nucleotide BLAST search with partial sequence data generated from the clone yielded a perfect 470 bp match to Arabidopsis thaliana cyclic 5 nucleotide-gated cation channel AtCNGC2 mRNA (Accession ATY 1628). This cDNA was obtained by screening an Arabidopsis EST database with the cyclic nucleotide binding domain of a mammalian ion channel [Kohler et al. (1998) The Plant Journal 18(1):97-104]. It has a 2178 bp open reading frame that encodes a 726 amino acid protein marked by a cyclic nucleotide binding domain in the C-terminus, a putative calmodulin binding site, and 10 hydrophobic regions at the N-terminus (Figs. 6 and 7). Sequencing of the genomic DNA spanning the AtCNGC2 cDNA revealed that DND1 (AtCNGC2) is a 3327 bp gene composed of 8 exons (Fig. 6). Subsequent cloning and sequencing identified the nature of the dndl mutation to be a G to A transition creating a premature stop codon at amino acid 120 (Fig. 6). 15 The dnd2 mutant was analyzed similarly according to the procedure established for characterizing the dndl mutant as disclosed herein and found to be similar to the dndl mutant in most aspects; whole plant phenotypic data for dnd 1 were representative of similar data collected for dnd 1 plants. The dnd2 mutation was recessive to wild type, and homozygous dnd2/dnd2 mutant plants exhibited an extreme reduction in the extent of HR cell death in 20 response to avirulent P. syringae. The dnd2 mutant plants also exhibited a dwarf (smaller sized) plant growth habit, constitutively elevated levels of free- and conjugated-salicylic acid in leaf tissues, and a constitutive broad spectrum defense phenotype that resembles plants induced for systemic acquired resistance. The phenotypes of dnd2 mutant plants cosegregated as a single Mendelian locus in the F2 progeny of crosses to wild type. However, it was noted 25 that dnd2 plants do differ from dndl plants in one phenotypic respect; they tend to become chlorotic or yellowed at the leaf tips and distal lateral margins of leaves at a time when most leaves of wild type Arabidopsis or dndl Arabidopsis do not show this yellowing. While DND1 maps to the upper arm of Arabidopsis chromosome 5, DND2 maps to the 30 lower arm of that chromosome 5. The DND2 gene maps to the genetic interval flanked by the 20 WO 01/07596 PCT/USOO/20216 available PCR based, polymorphism-detecting genetic markers ngal29 and LFY3 (www.arabidopsis.org). Further genetic mapping of the DND2 locus using F2 individuals and F3 families from 5 a cross of Col-0 dnd2-1/dnd2-1 to ecotype No-O has refined the site of the DND2 locus to the genetic interval between g4130 and K19P17. This corresponds to a genetic size approximately 2.5 centiMorgans, spanned by six overlapping BAC clones, covering approximately 400 kb of Arabidopsis genome. It was noted that the Arabidopsis genome within this interval has recently been sequenced, annotated, and released to Genbank. A survey of the genes encoded 10 within this interval revealed a putative cyclic nucleotide-gated ion channel (CNGC) encoding gene, termed AtCNGC1 (Kohler and Neuhaus 1998, supra). PCR primers were designed to amplify the segment of Arabidopsis ecotype Col-0 wild type genomic DNA shown in Fig. 13 and SEQ ID NO:4. The primer sequences were: 15 MFH813.9X (SEQ ID NO:7), 5'-ATCCGCTCGAGTGATTGGTTTCGTCTTGTCC-3'; and MFH819.9B (SEQ ID NO:8), 5'-TTCGCGGATCCTATGCACTGTGCCTGTGTGA-3'. The resulting PCR product DNA spanned the entire AtCNGC1-coding sequence (see Fig. 14 and SEQ ID NO:5) as well as roughly 2 kb of upstream DNA (putative promoter region) and roughly 0.5 kb of downstream DNA (putative terminator region). High-fidelity DNA 20 polymerase (Taq polymerase, Stratagene Co. La Jolla, CA) was used in the polymerase chain reaction together with the above primers and template to generate the expected product. This product was cloned into the Agrobacterium/plant transformation-competent plasmid vector pCLD04541 [Jones, J.D.G. et al. (1992) Transgenic Research 1:285-297]. The resulting products (from three independent PCR reactions), named pACol-01-la, pSCol-07-23a, and 25 pZCol-08-27c, were moved in to Agrobacterium tumefaciens and used to genetically transform Arabidopsis Col-0 dnd2-1/dnd2-1 plants, using the "floral dip" method [Clough and Bent (1998) Plant J. 16:735-743]). Putative transformants were identified by selection on kanamycin plates using standard methods. These putative transformants were then transplanted to soil while still very young (roughly ten days old). After growth for an additional few 30 weeks, it became apparent that with all three plasmid constructs, the transformed dnd2 mutant plants had been phenotypically complemented and resembled wild-type rather than dnd2. As 21 WO 01/07596 PCT/USOO/20216 in the successful positional cloning of DND1, this was initially determined by observation of plant size [Clough et al. (2000) Proc.Natl.Acad. Sci. (USA) in press]. Control dnd2 plants transformed with pCLD04541 vector that does not contain AtCNGC1-spanning DNA did not exhibit phenotypic complementation (see Fig. 16). 5 In summary, the DND1 and DND2 genes discovered initially by their phenotypic characteristics, i.e., enhanced disease resistance and suppression of HR cell death, both encode protein products with clear similarity to mammalian and other metazoan cyclic nucleotide-gated ion channels [Kohler and Neuhaus (1998) Supra; Kohler et al. (1999) Plant 10 J. 18:97-104; Leng et al. (1999) Plant Physiol 121:753-761]. cDNAs derived from these loci have been studied by other groups. Recent studies by Leng et al. demonstrated that the product of AtCNGC2 is indeed a functional ion channel that is gated by cyclic nucleotides. However, the present invention is the first disclosure that makes the critical connection between cyclic nucleotide-gated ion channel genes and the disease resistance/suppression of 15 cell death functions of the mutated DND genes. Accordingly, this invention provides methods of making disease resistant plants by manipulating either a DND gene (or gene product) or a cyclic nucleotide-gated ion channel gene (or gene product). The discovery of the AtCNGC2/DND1 and AtCNGC1/DND2 genes as regulators of 20 disease resistance together with availability of the genomic sequence information make it possible that plant disease resistance or cell death can be manipulated by the recombinant DNA technology well known in the art. For example, one skilled in the art can use the nucleotide sequences of the AtCNGC/DND genes disclosed herein to isolate related genes in other plants. The DND 1 and 2 genes share about 46% sequence identity at the nucleotide level in the 25 coding region. It is likely that a functional or structural homolog of the AtCNGC2/DND1 and AtCNGC1/DND2 genes would share similar sequence homology. Once identified, these genes can be employed to improve disease resistance. The CNGC/DND protein or a homolog thereof can be modified by substitution of amino acid residues, deletions, additions, and the like. Mutants generated may exhibit diverse phenotypes in addition to varying degrees of 30 pathogen resistance. A mutant (or mutants) exhibiting an enhanced disease resistance without a dwarfed stature can be isolated. Methods for mutagenesis and nucleotide sequence alterations 21.

