WO2001007596A1 - Arabidopsis thaliana cyclic nucleotide-gated ion channel/dnd genes; regulators of plant disease resistance and cell death - Google Patents

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

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WO2001007596A1
WO2001007596A1 PCT/US2000/020216 US0020216W WO0107596A1 WO 2001007596 A1 WO2001007596 A1 WO 2001007596A1 US 0020216 W US0020216 W US 0020216W WO 0107596 A1 WO0107596 A1 WO 0107596A1
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gene
cngc
dnd
plant
seq
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Andrew F. Bent
I-Ching Yu
Steven J. Clough
Kevin A. Fengler
Robert K. Smith, Jr.
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Wisconsin Alumni Research Foundation
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Priority to AU63733/00A priority patent/AU6373300A/en
Priority to CA002378107A priority patent/CA2378107A1/en
Publication of WO2001007596A1 publication Critical patent/WO2001007596A1/en

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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance

Definitions

  • This invention relates to plant physiology, in particular, plant genes, termed cyclic nucleotide-gated ion channel genes or DND (Defense, No Death) genes as regulators for plant diseases and methods for controlling plant diseases.
  • DND cyclic nucleotide-gated ion channel genes
  • 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 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. 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, 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 [Mclntosh, R.A., et al., (1995) Wheat Rusts: An Atlas of 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] .
  • 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. (1941) Phytopathology 32:653-669] .
  • 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].
  • 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, (1996) supra; Dangl, J.L. et al. (1996) Plant Cell 8: 1793-1807] .
  • HR cell 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].
  • HR cell 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].
  • 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].
  • SAR systemic acquired resistance
  • Other defense pathways have been identified that are apparently independent of salicylic acid, such as many jasmonic acid-dependant defense responses [Penninckx, LA. et al. (1996) Plant Cell 8:1809-1819].
  • Multigenically controlled defense pathways form important barriers to infection, and plant breeding efforts are often devoted to improvement of these "quantitative" types of resistance [Agrios, (1997) supra].
  • Mol. Plant-Microbe Interactions 13:277-286 identified two Arabidopsis mutants, dndl and 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 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.
  • 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 (cDNAs of AtCNGC2 and AtCNGCl, respectively) [Kohler, C. etal (1999) Plant J. 18:97-104;
  • DND1 and DND2 as used herein are intended to be synonymous with tC7VGC2 and AtCNGCl , respectively. Note, however, that in this previous work by others on AtCNGC2 and AtCNGCl, no association was made with plant disease 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.
  • 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 plants.
  • the nucleotide coding sequences of the DN 1 and 2 genes of the present invention are identical to previously known cD ⁇ A molecules that encode proteins that function as cyclic nucleotide-gated ion channels 2 (AtCNGC2) and 1 (AtCNGCl), respectively. Such cyclic nucleotide-gated ion channels are ubiquitous in plants, generally.
  • AtCNGC/DNDl Plants that do not express AtCNGC/DND genes 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 Bl toxin. Therefore, this invention discloses various methods for improving disease resistance by modifying the AtCNGC2/DNDl or AtCNGCl IDND2 gene or gene product, or genes or gene products in other plants that share substantial structural or functional similarity to the AtCNGC2/DNDl or
  • AtCNGCl/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, sense-strand suppression, virus-induced gene silencing, double strand R ⁇ A 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.
  • the invention discloses methods for identifying other AtCNGC2/DNDl or AtCNGCl/DND2 related disease resistance genes or structural or functional homologs thereof.
  • the AtCNGC2/DNDl or AtCNGCl IDND2 related genes or homologs or gene products thereof thus identified can be modified as described herein to improve disease resistance.
  • These genes 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 of their similarity to or close genetic proximity to AtCNGC2/DNDl or AtCNGCl IDND2 or their proximity to homologs of AtCNGC2/DNDl or AtCNGCl IDND2.
  • 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.
  • 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 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.
  • 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.
  • the invention provides transformed or genetically modified plant, plant tissue or seed made by the described method.
