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WO2000071696A1 - Gene permettant de reguler la resistance aux maladies chez les plantes - Google Patents

Gene permettant de reguler la resistance aux maladies chez les plantes Download PDF

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WO2000071696A1
WO2000071696A1 PCT/US2000/014718 US0014718W WO0071696A1 WO 2000071696 A1 WO2000071696 A1 WO 2000071696A1 US 0014718 W US0014718 W US 0014718W WO 0071696 A1 WO0071696 A1 WO 0071696A1
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edrl
plant
plants
gene
seq
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Roger W. Innes
Catherine A. Frye
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Indiana University Research and Technology Corp
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Indiana University Research and Technology Corp
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N15/8281—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
    • 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
    • C12N15/8282—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

Definitions

  • This invention relates to the field of plant pathology and disease resistance.
  • this invention relates to a novel gene and protein involved in the resistance of plants to various microbial pathogens.
  • HR hypersensitive resistance
  • SAR systemic acquired resistance
  • the HR can be induced by the interaction between a plant resistance gene and a matching pathogen avirulence gene.
  • Such "gene-for-gene” interactions provide a narrow range of resistance as they differentiate between races of a pathogen based on expression of a specific avirulence gene.
  • Resistance gene products are thought to function as receptors for ligands produced directly or indirectly by avirulence genes.
  • Multiple biochemical events are associated with the HR, including an oxidative burst, K/Cl ion exchange, deposition of autofluorescent compounds and callose in the cell wall, synthesis of antimicrobial phytoalexins, and cell death.
  • PR-1 PR-1
  • PR- 2 ⁇ -glucanase, also known as PR- 2
  • PR-5 a thaumatin-like protein
  • Arabidopsis mutants identified based upon constitutive expression of PR genes cprl and cpr5 are resistant to the fungal pathogen Peronospora parasi tica and the bacterial pathogen Pseudomonas syringae pv maculicola .
  • Other mutants that constitutively express PR genes have been isolated based upon the development of spontaneous leaf lesions that are similar in appearance to the lesions of an HR. These lesion mimic mutants also show resistance to both fungal and bacterial pathogens .
  • Methyl jasmonate and ethylene may induce a defense pathway that is independent of SA. Wounding as well as pathogen attack induce the production of jasmomc acid, which m turn induces defense genes other than those associated with SAR, including genes that encode defensms and thionms . Defensms and thionms are low molecular weight polypeptides that have potent antimicrobial activity m vi tro . Arabidopsis plants over-expressing endogenous thionin have increased resistance to the fungal pathogen Fusari u oxysporum .
  • Transgenic Arabidopsis plants unable to accumulate SA and thus unable to express SAR are able to respond to the jasmomc acid signal and express both defensm and thionm genes. Mutants that constitutively express the proposed jasmomc acid pathway, but not the SA pathway have not been reported; however, the cpr5 and acd2 mutants of Arabidopsis constitutively expresses both PR genes and defensm.
  • ISR induced systemic resistance
  • the mlo mutation of barley mediates resistance to all common races of the powdery mildew fungus Erysiphe graminis f sp hordei and, thus, provides a broader spectrum resistance than do the gene-for-gene type of resistance genes (Jorgensen, 1992, Euphytica 63:141-152). Resistance in mlo mutants correlates with the formation of cell wall appositions that may prevent fungal penetration (Jorgensen, 1992, supra ; Wolter et al . , 1993, Mol. Gen. Genet. 239:122-128) and with plant cell death (Peterhansel et al . , 1997, Plant Cell 9:1397-1409).
  • novel mutants and genes that control defense responses will broaden the range of affected pathogens. It would be particularly advantageous to isolate mutants or genes involved in inducibly enhanced disease resistance without spontaneously occurring abnormal defense responses. Novel regulatory mutants are likely to have distinct pathogen ranges due to differential induction of unique subsets of genes.