WO 01/07596 PCT/USOO/20216 are well known in the art. See, for example, Kunkel, T. (1985) Proc. Nati. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382. Alternatively, the disease resistance can be enhanced by inactivating or downregulating 5 the CNGC/DND gene or a homolog thereof. The DND1 genomic sequence shown in Fig. 6 contains about 1.6 kb 5' flanking sequence in addition to introns and exons, and about 700 nucleotides of 3' flanking region. The DND2 genomic sequence shown in Fig. 13 contains about 2 kb 5' flanking sequence and about 0.5 kb 3' flanking sequence in addition to the coding sequence for AtCNGC1. The flanking sequences surrounding the gene generally contain 10 various regulatory sequences which control expression of the gene, either transcriptionally or translationally. Therefore, the AtCNGC2/DND1 or AtCNGC1/DND2 gene expression can be down-regulated or inactivated by either transcriptionally or translationally. Similarly, one skilled in the art can derive any antisense molecule based on the sequences shown herein including the splice sites (i.e. intron-exon junction) to inactivate or down-regulate the 15 CNGC/DND gene. Sense-strand suppression, virus-induced gene silencing, double-strand RNA and other inactivation methods are also applicable [Hamilton and Baulcombe (1999) Science 286:950-952; Somerville, C. et al. (1999) Science 285:380-383; Jorgensen, et al. U.S. Patent No. 5,283,184]. The flanking sequences containing regulatory elements for transcription can also be used to identify compositions which inhibit CNGC/DND gene 20 expression. The DND1 and DND2 genes are highly related as evidenced by the sequence homology (~46% identity at the nucleotide level). Kohler et al (1999) reported a gene family of 6 putative CNGCs in Arabidopsis thaliana which share significant structural homology. One 25 skilled in the art can easily utilize the nucleotide sequences encoding the DND1 and DND2 genes provided herein to isolate additional potential disease resistance genes. The nucleotide sequences encoding the AtCNGC2/DND1 and AtCNGC1/DND2 can be utilized to isolate homologous genes from other plants including sorghum, Brassica, 30 wheat, tobacco, cotton, barley, sunflower, cucumber, alfalfa, soybeans, sorghum etc. Coding 23 WO 01/07596 PCT/USOO/20216 sequences from other plants may be isolated according to well known techniques based on their sequence homology to the AtCNGC2/DNDJ or AtCNGC1/DND2 coding sequences set forth herein SEQ ID NOs: 2 and 5. In these techniques all or part of the known coding sequence is used as a probe which selectively hybridizes to other disease resistance coding sequences 5 present in genomic or cDNA libraries from a chosen organism, or genomic sequence, or coding sequences are used to design PCR primers for the same purpose. Alternatively, homologous genes can be identified from the EST or genomic sequence databases using AtCNGC2/DND1 or AtCNGC1/DND2 genomic or cDNA sequences. Similarly, searching can utilize the entire AtCNGC2/DND1 or AtCNGC1/DND2 gene or derived amino acid sequence, 10 or subdomains thereof. Methods for similarly searching can be found in Brenner, S. and Lewitter, F., editors (1998) Trends Guide to Bioinformatics., Elsevier Science Ltd., Oxford, U.K. Identification of AtCNGC2/DND1 or AtCNGC1/DND2 and their homologs in other plants may facilitate identification of effector genes that interact with AtCNGC2/DND1 or AtCNGC1/DND2 or their homolog gene or gene product; or the identification of effector 15 chemicals or other interventions that alter AtCNGC2/DND1 or AtCNGC1/DND2 function in a desirable fashion. A detailed protocol for these experiments including hybridization screening of plated DNA libraries can be found in Sambrook et al., Molecular Cloning, eds., Cold Spring Harbor Laboratory Press (1989); Ausubel, F.M. et al. (1997) "Current Protocols in Molecular Biology" Wiley, New York. 20 For example, hybridization of such sequences may be carried out under conditions of reduced stringency, medium stringency or even high stringency conditions (e.g., conditions represented by a wash stringency of 35-40% Formamide with 5x Denhardt's solution, 0.5% SDS and lx SSPE at 37 C; conditions represented by a wash stringency of 40-45 % Formamide 25 with 5x Denhardt's solution, 0.5% SDS and 1x SSPE at 42'C; and conditions represented by a wash stringency of 50% Formamide with 5x Denhardt's solution, 0.5% SDS and 1x SSPE at 42'C, respectively), to DNA encoding the disease resistance genes disclosed herein in a standard hybridization assay. 30 Mutation of a cyclic nucleotide-gated ion channel gene in plants other than Arabidopsis can be used in a conventional plant breeding program to introduce a dnd phenotype into an elite 24 WO 01/07596 PCT/USOO/20216 variety. Such mutations can be identified as described herein for Arabidopsis. The breeding is facilitated by identifying one or more markers linked to the DAD gene. Such markers can include conventional markers or molecular markers such as RFLP or SSR markers. For example, SSR (simple sequence repeat) markers have been mapped for the entire soybean 5 genome and are publicly available from USDA (see http:SoyBase.agron.iastate.edu) or from Research Genetics Inc., Huntsville, AL. Conventional mapping methods are used to identify one or more SSR markers linked to the DND locus. Similar molecular markers are available for most agronomic crops. By conventional breeding, a suitable DND mutant allele can be introgressed into a desired commercial soybean line by following an appropriate linked SSR 10 marker during crossing and backcrossing, as is known in the art. The same process outlined above can be used, with appropriate markers, for crossing DND mutations into other plant varieties. The methods of the present invention and methods known in the art can be used to 15 transform any plant. In this manner, genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained. Transformation protocols may vary depending on the type of plant or plant cell, i.e. monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection [Crossway et al. (1986) Biotechniques 4:320 334]; electroporation [Riggs et al. Proc. Natl. Acad. Sci. USA. 83:5602-5606]; Agrobacterium 20 mediated transformation [Hinchee et al. Biotechnology 6:915-921]; direct gene transfer [Paszkowski et al. (1984) EMBO J. 3:2717-2722]; and ballistic particle acceleration [see, for example, Sanford et al., U.S. patent 4,945,050; and McCabe et al. (1988) Biotechnology 6:923-926]. Also see Weissinger et al. (1988) Annual Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant 25 Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA. 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839; and Tomes et al. "Direct DNA transfer into intact plant cells via microprojectile bombardment" In 30 Gamborg and Phillips (Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods, Springer-Verlag, Berlin (1995); Hooydaas-Van Slogteren & Hooykaas (1984) Nature (London), 25 WO 01/07596 PCT/USOO/20216 311:763-764; Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA. 84:5345-5349 (liliaceae); De Wet et al. (1985) In The Experimental Manipulation of Ovule Tissues, ed. G.P. Chapman et al., pp. 197-209; Longman, NY (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418; and Kaeppler et al. (1992) Theor. Apple. Genet. 84:560-566 (whisker-mediated transformation); 5 D'Halluin et al. (1992) Plant Cell 4:1495;1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference. 10 The cells which have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic 15 characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved. Efficient regeneration of plants from single cells or protoplasts is essential in the genetic manipulation of plants using various gene transfer technologies. The detailed protocols for 20 such procedures can be found in the following references: Li, H.Q. et al., (1996) Nat. Biotechnol. 14(6):736-740; Ghosh Biswas, G.C. et al. (1994) J. Biotechnol. 32(1): 1-10; Datta, S.K. et al. (1992) Plant Mol. Biol. 20(4):619-629; and Lorz, H. et al. (1979) Planta. Med. 36(1):21-29. 