  • the invention further provides a method for identifying a disease-resistance gene by screening for a cyclic nucleotide-gated ion channel gene, including AtCNGC2/DNDl and AtCNGCl IDND2 genes.
  • Figs. 1A-1D shows HR cell death defect in dndl mutant. Leaves of wild-type parent
  • Figs. 2A-2B illustrate the growth of bacteria within plant leaves.
  • Fig. 2A shows Arabidopsis lines Col (Col-0 wild-type, RPS2/RPS2; DNDl/DNDl), rps2 (Col-0 rps2- 201/rps2-201; DNDl/DNDl), and dndl (Col-0 and RPS2/RPS2; dndl /dndl) inoculated with P. syringae pv. tomato DC3000 pV288 (avrRpt2 + ).
  • Fig. 2B shows Arabidopsis lines Col-0 and dndl inoculated with isogenic P. syringae pv.
  • tomato DC3000 differing by the presence (pAvrRpml, filled symbols) or absence (pVSP ⁇ l, open symbols) of avirulence gene avrRpml carried on plasmid pVSP ⁇ l . Both plant lines are RRMl/RPMl genotype. All data points are mean + SD.
  • Figs. 3A-3C show pathogenesis-related gene expression monitored by RNA blot analysis of Col-0 wild-type (Col) and Col-0 dndl/dndl mutant (dndl) plants.
  • Fig. 3A illustrates ⁇ -glucanase expression 72 h after treatment of leaves with 10 mM MgCl 2 containing no pathogen ( ⁇ ), the nonavirulent control strain P. syringae pv. tomato DC3000 pVSP ⁇ l (vir), or the isogenic ⁇ vri?/?2-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 level of constitutive ⁇ -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-0 dndl-ldndl-1 or Col-0 dnd2-l/dnd2-l plants.
  • Figs. 5 A and 5B illustrate that the dndl and dnd2 mutant plants show more resistance to cell death induced by Fumonisin Bl (an inhibitor of ceramide synthase) compared to the wild type Arabidopsis.
  • Fig. 5A is a dose-response curve of Fumonisin Bl generated using control (Col) and dndl mutant plants.
  • Fig. 5B shows the delayed response of the dndl mutant plants after Fumonisin treatment compared to the wild type Arabidopsis.
  • Fig. 5A is a dose-response curve of Fumonisin Bl generated using control (Col) and dndl mutant plants.
  • Fig. 5B shows the delayed response of the dndl mutant plants after Fumonisin treatment compared to the wild type Arabidops
  • nt 1632 shows nucleotide sequences of the genomic region cont ⁇ ining /ltCNGC2/ ND 1 gene, 5,897 nucleotides in length.
  • the notable features are as follows: nt 1632; 5' end of the AtCNGC2/DNDl cD ⁇ A, nt 1663; ATG putative start codon, nt 1716; end of exon 1 of AtCNGC2/DNDl gene, nt 2088-2763; exon 2, nt 2928-3143 ;exon 3, nt 3333-3652; exon 4, nt 3747-3863; exon 5.
  • nt3953-4192 exon 6, nt 4275-4363; exon 7, nt 4478-5153; 3' end of
  • 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 ⁇ O:l
  • Fig. 7 shows the amino acid sequence (SEQ ID NO:3) of the protein encoded by the
  • Fig. 8 shows the nucleotide sequence of AtCNGC2IDNDl cDNA (SEQ ID NO: 2).
  • 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.
  • 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 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).
  • Fig. 11 shows growth of virulent P. syringae pv. tomato (pst) DC3000 in Col-0 dndl /dndl plants transformed with cosmid 1H3.
  • bacterial growth was sampled only at 3 days post inoculation (depicted by the X).
  • 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 Xbal, except where noted.
  • Fig 13 shows the nucleotide sequence of the genomic region containing the Arabidopsis DND2 (AtCNGCl) gene (SEQ ID NO:4).
  • Fig. 14 shows the nucleotide sequence of DND2 (AtCNGCl) cDNA (SEQ ID NO:5).