  • the different enhanced response may yield resistance responses that are spatially distinct (i.e. epidermal v. mesophyll cells), temporally distinct (i.e. affecting early v. late stages of infection), and/or comprised of a distinct subset of defense mechanisms (i.e. formation of necrotic lesions, deposition of callose , synthesis of antimicrobial Phytoalexins and others).
  • the isolation of mutants will yield the critical gene(s), which can be used to transgenically transfer the enhanced resistance trait to new species.
  • EDRl a novel gene
  • the invention further provides transgenic plants and mutants having an enhanced resistance to plant pathogens.
  • disease resistance is enhanced in a manner that does not involve constitutive expression of pathogenesis-related (PR) genes.
  • PR pathogenesis-related
  • a disease resistance gene EDRl
  • the EDRl gene is located on Arabidopsis thaliana chromosome 1 between the ATEAT and NCC1 markers.
  • the disruption of the EDRl gene is associated with enhanced resistance to plant pathogens or other disease-causing agents.
  • this gene encodes a protein with a kinase domain.
  • the gene contains exons that encode a protein that is 900-1000 amino acids in length.
  • the nucleic acid molecule contains an open reading frame that encodes a protein that is at least 50% identical over its full length to SEQ ID NO:2, and in a particularly preferred embodiment encodes SEQ ID NO : 2.
  • the nucleic acid molecule is comprised of SEQ ID NO:l.
  • a cDNA molecule comprising the exons of the gene which encode a polypeptide 900-1000 amino acids in length.
  • a nucleic acid molecule of at least 15 nucleotides in length preferably at least 20 nucleotides in length, and most preferably at least 27 nucleotides in length, that is identical in sequence to a portion of the EDRl gene located on Arabidopsis thaliana chromosome 1.
  • the invention provides a nucleic acid molecule of at least 15, preferably 20, and most preferably 27 nucleotides in length, that is identical to or complementary to a consecutive 15, 20 or 27 nucleotide portion, respectively, of the sequence set out in SEQ ID NO:l.
  • an isolated nucleic acid is provided that is a plant gene and whose disruption is associated with enhanced resistance to plant pathogens or other disease-causing agents.
  • This nucleic acid has a sequence that is selected from SEQ ID NOS:l, 5 or 9, a nucleic acid sequence that is at least 60% identical to SEQ ID NOS:l, 5 or 9 a nucleic acid encoding any of SEQ ID NOS : 2 , 4, 6 or 10, and a nucleic acid encoding a sequence that is at least 50% identical to any of SEQ ID NOS : 2 , 4, 6 or 10.
  • a polypeptide that is produced by the expression of the isolated nucleic acid molecule, and antibodies immunologically specific for the polypeptide.
  • a nucleic acid molecule of, preferably, at least 27 nucleotides in length, more preferably, at least 20 nucleotides in length and, most preferably, 15 nucleotides in length that is identical in sequence to a consecutive 27, 20 or 15 base pair portion, respectively, of the sequence of the isolated nucleic acid molecule set forth above.
  • a method to enhance resistance of a plant to plant pathogens or other disease agents.
  • This method comprises the removal of the EDRl function from the plant cell.
  • the removal of EDRl function is accomplished by the addition of transgenes.
  • the transgene preferably is comprised of at least 15 nucleotides of the gene on chromosome 1 of Arabidopsis thaliana .
  • the transgene expresses a mutant version of the EDRl protein that interferes with the function of the native version of the protein (i.e., a "dominant negative" form).
  • the nucleic acid is mutated in the portion encoding the kinase domain of EDRl.
  • the nucleic acid molecule is SEQ ID NO:l and the coding sequence for residue 696 is mutated.
  • the transgene is comprised of at least 15 nucleotides of SEQ ID NO:l, sequences encoding SEQ ID NO:2, SEQ ID N0:4, SEQ ID NO:6 or SEQ ID NO : 7 , or variants thereof.