25 As noted earlier, the nucleotide sequences of the invention can be utilized to protect plants from disease, particularly those caused by plant pathogens. Pathogens of the invention include, but are not limited to, viruses or viroids, bacteria, fungi, and the like. Specific examples of these pathogens include, but are not limited to, the pathogens listed in Table I. 30 The identification of the DND1 and DND2 genes as cyclic nucleotide-gated channel genes in Arabidopsis offers additional means to identify compositions which can enhance 26 WO 01/07596 PCT/USOO/20216 disease resistance in plants. Plant tissue cultures and recombinant plant cells containing the proteins and nucleotide sequences of CNGC/DND gene, or transgenic cells of other species such as Eschericha coli or Saccharomyces cerevisae or Xenopus laevis that express the CNGC/DND protein or the purified CNGC/DND protein may be used in an assay to screen 5 compositions which inhibit the function of the cyclic nucleotide-gated channel protein. Such an assay is useful as a general screen to identify compositions which inhibit AtCNGC2/DND1 or AtCNGC1/DND2 protein activity. The detailed assay protocol for measuring the channel activity can be found in Leng et al. (1999) Plant Physiol. 121:753-761. A composition that results in less channel activity upon addition to the assay, compared to that of control, is 10 defined as an inhibitor. If such a composition is found, it would be useful to enhance disease resistance in plants. As discussed, the genes of the invention can be manipulated to enhance disease resistance and/or cell death in plants. In this manner, the expression or activity of the 15 AtCNGC2/DND1 (or AtCNGC1/DND2) or other disease resistance genes can be altered. Such means for alteration of the gene include co-suppression, antisense, mutagenesis, alteration of the sub-cellular localization of the protein, etc. In some instances, it may be beneficial to express the gene from an inducible promoter, particularly from a pathogen inducible promoter or from a tissue-specific or growth-stage-specific promoter, or by a chemical-spray induced 20 fashion (see U.S. 5,689,042, U.S. 6,008,436, U.S. 5,589,622, and U.S. 5,789,214). Such promoters include those from pathogenesis-related proteins (PR proteins) which are induced following infection by a pathogen; e.g. PR proteins, SAR proteins, beta-1, 3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) The Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111 25 116. Plants homozygous for the dndl or dnd2 mutation exhibit substantial suppression of "hypersensitive response" (HR) cell death, a form of localized cell death associated with "gene for-gene" plant disease resistance and also associated with some instances of pathogen-induced 30 necrosis as part of disease damage or susceptibility. This cell death is beneficial to the plant in some instances but is deleterious in others. Plant cell death can be partially or completely 27 WO 01/07596 PCT/USOO/20216 controlled by similar modifications of the AtCNGC2/DND1 or AtCNGCJ/DND2 gene or homolog thereof as disclosed in the present invention. EXAMPLES 5 The following examples are provided for illustrative purposes, and are not intended to limit the scope of the invention as claimed herein. Any variations in the exemplified articles which occur to the skilled artisan are intended to fall within the scope of the present invention. 10 Example 1. Inoculations with P. syringae. Original mutants and their progeny were tested for the HR by pipet inoculation of individual leaves with P. syringae pv. glycinia Race 4 pV288 (asvrRp2+) or Race 4 p VSP61 (no avr gene) at ~2 x 108 colony forming units (cfu)/ml (19, 20). Additional P. syringae strains used to test for gene-for-gene HR included P. syringae pv glycinia Race 4 pAvrRpml 15 (avrRpml*) and Race 4 pVB01 (avrB*) [Kunkel, (1993) supra; Bisgrove, (1994) supra]. Positive and negative Arabidopsis controls included the use of wild-type Col-0, Col-0 rps 201/rps2-201, and Col-0 rpm/rpm] ("rps3-1 ") mutants [Kunkel, (1993) supra; Bisgrove, (1994) supra.] For bacterial growth experiments and for gene expression studies, P. syringae pv. tomato strain DC3000 and P. syringae pv. maculicola strain 4326 were used with the 20 above plasmids or with pKec218 (avrRps4 4 ) [Hinsch, M. et al. (1996) Mol. Plant-Microbe Interact. 9:55-61]. Quantitative determinations of bacterial growth in leaves were performed by dilution plating of homogenized leaf tissue on selective media, as described in Whalen, (1991) supra. 25 Example 2. Mutant Screen and Crossing. Arabidopsis thaliana ecotype Col-0 seeds were mutagenized with ethyl methane sulfonate; M2 populations were obtained from Lehle Seeds (Round Rock, TX). To test for activation of the HR, P. syringae pv. glycinia Race 4 pV288 (avrRpt*), at a concentration of =2 x 108 cfu/ml in 10 mM MgCl,, was introduced by vacuum infiltration into leaf mesophyll 30 tissue of z 11,000 M2 seedlings. Leaves were observed 24 and 40 h after infiltration, and plants with reduced, delayed, or no leaf collapse were saved for further analysis. Lines of 28 WO 01/07596 PCT/USOO/20216 potential interest were crossed with the wild-type Col-0 parent to initiate backcrossing and with ecotype No-O to initiate genetic mapping. For complementation tests, Arabidopsis Col-0 dnd/dndl plants were crossed to homozygous cprl and cpr5 mutants, which also display a reduced rosette size [Bowling, S.A. et al. 1997) Plant Cell 9:1573-1584; Cao, (1994) 5 Dominance/recessiveness and genetic complementation were deduced by observation that all F1 plants were silt-type in appearance and displayed the HR after inoculation with P. syringae pv glycinia Race 4 pV288. Example 3. Microscopy. 10 To monitor HR cell death at the cellular level, pipet infiltration was used to introduceP. syringae pv. glycinia Race 4 pV288 (avrRpt*) or Race 4 pVSP61 (no avr gene) into 40-=70% of the mesophyll space of individual leaves, at the bacterial concentrations indicated. Leaves were removed from plants after 24 h, fixed in 2% formaldehyde, 5% acetic acid, and 40% ethanol for 30 min, and then cleared sequentially in 50% ethanol and 95% ethanol [Yu, (1993) 15 supra]. Leaf parenchyma cells then were examined for HR-associated autofluorescence by using fluorescence microscopy with a fluorescein filter set (Ex 495 + 20 nm, Em > 505 nm [Klement, (1990) supra]. Alternatively, Evan's Blue (Sigma) was infiltrated into leaves as a 1 % aqueous solution 22-26 h after pathogen inoculation [Klement, (1990) supra]. After at least 10 min of staining, leaves were removed from plants, a portion of the epidermis was peeled 20 back, and leaves were rinsed in H 2 0, mounted in H 2 0, and observed by light microscopy. Leaf areas damaged by physical handling were not considered when evaluating the proportion of dead and living cells. Example 4. Genetic Mapping. 25 F2 populations from a No-0 x Col-0 dndl/dndl cross were used for mapping. The HR phenotype was assessed visually 24 and 48 h after pipet inoculation of leaves with P. syringae pv. glycinia Race 4 pV288 (avrRpt*) resuspended to ~1 x 10' cfu/ml in 10 mM MgCl 2 Informative F2 lines were retested for HR in selfed F3 families. PCR-based cleaved amplified polymorphic sequence and microsatellite markers were used as described in Bell, C.J. et al. 30 (1994) Genomics 19:137-144; and Konieczny, A. et al. (1993) Plant J. 4:403-410; a set of 17 markers spanning all five Arabidopsis chromosomes was used for initial linkage analysis. 29 WO 01/07596 PCT/USOO/20216 Example 5. Inoculations with Other Pathogens. Tobacco ringspot virus grape strain was applied to plants, and virus multiplication was monitored by using ELISA as described in Lee, (1996) supra. Xanthomonas campestris pv. campestris strain 2669 [Parker, J.E. et al. (1993) Mol. Plant-Microbe Interact. 6:216-224] 5 were applied at a concentration of ~ 1 x 10 7 cfu/ml and monitored as described in Parker, (1993) supra. Peronospora parasitica isolate Noco2 was applied and monitored as described in Parker, J.E. et al. (1997) Trends Biochem. Sci. 22:291-296. For all experiments, Arabidopsis ecotype Col-0 served as a susceptible control for pathogen multiplication and virulence. 10 Example 6. Gene Expression Studies. P. syringae pv. tomato strains DC3000 (pV288) or DC3000 (pVSP61) were introduced into leaf mesophyll of intact plants by vacuum infiltration (as above), typically at a dose of 5 x 10 4 cfu/ml. Total RNA was extracted from leaf material and equal quantities of RNA from each sample were separated in agarose-formaldehyde gels, blotted, and hybridized with 1 2