  • Fig. 15 shows the amino acid sequence of the protein encoded by the DND2 (AtCNGCl) gene (SEQ ID NO: 6).
  • Fig. 16 shows the results of the complementation studies of dndl small rosette size phenotype by transformation with the Arabidopsis genomic DNA fragment shown in Fig. 13 (SEQ ID NO: 4), encoding AtCNGCl /DND2.
  • Cold + vector represents the wild type plants transformed with vector only
  • dnd2 -I- vector represents the dndl mutant plants transformed with vector only
  • dnd2+ AtCNGCl represents the dnd mutant plants transformed with a vector containing the Arabidopsis genomic DNA frgment shown in Fig. 13 (SEQ ID NO:4), encodidng AtCNGCl IDND2.
  • down-regulation refers to a general method of reducing the level of gene products (RNA or protein).
  • RNA or protein gene products
  • down-regulation of a gene may be achieved either transcriptionally or translationally.
  • an antisense molecule may be introduced into a cell or tissue to down-regulate the gene from which the antisense molecule is derived.
  • mutation 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.
  • 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.
  • 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.
  • 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.
  • various laboratory tests have been 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, measurement of pathogen growth in the infected plant, measurements of extent of necrosis, plant cell death and hypersensitivity response.
  • 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.
  • an inoculated pathogen i.e. P. syringae pv. tomato
  • gene refers to a deoxynucleic acid molecule that encodes a protein or peptide upon transcription and translation.
  • gene product refers to either an RNA molecule or protein which is generated by expression of a given gene.
  • 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 invention. Accordingly, it includes not only the AtCNGC2/DNDl or AtCNGCl IDND2 gene of Arabidopsis but also those corresponding or related genes of other plant species which have structural or functional homology with the AtCNGC2/DNDl or AtCNGCl/DND2 gene disclosed herein.
  • AtCNGC2/DNDl or AtCNGCl IDND2 can exist in other plants, and that such variants can be identified, as herein described, by structural homology, by functional homology, or by similarity of phenotype in genetic analyses, or by any combination of the foregoing.
  • a structural homolog of the CNGC/DND gene is defined as one hybridizing with the Arabidopsis AtCNGC2/DNDl (SEQ ID NO: 2) or CNGC1/DND2 (SEQ 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 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 [Zaelles and Siegelbaum (1996) Ann. Rev. Neurosci. 19:235-263; Kohler, et al. (1999) supra].
  • a functional homolog of a N gene product is a cyclic nucleotide-gated ion 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.
  • 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 he DND 1 orDND2 gene offers new possibilities of controlling various plant diseases.
  • dndl and dnd2 mutants 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 mutant plants show an extreme reduction in the extent of cell death in response to avirulent P. syringae, and dwarfism. Because dndl and dndl 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 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. 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 dndl line is disrupted in a common component of the plant defense response that is shared by initially distinct gene-for-gene signal transduction pathways.
  • leaves from the wild-type parental line infected at this dose with P. syringae expressing avrRP2 contained numerous isolated autofluorescent cells.
  • 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.
  • Fig. 2B shows the growth of the virulent P. syringae pv. tomato strain DC3000 (pVSP ⁇ l) in wild-type Col-0 and in Col-0 dndl I 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 in studies with the virulent P. syringae pv. maculicola strain 4326.
  • the dndl plants express a level of resistance to virulent P.
  • 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 observed if the otherwise virulent P. syringae strains DC300 or 4326 expressed avirulence genes avrRpml 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.
  • Xanthomonas campestris pv. campestris andX c. pv. raphani (bacteria) only produced mild yellowing on dndl rather than the necrotic lesions produced on Col-0.
  • Peronospora parasitica (oomycete) produced threefold fewer spores on dndl as opposed to Col-0 [3.0 ⁇ 2.2 vs. 10.7 ⁇ 3,1 mean ⁇ SE if (spores x 10 3 ) per leaf].