  • the transgene expresses the antisense strand of the nucleic acid molecule encoding EDRl.
  • the transgene expresses the sense strand of the nucleic acid molecule.
  • transgenes express both the sense and anti- sense strands of the nucleic acid molecule.
  • This aspect of the invention also provides a plant made from this method, and a reproductive unit from the plant, e.g., a seed.
  • the plant is Arabidopsis thaliana .
  • a method for screening plants for mutations conferring enhanced disease resistance by pathways that do not involve constitutive PR gene expression.
  • the method comprises the following basic steps: (a) provide a plant variety and a pathogen to which the plant normally is not resistant; (b) inoculate a population of the plant variety with the pathogen; (c) select a sub- population of the plants that exhibit a disease- resistance response; and (d) eliminate from the sub- population any plants exhibiting constitutive PR gene expression.
  • the above method can be modified or optimized m a variety of ways, including, but not limited to: (1) using Arabidopsis as the plant for screening; most preferably using variety Col-0 and the pathogen Pseudomonas sy ⁇ ngae, to which Col-0 is sensitive; (2) optionally, treating the plants (seeds) with a mutagemzmg agent (such as fast-neutron bombardment, exposure to ethyl methanesulfonate, or exposure to gamma irradiation) prior to inoculation with the pathogen; (3) n the sub-population of plants exhibiting a disease resistance response, eliminating individuals exhibiting a hypersensitive response (HR) ; and (4) challenging the selected resistant plants to other plant pathogens to further select for broad spectrum resistance.
  • a mutagemzmg agent such as fast-neutron bombardment, exposure to ethyl methanesulfonate, or exposure to gamma irradiation
  • the selected resistant plants are resistant to several classes of plant pathogens. Accordingly, if the initial pathogen used in the screen was a bacterium, for example, the secondary screen might be against a fungal or viral pathogen.
  • mutant Arabidopsis plants produced by the above-described method are provided.
  • mutants edrl , edr2, edr3 and edr4 are provided. These mutants each comprise mutations in different genes, the mutations resulting in the plants' enhanced disease resistance.
  • the wild-type genes, EDRl (described in greater detail below) , EDR2 , EDR3 and EDR4, corresponding to the mutated genes in the aforementioned edrl , edr2 , edr3 and edr4 Arabidopsis mutants, respectively, are also provided.
  • FIG. 1 Response of edrl plants to P. syringae .
  • Arabidopsis plants were vacuum infiltrated with P. syringae and bacterial growth in plant leaves monitored over 4 days. Data points represent the mean ⁇ SE of three samples. Each graph is representative of at least three experiments.
  • Fig IA Time course of P. s . toma to DC3000 growth in Arabidopsis leaves.
  • Fig. IB Time course of P. S . maculi cola M4 growth in Arabidopsis leaves.
  • FIG. 1 Alignment of the EDRl and CTR1 proteins. Amino acid sequences were aligned using the GCG GAP program with a gap creation penalty of 12 and a gap extension penalty of 2. Vertical bars indicate amino acid identities, dots indicate functionally similar amino acids. The highly similar region from residue 668 to the end of EDRl (residue 933) corresponds to the kinase domain.
  • FIG. 4 Alignment of the kinase domains of the EDRl, TCTR2, Rice EST and CTR1 proteins. Black boxes indicate identical amino acids. Arrows indicate regions that distinguish CTR1 from the other three genes. These regions can be used to develop PCR primers that will specifically amplify I.D.R---homologous genes from both monocot and dicot species.
  • EDRl and " edrl” are used.
  • the term “EDRl” is used to designate the naturally-occurring or wild-type genotype. This genotype has the phenotype of naturally- occurring spectrum of disease resistance and susceptibility.
  • the term “ edrl” refers to a genotype having recessive mutation (s) in the wild-type EDRl gene. The phenotype of edrl individuals is enhanced disease resistance.
  • EDRl refers to the protein product of the EDRl gene.