P

15 radiolabeled probe essentially as described in Ausubel, (1997) supra. DNA probes were from Cao et al. [Cao, (1994) supra]. Hybridization was quantified by using a storage phosphor imaging system according to the manufacturer's instructions (Molecular Dynamics). Signal for PR-1 or p-glutanase in each lane was normalized to the control P-ATPase signal for that lane to correct for slight differences in gel loading, and normalized signals then were divided 20 by the signal for the Col-0/no-pathogen sample to establish a relative scale. Example 7. Salicylic Acid Determinations. Salicylic acid determinations were performed as described in Uknes, S. et al. (1993) Mol. Plant-Microbe Interact. 6:692-698 on leaf material from uninoculated 6-week-old plants. 25 Example 8. Functional Complementation of the dndl phenotype. Tri-parental mating: In order to transform the cosmids into Arabidopsis for complementation studies, the cosmids were put into Agrobacterium. Members of the cosmid library were transferred to Agrobacterium tumefaciens strain GV3 101 via a tri-parental mating. 30 Liquid cultures were prepared for each of the parents: GV3101 (pMP90), E. coli strain HB101 containing the mating helper plasmid pRK2013, and the cosmid-bearing E. coli XL-1 donor. 30 WO 01/07596 PCT/USOO/20216 Cultures were spotted on LBA media (no antibiotics) such that an approximate 5:1:1 ratio of recipient, helper, and donor was achieved within a single spot for each cosmid. These mating spots were grown overnight at 28 C. The next day each mating spot was re-streaked onto low salt LB media (10g tryptone, 5 g yeast extract, and 5 g NaCl/liter) + tetracycline (2.5 ig/ml) 5 + rifampicin (100 yg/ml) + gentamycin (50 tg/ml) and grown at 28'C for two days. Colonies were picked from these plates and re-streaked unto low-salt LBA (1.5% agar) containing rifampicin (100 yg/ml) and kanamycin (25 tg/ml) to select for Agrobacterium colonies containing a cosmid vector. 10 Arabidopsis transformation: Mutant dndl plants were transformed with Agrobacterium harboring cosmid clones via the floral dip method (Clough and Bent, 1998). bacterial cultures (150 ml) of each cosmid were grown overnight at 28'C in low-salt LB +kanamycin (25 ytg/ml), spun at 6,000 rpm for 15 minutes. Bacteria were resuspended in 5% sucrose spiked with .05 % surfactant Silwet L-77 (Osi Specialities, Inc.). Mutant dnd1 plants were grown in 15 3.5 inch pots with mounded soil fettered with tulle under 8-15 hr. light in the greenhouse until they displayed primary bolts. Plants were dipped into the Agrobacterium solution for 2-5 sec. and then placed under a dome overnight. At this time, plants were moved to 15 hr. light. Seeds were harvested 3-5 weeks later, once the siliques were brown and thoroughly dry. After 2-3 days of additional drying in 1.5 ml micro-centrifuge tubes on the lab bench, seeds were 20 sterilized either by liquid or vapor-phase sterilization as described (Appendix 4). Sterilized seeds were re-suspended in sterile .1 % agarose and plated on kanamycin (50 ug/ml) selection plates. Typically, -3000 seeds were plated per 150 x 15 mm petri plate. After 7-10 days of growth under 24 hr. light, kanamycin resistant seedlings with green leaves and well-established root systems were deemed putative transformants and were transplanted to soil for further 25 analysis. Because dnd mutants are 10OX more recalcitrant to transformation than wild-type, several plates of seeds (sometimes 5-10) were screened in order to obtain a few putative transformants. After putative transformants were obtained and transplanted to soil, they were grown 30 for an additional 3-4 weeks in a growth chamber under 8 hr. light. At this time, the sizes of individual transformants were compared to that of a wild-type Col-0 control (line A2 1, a vector 31 WO 01/07596 PCT/US00/20216 control transformant that is wild-type size and kanamycin resistant), that was similarly selected on kanamycin plates and transplanted to soil. Putatively complementing cosmids were identified based on size and were allowed to self and T2 seeds were harvested from these plants for further analysis. 5 Bacterial Growth Curves: In addition to affecting plant size, the dndl mutation also affects pathogen growth and the ability to produce a HR in response to avirulent pathogen. To verify that cosmids complementing the dwarf phenotype of dndl actually complemented the DND1 locus, reversion of these other characteristic phenotypes of dndl were also examined 10 in these plants. Growth curves were performed on T2 plants transformed with a complementing cosmid, as well as on Col-0 and dndl controls. Plants were vacuum-infiltrated with a p.s. pv. tomato DC3000 (Pst DC3000) carrying either avrRP2 (pV288) or no avr gene vector only). Approximately 5 x 104 cfu/ml bacteria were used for each inoculation (O.D.6,+.005). This level of pathogen effectively mimics the low pathogen levels that occur 15 during natural infections. For each sample, two leaf discs were taken from each of two plants, in triplicate, using a #1 cork borer. Thus, for each plant/pathogen combination 12 leaf discs were sampled per time point. Samples were collected at 0, 2, and 4 days post inoculation (or just at 3 days). Leaf discs were harvested into a 1.5 ml micro-centrifuge tubes with 200 PI 10 mM MgCl, ground with a pestle, and diluted serially onto NYGA (5g Bacto-peptone, 3 g 20 yeast extract, 20 ml glycerol, and 15 g agar/liter) + rifampicin (100 stg/ml) + cycloheximide (50 pg/ml), then grown for two days at 28'C. Colonies were counted and the data was analyzed using Sigma Plot (Jandel Scientific, CO). HR Assay: HR assays were performed on T2 from two complementing cosmids, in 25 addition to Col-0 and dndl controls. Inoculation with high levels of P.s. glycinia Race 4 (Psg R4) (obtained from N.T. Keen, Univ. of California-Riverside) carrying avrRpt2 induces the HR (visible leaf collapse) in incompatible reactions. Plants were inoculated with 2 x 108 cfu/ml (O.D.6, =.2) bacteria with a syringe and were scored for visible leaf collapse 24 hours after inoculation. The severity of HR was rated on a 0-5 scale (0 = no collapse, 5 = total 30 collapse). For each plant, three leaves were inoculated with Psg R4 (pV288) (with avr gene) and one leaf was inoculated with Psg R4 (pVSP61) (no avr gene). 