  • Microscopy of leaves infected with virulent P. parasitica confirmed 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 typically had formed haustoria on 2-10 host cells in dndl plants, whereas in wild-type Col-0 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.
  • 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 ⁇ -glucanase and PR-1 (Figs. 3A and 3B) [Cao, H. et al. (1994) Plant Cell 6: 1583-1592; Ausubel, F.M. et al. (1997) Current Protocols In Molecular Biology (Wiley, New York)].
  • PR pathogenesis-related
  • dndl is a separate locus from the two published cpr loci, CPR1 and CPR5 (see Examples section).
  • 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 mutants such as dominant or semi-dominant behavior, very low fertility, glabrousness, or distorted leaf shape.
  • 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.
  • 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 dndl mutants exhibited delayed response and reduced sensitivity to Fumonisin Bl -induced cell death compared to the wild type Arabidopsis, indicating that the dnd mutants may have more general suppression of programmed cell death. Fumonisin B 1 is a known inhibitor of ceramide synthase which induces apoptosis in diverse organisms.
  • BACs, cosmid library, and the use of RFLPs are well known in the art and can be found in Ausubel, (1997).
  • each clone was tested for the capacity to functionally complement the dndl mutation. This was accomplished by transforming mutant dndl plants with each of the cosmids via Agrobacterium-medizted 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 DNDl locus.
  • dndl plants exhibited a significantly lower transformation rate, 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 for the size.
  • Tl 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).
  • complementation data delimited the location of the DNDl locus and demonstrated that the gene encoded in the region is responsible for the loss of function, i.e. dwarfism, in the dndl plants.
  • T2 plants of wild-type size exhibited defense responses similar to that of
  • 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.
  • dndl plants transformed with cosmid 1A8 or 1H2 displayed a strong HR response to Psg R4(avrRpt2 + ) (Fig. 10).
  • dwarf T2 plants were defective in HR cell death indicating that these dndl plants did not contain a complementing cosmid transgene (Fig. 10).
  • T2 plants transformed with cosmid 1H3 that were wild-type in size were susceptible to Pst DC3000 (i.e. their response mirrored that of Col-0).
  • Pst DC3000 i.e. their response mirrored that of Col-0.
  • a day three growth analysis of dwarf T2 plants provided data to indicate that these plants retained elevated resistance characteristic of the dndl mutation. Thus these plants did not contain a complementing cosmid transgene.
  • 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 hydrophobic regions at the N-terminus (Figs. 6 and 7). Sequencing of the genomic DNA spanning the AtCNGC2 cDNA revealed that DNDl (AtCNGCl) 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).
  • the dndl 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 dndl mutation was recessive to wild type, and homozygous dnd2ldndl mutant plants exhibited an extreme reduction in the extent of HR cell death in response to avirulent P. syringae.
  • the dndl 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.
  • dndl 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.
  • DNDl maps to the upper arm of Arabidopsis chromosome 5
  • DND2 maps to the lower arm of that chromosome 5.
  • the DND2 gene maps to the genetic interval flanked by the available PCR based, polymorphism-detecting genetic markers ngal29 and LFY3 (www . arabidopsis . org) .
  • 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: 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 AtCNGCl -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 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.
  • DNDl 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 ⁇ euhaus (1998) Supra; Kohler et al. (1999) Plant J. 18:97-104; Leng et al. (1999) Plant Physiol 121:753-761].
  • cD ⁇ As 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.
  • 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 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).
  • AtCNGC2/DNDl and AtCNGCl IDND2 genes as regulators of 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 D ⁇ A technology well known in the art.
  • 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 coding region. It is likely that a functional or structural homolog of the AtCNGC2/DNDl and
  • AtCNGCl IDND2 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 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 are well known in the art. See, for example, Kunkel, T. (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382.
  • the disease resistance can be enhanced by inactivating or downregulating the CNGC/DND gene or a homolog thereof.
  • the DNDl 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 AtCNGCl .
  • the flanking sequences surrounding the gene generally contain various regulatory sequences which control expression of the gene, either transcriptionally or translationally.
  • AtCNGC2/DNDl or AtCNGCl/DND2 gene expression can be down-regulated or inactivated by either transcriptionally or translationally.
  • 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 CNGC/DND gene.
  • flanking sequences containing regulatory elements for transcription can also be used to identify compositions which inhibit CNGC/DND gene expression.
  • the DNDl and DND2 genes are highly related as evidenced by the sequence homology
  • the nucleotide sequences encoding the AtCNGC2/DNDl and AtCNGCl IDND2 can be utilized to isolate homologous genes from other plants including sorghum, Brassica, wheat, tobacco, cotton, barley, sunflower, cucumber, alfalfa, soybeans, sorghum etc. Coding sequences from other plants may be isolated according to well known techniques based on their sequence homology to the AtCNGC2/DNDl or AtCNGCl/DND2 coding sequences set forth herein SEQ ID NOs: 2 and 5.
  • coding sequence is used as a probe which selectively hybridizes to other disease resistance coding sequences 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.
  • homologous genes can be identified from the EST or genomic sequence databases using AtCNGC2/DNDl or AtCNGCl IDND2 genomic or cDNA sequences.
  • searching can utilize the entire AtCNGC2/DNDl or AtCNGCl IDND2 gene or derived amino acid sequence, or subdomains thereof. Methods for similarly searching can be found in Brenner, S. and
  • AtCNGC2/DNDl or AtCNGCl/DND2 and their homologs in other plants may facilitate identification of effector genes that interact with AtCNGC2/DNDl or AtCNGCl/DND2 or their homolog gene or gene product; or the identification of effector chemicals or other interventions that alter AtCNGC2/DNDl or AtCNGCl IDND2 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.
  • 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 1 x SSPE at 37 ° C ; conditions represented by a wash stringency of 40-45 % Formamide with 5x Denhardt's solution, 0.5 % SDS and lx SSPE at 42°C; and conditions represented by a wash stringency of 50% Formamide with 5x Denhardt's solution, 0.5% SDS and lx SSPE at 42 °C, respectively), to DNA encoding the disease resistance genes disclosed herein in a standard hybridization assay.
  • conditions represented by a wash stringency of 35-40% Formamide with 5x Denhardt's solution, 0.5 % SDS and 1 x SSPE at 37 ° C conditions represented by a wash stringency of 40-45 % Formamide with 5x Denhardt's solution,
  • 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 variety. Such mutations can be identified as described herein for Arabidopsis.
  • the breeding is facilitated by identifying one or more markers linked to the DND gene.
  • markers can include conventional markers or molecular markers such as RFLP or SSR markers.
  • SSR simple sequence repeat
  • 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 micro injection [Crossway et al. (1986) Biotechniques 4:320- 334]; electroporation [Riggs etal. Proc. Natl. Acad. Sci. USA. 83:5602-5606]; Agrobacterium mediated transformation [Hinchee et al. Biotechnology 6:915-921]; direct gene transfer
  • 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 characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.
  • 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.
  • DNDl and DND2 genes as cyclic nucleotide-gated channel genes in Arabidopsis offers additional means to identify compositions which can enhance 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 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/DNDl or AtCNGCl /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 defined as an inhibitor. If such a composition is found, it would be useful to enhance disease resistance in plants.
  • the genes of the invention can be manipulated to enhance disease resistance and/or cell death in plants.
  • the expression or activity of the AtCNGC2/DNDl (or AtCNGCl /DNDl) 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.
  • 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- 116.
  • PR proteins pathogenesis-related proteins
  • 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 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 controlled by similar modifications of the AtCNGC2/DNDl or AtCNGCl/DND2 gene or homolog thereof as disclosed in the present invention.
  • HR hypersensitive response
  • Example 1 Inoculations with P. syringae.
  • Arabidopsis thaliana ecotype Col-0 seeds were mutagenized with ethyl methane sulfonate; M2 populations were obtained from Lehle Seeds (Round Rock, TX).