32 WO 01/07596 PCTUSOO/20216 Example 9. Modification of Soybean Plants to enhance Disease Resistance and/or reduced Cell Death. As one example of a use of the present invention, soybean plants can be engineered to exhibit enhanced disease resistance and/or reduced cell death following infection by a 5 pathogen. A soybean DNA sequence encoding a cyclic nucleotide-gated ion channel homologous to one of the DNA sequences described herein can be obtained from information and materials available in the art, without undue experimentation. Information currently available in genomic sequence databases include EST DNA sequences for cDNAs isolated from soybean. Recently, an EST clone (Genbank Accession AW 781088) was identified as a 10 putative CNGC of soybean. Similarly, multiple EST clones have been identified to encode putative CNGC proteins of other plant species including lotus japonicus, tomato, cotton, and watermelon. Using computer-assisted methods, one skilled in the art can derive a probable amino acid sequence encoded by a given cDNA. A researcher can readily identify within sequence databases a soybean DNA sequence that encodes a cyclic nucleotide binding domain 15 or other derived amino acid sequence motifs characteristic of cyclic nucleotide-gated ion channels. The complete DNA sequence for that cDNA, or for the corresponding region of soybean genomic DNA, is then determined, to complete the identification of the sequence as a DND/cyclic nucleotide-gated ion channel gene. An expression cassette is constructed for pathogen-induced expression of an antisense or sense gene. For this purpose, many different 20 pathogen-induced genes can serve as the source of a suitable promoter. For example, the promoter region of the pathogenesis-induced soybean PR-1 gene [Genbank accession AF136636, see also Ryals et al. (1996) Plant Cell 8:1809-1819; Raymond et al. (2000) Plant Cell 12:707-720] or another infection-induced promoter, is fused to a small (25-100 bp), medium (101-500 bp) or large (501 bp to full gene-length) segment of the soybean DND gene, 25 in sense or antisense-orientation relative to the promoter [Hamilton and Baulcombe (1999) supra; Jorgensen, et al. U.S. Patent 5,283,184; and, Bridges et al., U.S. 5,073,676]. This is followed by a standard transcriptional terminator such as the Agrobacterium tumefaciens nopaline synthase 3' terminator region. Using methods well-known to skilled artisans, the PR 1 promoter/antisense DND/nos terminator DNA or PR-1 promoter/sense DND/nos terminator 30 DNA construct is placed in a vector suitable for biolistic or Agrobacterium-mediated transformation of soybean, and then used to transform an agriculturally suitable soybean variety. Transformants are identified by the use of a co-transformed marker gene, using either 33 WO 01/07596 PCT/USOO/20216 a selectable marker such as kanamycin-resistance, or a screenable marker such as GUS. Transformants are regenerated following techniques known in the art, to produce mature plants. Fertile productive transgenic soybean lines carrying these DNA constructs are thereby created and identified. Plants are tested for pathogen-inducible expression of the PR-i 5 promoter/antisense DND/nos terminator DNA or PR-1 promoter/sense DND/nos terminator DNA construct. Plants can be further tested for transcriptional or translational silencing of expression of the endogenous soybean DND/cyclic nucleotide-gated ion channel gene. The silencing may arise only in infected tissues, or may arise systemically throughout much of the plant, and may arise due to a variety of molecular mechanisms. Resistance to pathogens or 10 pathogen-induced cell death can be assayed in the transgenic plants. Resistance may occur locally at the site of infection, or may extend systemically to many other portions of the infected plant, and may arise due to a variety of molecular mechanisms. Note that, in keeping with the epidemiology of many plant diseases, initial infections will often occur at a limited number of sites on the plant, so that induction of resistance at an early stage after infection can 15 reduce the spread of infection to other sites on the infected plant and can also reduce the spread of pathogen to other plants. As a second example of the present invention, soybean plants are engineered to exhibit enhanced disease resistance and/or reduced cell death induced by treatment with an inducing 20 chemical. A soybean DND/cyclic nucleotide-gated ion channel gene can be identified by the methods described in the previous paragraph or by other methods discussed herein. DNA constructs are created that contain a chemically inducible promoter such as that disclosed by Ryals et al. U.S. patent 5,789,214 fused to a small (25-100 bp), medium (101-500 bp) or large (501 bp to full gene-length) segment of the soybean DND gene, in sense or antisense 25 orientation relative to the promoter [Hamilton and Baulcombe (1999) supra; (Jorgensen, et al. U.S. Patent 5,283,184; Bridges, et al. supra]. This is followed by a standard transcriptional terminator such as the Agrobacterium tumefaciens nopaline synthase 3' terminator region. Using methods well-known to skilled artisans, the promoter/antisense DND/nos terminator DNA or the promoter/sense DND/nos terminator DNA construct is placed in a vector suitable 30 for biolistic or Agrobacterium-mediated transformation of soybean, and then used to transform an agriculturally suitable soybean variety. Identification and regeneration of transformants is 34 WO 01/07596 PCT/USOO/20216 carried out as described previously. Fertile productive transgenic soybean lines carrying this DNA construct are thereby created and identified. Plants are tested for chemically-inducible expression of the promoter/antisense DND/nos terminator DNA or the promoter/sense DND/nos terminator DNA construct. Plants can be further tested for transcriptional or 5 translational silencing of expression of the endogenous soybean DND/cyclic nucleotide-gated ion channel gene. The silencing may arise only in infected tissues, or may arise systemically throughout much of the plant, and may arise due to a variety of molecular mechanisms. Resistance to pathogens or pathogen-induced cell death can be assayed in the transgenic plants. Resistance may occur locally at the site of infection, or may extend systemically to many other 10 portions of the infected plant, and may arise due to a variety of molecular mechanisms. Note that, in keeping with the epidemiology of many plant diseases, initial infections will often occur at a limited number of sites on the plant and on a limited number of plants in a given field, so that induction of resistance at an early stage after the initial infection can reduce the spread of infection to other sites on the infected plant and can also reduce the spread of 15 pathogen to other plants. Induction of resistance in plants by chemical treatment prior to infection can reduce the susceptibility to disease of at-risk plants prior to the occurrence of infections. Techniques and agents for introducing and selecting for the presence of heterologous 20 DNA in plant cells and/or tissue are well-known. Genetic markers allowing for the selection of heterologous DNA in plant cells are well-known, e.g., genes carrying resistance to an antibiotic such as kanamycin, hygromycin, gentamicin, or bleomycin. The marker allows for selection of successfully transformed plant cells growing in the medium containing the appropriate antibiotic because they will carry the corresponding resistance gene. In most cases 25 the heterologous DNA which is inserted into plant cells contains a gene which encodes a selectable marker such as an antibiotic resistance marker, but this is not mandatory. An exemplary drug resistance marker is the gene whose expression results in kanamycin resistance, i.e., the chimeric gene containing nopaline synthetase promoter, Tn5 neomycin phosphotransferase II and nopaline synthetase 3' non-translated region described by Rogers et 30 al., Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press, Inc., San Diego, CA (1988). 35 WO 01/07596 PCTIUSOO/20216 Techniques for genetically engineering plant cells and/or tissue with an expression cassette comprising an inducible promoter or chimeric promoter fused to a heterologous coding sequence, including possibly an antisense DNA construct and/or a DNA construct designed to elicit double-stranded RNA-mediated gene silencing, followed by a transcription termination 5 sequence are to be introduced into the plant cell or tissue by Agrobacterium- mediated transformation, electroporation. microinjection, particle bombardment or other techniques known to the art. The expression cassette advantageously further contains a marker allowing selection of the heterologous DNA in the plant cell, e.g., a gene carrying resistance to an antibiotic such as kanamycin, hygromycin, gentamicin, or bleomycin. 10 A DNA construct carrying a plant-expressible gene or other DNA of interest can be inserted into the genome of a plant by any suitable method. Such methods may involve, for example, the use of liposomes, electroporation, diffusion, particle bombardment, microinjection, gene gun, chemicals that increase free DNA uptake, e.g., calcium phosphate 15 coprecipitation, viral vectors, and other techniques practiced in the art. Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, such as those disclosed by Herrera-Estrella (1983), Bevan (1983), Klee (1985) and EPO publication 120,516 (Schilperoort et al.). In addition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternative methods can be used 20 to insert the DNA constructs of this invention into plant cells. The choice of vector in which the DNA of interest is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g., replication, protein expression, and the host cell to be transformed, these being limitations inherent in the 25 art of constructing recombinant DNA molecules. The vector desirably includes a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally when introduced into a prokaryotic host cell, such as a bacterial host cell. Such replicons are well known in the art. In addition, preferred embodiments that include a prokaryotic replicon also include a gene 30 whose expression confers a selective advantage, such as a drug resistance, to the bacterial host cell when introduced into those transformed cells. 36 WO 01/07596 PCT/USOO/20216 Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline, among other selective agents. The neomycin phosphotransferase gene has the advantage that it is expressed in eukaryotic as well as prokaryotic cells. Those vectors that include a prokaryotic replicon also typically include convenient restriction sites for insertion of a recombinant DNA molecule of the present invention. Typical of such vector plasmids are pUC8, pUC9, pBR322, and pBR329 available from BioRad Laboratories (Richmond, CA) and pPL, pK and K223 available from Pharmacia (Piscataway, NJ), and pBLUESCRIPT and pBS available from Stratagene (La Jolla, CA). A vector of the 10 present invention may also be a Lambda phage vector including those Lambda vectors described in Molecular Cloning: A Laboratory Manual, Second Edition, Maniatis et al., eds., Cold Spring Harbor Press (1989) and the Lambda ZAP vectors available from Stratagene (La Jolla, CA). Other exemplary vectors include pCMU [Nilsson et al. (1989) Cell 58:707]. Other appropriate vectors may also be synthesized, according to known methods; for example, 15 vectors pCMU/Kb and pCMUII used in various applications herein are modifications of pCMUIV [Nilsson, (1989) supra]. Typical expression vectors capable of expressing a recombinant nucleic acid sequence in plant cells and capable of directing stable integration within the host plant cell include 20 vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al. (1987) Meth. in Enzymol. 153:253-277, and several other expression vector systems known to function in plants. See for example, Verma et al., No. W087/00551; Cocking and Davey (1987) Science 236:1259-1262. 25 A transgenic plant can be produced by any means known to the art, including but not limited to Agrobacterium tumefaciens-mediated DNA transfer, preferably with a disarmed T-DNA vector, electroporation, direct DNA transfer, and particle bombardment [See Davey et al. (1989) Plant Mol. Biol. 13:275; Walden and Schell (1990) Eur. J. Biochem. 192:563: Joersbo and Burnstedt (1991) Physiol. Plant. 81:256; Potrykus (1991) Annu. Rev. Plant 30 Physiol. Plant Mol. Biol. 42:205; Gasser and Fraley (1989) Science 244:1293; Leemans (1993) Bio/Technology 11:522; Beck et al. (1993) Bio/Technology 11:1524; Koziel et al. (1993) Bio/Technology 11:194; Vasil et al. (1993) Bio/Technology 11:1533 and Gelvin, S.B. (1999) Curr. Opin. Biotech. 9:227-232]. Techniques are well-known to the art for the introduction 37 WO 01/07596 PCT/USOO/20216 of DNA into monocots as well as dicots, as are the techniques for culturing such plant tissues and regenerating those tissues. Many of the procedures useful for practicing the present invention, whether or not 5 described herein in detail, are well known to those skilled in the art of plant molecular biology. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989) Molecular 10 Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, New York; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, New York; Wu (ed.) (1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold 15 Spring Harbor, New York; Old and Primrose (1981) Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, 20 Plenum Press, New York, Kaufman (1987) in Genetic Engineering Principles and Methods, J.K. Setlow, ed., Plenum Press, NY, pp. 155-198; Fitchen et al. (1993) Annu. Rev. Microbiol. 47:739-764; Tolstoshev et al. (1993) in Genomic Research in Molecular Medicine and Virology, Academic Press; Ausubel, F.M. et al. (1997) "Current Protocols in Molecular Biology" Wiley, New York. Abbreviations and nomenclature, where employed, are deemed 25 standard in the field and commonly used in professional journals such as those cited herein. All references cited in the present application are incorporated in their entirety herein by reference to the extent not inconsistent herewith. 30 38 WO 01/07596 PCTUSOO/2021 6 TABLE I Specific pathogens for the major crops Soybeans: Phytophthora megasperma fsp. glycinia, Macrophornina pt2aseo~llzafa, Rhizocto2a Soianl.., 50ierot2222a sclerociorun, .Fusariurn oxysporum, Diaporthe phaseolorum var. sojae (Phornoosis sojaell, -74anorrhe phasce.2orurn var-. cauiLivora, scieroziun rolfsj4-, Cercospora kikuchii, Cercospcra sojinja, Peronospora rnanshurica, Co!I.eucrichun demna:2urn, (Collecotichun :uncacun) , Corynespora cassiic.cla, Septorja glvcines, Phvllos.icta soJ-4co2.a, Aternar4La a-, ternaza, Pseudornonas syrincgae p.v,,. glycinea, Xan:hornonas carnoes~r-s p .v. chaseoli, -Alicrosrhaera di-Ffusa, Fusarur semi tect:U'm, Phialophora gregata, Soybean mosaic virus, Gornereiia gvcines, Tobacco Ring spot vir-as, Tob:acco Streak virus, Phakopsora pachyrhizi, Py-!iurn aphanidermaun Py'hi um ultinu, Pythi=r debaryanun, Tomato spotted wilt virus, H-eterodera glyc--:es Fusariurn solani; Canola: Albugo candida, Alternari1a brassicae, Leptoschaeria maculans, Rhiizocton.a solani, Sclerotirnia scleroziorun, !yoshafel brassiccola, Pythiun ultinur, Peronospora parasitica, F'usariun roseun, Alcernaria alt:ernaca; Aklial-fa: Claviba~er -nichiganese subsp. insidiosr, Pythiumn ultirnum, Pyhir irrec22lare, Pythiun splendens, Pythiuznr debaryanrn, Pythium aphani derra turn, Phycoph thcra mega sperra, Peronospora trifoliorun, Phoma rnedicaginis var. -ned--icaginis, Cercospora medicaginis, Pseudopeziza medicagin-is, Leptorochiia medicaginis, Filsariun oxysporun, :Rhizoc:onia solani, Urornyces scriatus, Coll etcnr cri-folli race -- and race 2, Lepzosphaer-ulLn- briosi-ana, Sternphlizrn botryosun, Stagcnospor-a meliloci, Scleroti-.n2C :rifolirun, Alfalfa Mosaic Virus, Verticil-iurah-ru, Xanth.crnonas camzpestris p.v. alfalfae, Aohanomryces eutei-ches, Sztemphylium herbarun, Scemrhyiumr alf~alfae; Wheat-: Pseudomonas syr-inge P.v. acro-faciens, Urocystis agropyri, Xanthornonas campestri's 39 WO 01/07596 PCT/USOO/20216 p.v. :ranslucens, Pseudormonas syringae P.v. syringae, Alcernaria alternata, Cladospori--m herbarur, Fusarium graminearurn, Fusariumn avenaceum, Pusarium7 culmorun, Us:zjlagc tricici, Ascochyca critici, Cephialosporiumn grmineumn, Collotecrichum grarniicola, Erysiphe araminis f.sp. crit:ici, Puccinia graminis f.sp. :ritici, Puccinia recondica f.sr. :ritici, Puccinia s:riiformis, Pyrenophora cfritici-repencis, Seporia nodorum, Septoria critic, Septoria avenae, Pseudocercosporefla herpocrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannornvces graminis var. critics, Pythium aphanidermatun, Pvthium arrhenomanes, Pvchium ultimum, Bipolaris sorokiniana, Barley Yellow Dwarf Vir:S, Brome Mosaic Virus, Soil Borne Wheat Mosaic Virus, Wheaz Streak Mosaic Virus, Wheat SDindle Streak Virus, American Wheat Striate Virus, Claviceps purDurea, Tilletia zritici, Tilletia laevis, Uscilago zritici, Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola, Pythium aphanidermacurn, High Plains Virus, European wheat striate virus; Sunflower: Plasmophora halstedii, Sclerotinia scierczrumn, Aster Yellows, Sercria helianthi, Phomoosis helianthi, Alternaria helianthi, Alternaria zinniae, Borrycis cinerea, -Phoma macdonaldii, Macrophomina phaseolina, E-rysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticilliurn dahliae, Erwinia carotovorum ov. carotovora, Cephalosporium acremonium, Phytophthcra cryptogea, Albugo tragopogonis; Corn: Fusarium mroniliforme var. subglutinans, Erwinia stewartii, Fusarium moniliforme, Gibberella zeae (Fusarium graminearun), Stenocarpella maydi (Diplodia maydis), Pythiurn irregulare, Pythium debarvanun, Pythium graminicola, Pythium splendens, Pythiun ulcimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis 0, T (Cochliobolus heterostrophus), Helrninthosporium carbonum I, II & II7 (Cochliobolus carbonum), Exserohilurn turcicum 7, 7I & 7II, Helminthospcrium pedicellaturn, Physoderma maydis, Phyllos:icta maydis, Kabatiella zeae, 40 WO 01/07596 PCT/USOO/20216 Colletotrichum graminicola, Cercosora zeae-maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina, Penicilliur oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganense subsp. nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chrysanthemi pv. zea, Erwinia corotovora, Cornstun: spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora maydis, Peronosclerospora sacchar2, Spacelotheca reiliana, Physopella zeae, Cephalosporiurm mavdis, Caphalosporium acremonium, Maize Chlorotic Mo:tle Virus, High Plains Virus, Maize Mosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize Stripe Virus, Maize Rough Dwarf Virus; Sorghum: Exserohilum curcicum, Colletotrichum graminicola (Clomerella graminicola), Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas svringae p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium moniliiforme, Alternaria alternate, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunaca, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipi cans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani, Acremonium strictun, Sclerochthona macrospora, Peronoscl erospora sorghi, Peronoscl erospora philippinensis, Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum, Pythium arrhenomanes, Pythium graminicola, etc. 41