  • FI plants were silt-type in appearance and displayed the HR after inoculation with P. syringae pv glycinia Race 4 pV288.
  • 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].
  • Fluorescein filter set Ex 495 ⁇ 20 nm, Em > 505 nm [Klement, (1990) supra].
  • 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 back, and leaves were rinsed in H 2 O, mounted in H 2 O, and observed by light microscopy.
  • Tobacco ringspot virus grape strain was applied to plants, and virus multiplication was monitored by using ELISA as described in Lee, (1996) supra.
  • Peronospora parasitica isolate Noco2 was applied and monitored as described in Parker, J.E. et al. (1997) Trends Biochem. Sci. 22:291-296.
  • Arabidopsis ecotype Col-0 served as a susceptible control for pathogen multiplication and virulence.
  • Hybridization was quantified by using a storage phosphor imaging system according to the manufacturer's instructions (Molecular Dynamics). Signal for PR-1 or ⁇ -glutanase in each lane was normalized to the control ⁇ -ATPase signal for that lane to correct for slight differences in gel loading, and normalized signals then were divided by the signal for the Col-0/no-pathogen sample to establish a relative scale.
  • 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 GV3101 via a tri-parental mating. Liquid cultures were prepared for each of the parents : GV3101 (pMP90) , E. coli strain HB 101 containing the mating helper plasmid pRK2013, and the cosmid-bearing E. coli XL-1 donor. 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.
  • mating spots were grown overnight at 28 °C. The next day each mating spot was re-streaked onto low- salt LB media (lOg tryptone, 5 g yeast extract, and 5 g NaCl/liter) + tetracycline (2.5 ⁇ g/ml) + rifampicin (100 ⁇ g/ml) + gentamycin (50 ⁇ g/ml) and grown at 28 °C for two days.
  • low- salt LB media lOg tryptone, 5 g yeast extract, and 5 g NaCl/liter
  • tetracycline 2.5 ⁇ g/ml
  • rifampicin 100 ⁇ g/ml
  • gentamycin 50 ⁇ g/ml
  • Colonies were picked from these plates and re-streaked unto low-salt LBA (1.5% agar) containing rifampicin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml) to select for Agrobacterium colonies containing a cosmid vector.
  • 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 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 ⁇ g/ml) selection plates. Typically, -3000 seeds were plated per 150 x 15 mm petri plate. After 7-10 days of growth under 24 hr.
  • kanamycin resistant seedlings with green leaves and well-established root systems were deemed putative transformants and were transplanted to soil for further analysis. Because dnd mutants are - 100X more recalcitrant to transformation than wild-type, several plates of seeds (sometimes 5-10) were screened in order to obtain a few putative transformants.
  • putative transformants were obtained and transplanted to soil, they were grown 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 A21, a vector 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.
  • 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 DND7 locus, reversion of these other characteristic phenotypes of dndl were also examined 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).
  • Pst DC3000 carrying either avrRP2 (pV288) or no avr gene vector only.
  • Leaf discs were harvested into a 1.5 ml micro-centrifuge tubes with 200 ⁇ l 10 mM MgCl 2 , ground with a pestle, and diluted serially onto ⁇ YGA (5g Bacto-peptone, 3 g yeast extract, 20 ml glycerol, and 15 g agar/liter) + rifampicin (100 ⁇ g/ml) + cycloheximide
  • HR assays were performed on T2 from two complementing cosmids, in addition to Col-0 and dndl controls. Inoculation with high levels of P.s. glycinia Race 4 (Psg)
  • soybean plants can be engineered to exhibit enhanced disease resistance and/or reduced cell death following infection by a 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 putative CNGC of soybean.
  • An expression cassette is constructed for pathogen-induced expression of an antisense or sense gene.
  • many different pathogen-induced genes can serve as the source of a suitable promoter.
  • 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.
  • 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, 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.