Claims

1. A method for improving disease resistance in a plant by down-regulating, mutating or inactivating a cyclic nucleotide-gated ion channel (CNGC) DND gene or gene product.

2. The method of claim 1 wherein said disease is a result of a plant pathogen.

3. The method of claim 2 wherein said plant pathogen is selected from the group consisting of viruses, bacteria, and fungi.

4. The method of claim 3 wherein said pathogen is a virus selected from the nepovirus group including Tobacco ringspot virus.

5. The method of claim 3 wherein said pathogen is a gram-negative bacterium, including bacteria of the genus Pseudomonas or Xanthorionas, including Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. campestris.

6. The method of claim 3 wherein said pathogen is an ascomycete funcus, including fungi of the genus Erysiphe, including Eryshiphe orontii.

7. The method of claim 1 wherein said CNGC or DND gene is homologous to SEQ ID NO: 2.

8. The method of claim 7 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 2 under low stringency conditions.

9. The method of claim 7 wherein said CNGC or DATD gene is one hybridizing with SEQ ID NO: 2 under medium stringency conditions.

10. The method of claim 7 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 2 under high stringency conditions. 42 WO 01/07596 PCTIUSOO/20216

11. The method of claim 1 wherein said CNGC or DND gene is homologous to SEQ ID NO: 5.

12. The method of claim 11 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 5 under low stringency conditions.

13. The method of claim 11 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 5 under medium stringency conditions.

14. The method of claim 11 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 5 under high stringency conditions.

15. The method of claim 1 wherein said down regulation or inactivation is achieved by expressing a CNGC or DND antisense or sense molecule in said plant.

16. The method of claim 15 wherein the CNGC or DND antisense or sense molecule is expressed under control of an inducible promoter.

17. The method of claim 16 wherein the inducible promoter is a pathogen-inducible promoter.

18. A transformed plant or plant tissue or seed modified according to the method of claim 1.

19. A transformed plant or plant tissue or seed comprising a CNGC or DND antisense molecule.

20. A transformed plant or plant tissue or seed comprising a CNGC or DND sense molecule. 43 WO 01/07596 PCT/USOO/20216

21. A method for improving disease resistance in a plant by administering an inhibitor of CNGC activity into said plant.

22. A method for controlling cell death in a plant by down-regulating, mutating or inactivating a CNGC or DND gene or gene product.

23. The method of claim 22 wherein said CNGC or DND gene is homologous to SEQ ID NO:2.

24. The method of claim 23 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 2 under low stringency conditions.

25. The method of claim 23 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 2 under medium stringency conditions.

26. The method of claim 23 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 2 under high stringency conditions.

27. The method of claim 22 wherein said CNGC or DND gene is homologous to SEQ ID NO: 5.

28. The method of claim 27 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 5 under low stringency conditions.

29. The method of claim 27 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 5 under medium stringency conditions.

30. The method of claim 27 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 5 under high stringency conditions. 44 WO 01/07596 PCT/USOO/20216

31. The method of claim 22 wherein said down regulation or inactivation is achieved by using a CNGC or DND antisense or sense molecule.

32. The method of claim 31 wherein the sense or antisense molecule is expressed under control of an inducible promoter.

33. The method of claim 32 wherein the inducible promoter is a pathogen-inducible promoter.

34. A method for reducing hypersensitive response in response to a pathogen attack in a plant by down-regulating, mutating or inactivating a cyclic nucleotide-gated ion channel CNGC or DND gene or gene product.

35. The method of claim 34 wherein said pathogen is selected from the group consisting of viruses, bacteria, and fungi.

36. The method of claim 35 wherein said pathogen is a virus selected from the nepovirus group consisting of Tobacco ringspot virus.

37. The method of claim 35 wherein said pathogen is a gram-negative bacterium, including bacteria of the genus Pseudomonas or Xanthorionas, including Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. campestris.

38. The method of claim 35 wherein said pathogen is an ascomycete funcus, including fungi of the genus Erysiphe, including Eryshiphe orontii.

39. The method of claim 34 wherein said CNGC or DND gene is homologous to SEQ ID NO: 2.

40. The method of claim 39 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 2 under low stringency conditions. 45 WO 01/07596 PCTIUSOO/20216

41. The method of claim 39 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 2 under medium stringency conditions.

42. The method of claim 39 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 2 under high stringency conditions.

43. The method of claim 34 wherein said CNGC or DND gene is homologous to SEQ ID NO: 5.

44. The method of claim 43 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 5 under low stringency conditions.

45. The method of claim 43 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 5 under medium stringency conditions.

46. The method of claim 43 wherein said CNGC or DND gene is one hybridizing with SEQ ID NO: 5 under high stringency conditions.

47. The method of claim 34 wherein said down regulation or inactivation is achieved by expressing a CNGC or DND antisense or sense molecule in said plant.

48. The method of claim 47 wherein the sense or antisense molecule is expressed under control of an inducible promoter.

49. The method of claim 47 wherein the inducible promoter is a pathogen-inducible promoter.

50. A method for identifying a disease resistance gene in a plant by screening for a CNGC or DND gene. 46 WO 01/07596 PCT/USOO/20216

51. The method of claim 50 wherein said CNGC or DND gene is AtCNGC2/DND1 as given in SEQ ID NO:2.

52. The method of claim 50 wherein said CNGC or DND gene is AtCNGC1/DND2 as given in SEQ ID NO: 5. 47