  • the PR- 1 promoter/antisense DND/nos terminator D ⁇ A or PR-1 promoter/sense DND/nos terminator D ⁇ A 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 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-1 promoter/antisense DND/nos terminator D ⁇ A or PR-1 promoter/sense DND/nos terminator
  • 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 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.
  • soybean plants are engineered to exhibit enhanced disease resistance and/or reduced cell death induced by treatment with an inducing 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.
  • D ⁇ A constructs are created that contain a chemically inducible promoter such as that disclosed by Ryals et al. U.S.
  • 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 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 portions of the infected plant, and may arise due to a variety of molecular mechanisms.
  • 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 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.
  • heterologous 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 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 al., Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic
  • 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.
  • 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 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.).
  • Ri root-inducing
  • 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 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.
  • a prokaryotic host cell such as a bacterial host cell.
  • preferred embodiments that include a prokaryotic replicon also include a gene whose expression confers a selective advantage, such as a drug resistance, to the bacterial host cell when introduced into those transformed cells.
  • 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.
  • 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 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.
  • vectors pCMU [Nilsson et al. (1989) Cell 58:707].
  • Other appropriate vectors may also be synthesized, according to known methods; for example, 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 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. WO87/00551; Cocking and Davey (1987) Science 236: 1259-1262.
  • Ti tumor-inducing
  • a transgenic plant can be produced by any means known to the art, including but not limited to Agrobacterium tumefaciens-medizted 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 Physiol. Plant Mol. Biol. 42:205; Gasser and Fraley (1989) Science 244: 1293; Leemans (1993)
  • 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 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.
  • Soybeans Phytophthora megasperma fsp. glycinia, Macrophomina phased ma, Rhizoctonia ⁇ olani Sclerotinia sclerotiorum, Fusa ⁇ um oxyspcrurr, Diaportne phaseoloru var sojae (Phomopsis sojae) , Diaportne pnaseclorum var caulivora, Sclerozium rolfsn, Cercospora kikuchn , Cercospcra sojma, Percnospcra manshurica, Colletotricnuri dematiuir (Colletotichum zruncatum/ , Coryne ⁇ pcra cassncola, Septoria glycines , Phyllosticta soi icola, Alzernaria alternata, Pseudo onas syringae p.v glycmea , Xanth
  • medicagmis Cercospora medicagims , Pseudopeziza medicagmis , Leptotrochila medicagmis , Fusarium oxy ⁇ poru , Rnizoctoma scla.ni, Uromyce ⁇ striatu ⁇ , Colletctr cnum t ⁇ folii race L and race 2, Lepto ⁇ pnaerulma ⁇ rios ana, Szemphylium bctryosum, Stagonospora melilozi , Sclerotinia znfoliorum, Alfalfa Mosaic Virus, Verticillium alDo-atru , Xantnomona ⁇ campestris p.v.
  • syringae Alzernaria alternata , Cladc ⁇ porium herbarum, Fusarium graminearum, Fusarium ave aceum, Fusarium culmorum, Uszilagc trizici, Ascochyta zritici, Cephalosporium gramineu , Collotetrichum graminiccia, Erysiphe gramini ⁇ f . sp . tritici, Puccinia graminis f . sp . tritici, Puccinia recondita f . sp .
  • Physoderma maydis Phyllo ⁇ zicta maydis, Kabatielia zeae, Colletotrichum graminicola , Cercospora zeae-maydi ⁇ , Cercospora sorghi, U ⁇ tilago maydis, Puccinia sorghi, Puccinia polysora, Macrophomina pha ⁇ eolina, Penicilli m oxalicum, Nigro ⁇ pora oryzae , Clado ⁇ porium herbaru , Curvularia iunata , Curvuiaria inaequali ⁇ , Curv laria pallescens, Clavibacter michiganen ⁇ e subsp .
  • nebra ⁇ kense Trichoderma viride, Maize Dwarf Mosaic Virus A & B , Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonoma ⁇ avenae, Erwinia chry ⁇ anthemi pv.

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EP2045327A2 (en) 2005-03-08 2009-04-08 BASF Plant Science GmbH Expression enhancing intron sequences
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CN103789231A (zh) * 2014-01-07 2014-05-14 广西大学 一种利用离体叶脉片接种烟草青枯病的人工发病方法
CN103789215A (zh) * 2014-01-07 2014-05-14 广西大学 一种利用离体叶脉片接种烟草立枯病的人工发病方法
CN103805539A (zh) * 2014-01-07 2014-05-21 广西大学 一种烟草青枯病离体接种的人工发病方法
US20140283211A1 (en) * 2013-03-14 2014-09-18 Monsanto Technology Llc Methods and Compositions for Plant Pest Control
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US10000767B2 (en) 2013-01-28 2018-06-19 Monsanto Technology Llc Methods and compositions for plant pest control
CN108660141A (zh) * 2018-05-28 2018-10-16 贵州省烟草科学研究院 NtCNGC1基因在烟草抗青枯病中的应用
CN112011643A (zh) * 2020-09-30 2020-12-01 河南科技大学 葡萄的qRT-PCR内参基因及其引物与应用
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CN116590304A (zh) * 2023-04-06 2023-08-15 东北农业大学 一种洋葱AcCNGC2基因及其应用
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CN110607385A (zh) * 2019-09-23 2019-12-24 深圳大学 拟南芥叶片锯齿状边缘相关基因功能性分子标记及其应用
CN111394494B (zh) * 2020-02-12 2023-08-25 深圳大学 拟南芥叶片锯齿状边缘相关基因的功能性分子标记的应用

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EP2045327A2 (en) 2005-03-08 2009-04-08 BASF Plant Science GmbH Expression enhancing intron sequences
EP2166101A2 (en) 2005-03-08 2010-03-24 BASF Plant Science GmbH Expression enhancing intron sequences
EP2166102A2 (en) 2005-03-08 2010-03-24 BASF Plant Science GmbH Expression enhancing intron sequences
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EP2166100A2 (en) 2005-03-08 2010-03-24 BASF Plant Science GmbH Expression enhancing intron sequences
CN102747090B (zh) * 2012-07-02 2014-06-11 中山大学 水稻抗病基因OsLYP4及其应用
CN102747090A (zh) * 2012-07-02 2012-10-24 中山大学 水稻抗病基因OsLYP4及其应用
US10000767B2 (en) 2013-01-28 2018-06-19 Monsanto Technology Llc Methods and compositions for plant pest control
US10435701B2 (en) 2013-03-14 2019-10-08 Monsanto Technology Llc Methods and compositions for plant pest control
US20140283211A1 (en) * 2013-03-14 2014-09-18 Monsanto Technology Llc Methods and Compositions for Plant Pest Control
CN103789231A (zh) * 2014-01-07 2014-05-14 广西大学 一种利用离体叶脉片接种烟草青枯病的人工发病方法
CN103789215A (zh) * 2014-01-07 2014-05-14 广西大学 一种利用离体叶脉片接种烟草立枯病的人工发病方法
CN103805539A (zh) * 2014-01-07 2014-05-21 广西大学 一种烟草青枯病离体接种的人工发病方法
CN103789216A (zh) * 2014-01-07 2014-05-14 广西大学 一种烟草立枯病离体接种的人工发病方法
US20220106606A1 (en) * 2014-12-03 2022-04-07 Monsanto Technology Llc Transgenic plants with enhanced traits
CN106636128A (zh) * 2016-11-15 2017-05-10 内蒙古和盛生态育林有限公司 Cngc2基因和其编码的氨基酸序列在培育植物新品种中的应用
CN108660141A (zh) * 2018-05-28 2018-10-16 贵州省烟草科学研究院 NtCNGC1基因在烟草抗青枯病中的应用
CN108660141B (zh) * 2018-05-28 2021-08-03 贵州省烟草科学研究院 NtCNGC1基因在烟草抗青枯病中的应用
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CN116590304A (zh) * 2023-04-06 2023-08-15 东北农业大学 一种洋葱AcCNGC2基因及其应用
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