  • nucleic acid mutant is used to designate an organism or genomic DNA sequence with a mutation that causes the product of the EDRl gene to be non-functional or largely absent. Such mutations may occur in the coding and/or regulatory regions of the EDRl gene, and may be changes of individual residues, or insertions or deletions of regions of nucleic acids. These mutations may also occur in the coding and/or regulatory regions of other genes which may regulate or control the EDRl gene and/or the product of the EDRl gene so as to cause said gene product to be non-functional or largely absent. With reference to nucleic acids of the invention, the term “isolated nucleic acid" is sometimes used.
  • genomic DNA refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally-occurring genome of the organism from which it was derived.
  • isolated nucleic acid may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a procaryote or eukaryote.
  • isolated nucleic acid molecule may also comprise a cDNA molecule or a synthetic DNA molecule.
  • isolated nucleic acid primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above.
  • the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a "substantially pure” form .
  • Nucleic acid sequences and amino acid sequences can be compared using computer programs that align the similar sequences of the nucleic or amino acids thus define the differences.
  • the GAP program from the GCG Wisconsin Package Version 9.1 (available from the "Genetics Computer Group", Madison, Wisconsin) with a gap creation penalty of 12 and a gap extension penalty of 2 is used herein to compare sequence identity and similarity.
  • nucleic acid or amino acid sequences having sequence variations that do not materially affect the nature of the protein (i.e. the structure, thermostability characteristics and/or biological activity of the protein) .
  • nucleic acid sequences the term “substantially the same” is intended to refer to the coding region and to conserved sequences governing expression, and refers primarily to degenerate codons encoding the same amino acid, or alternate codons encoding conservative substitute amino acids in the encoded polypeptide.
  • amino acid sequences refers generally to conservative substitutions and/or variations in regions of the polypeptide not involved in determination of structure or function.
  • percent identical refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical amino acids in the compared amino acid sequence by a sequence analysis program.
  • Percent similar refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical or conserved amino acids. conserved ammo acids are those which differ in structure but are similar in physical properties such that the exchange of one for another would not appreciably change the tertiary structure of the resulting protein. Conservative substitutions are defined in Taylor (1986, J. Theor. Biol. 119:205).
  • percent identical refers to the percent of the nucleotides of the subject nucleic acid sequence that have been matched to identical nucleotides by a sequence analysis program.
  • isolated protein or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form.
  • antibodies of the invention the terms “immunologically specific”, “ immunospecific” or “specific” refer to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
  • the term “specifically hybridizing” refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under predetermined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single- stranded nucleic acids of non-complementary sequence.
  • promoter region refers generally to the transcriptional regulatory regions of a gene, which may be found at the 5' or 3 ' side of the coding region, or within the coding region, or within introns .
  • reporter gene refers to genetic sequences which may be operably linked to a promoter region forming a transgene, such that expression of the reporter gene coding region is regulated by the promoter and expression of the transgene is readily assayed.
  • selectable marker gene refers to a gene product which when expressed confers a selectable phenotype, such as antibiotic resistance, on a transformed cell or plant.
  • operably linked means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector.
  • transcription control elements e.g. promoters, enhancers, and termination elements
  • DNA construct refers to genetic sequences used to transform plants or other organisms (e.g., bacteria, yeast). When transforming plants, these constructs may be administered to plants in a viral or plasmid vector. Other methods of delivery such as Agrobacterium T-DNA mediated transformation and transformation using the biolistic process are also contemplated to be within the scope of the present invention.
  • the transforming DNA may be prepared according to standard protocols such as those set forth in "Current Protocols in Molecular Biology", eds. Frederick M. Ausubel et al., John Wiley & Sons, 2000.
  • a gene is provided that is a novel regulator of plant disease resistance.
  • This gene, EDRl was initially isolated from Arabidopsis thaliana . Its manner of regulating disease resistance is novel and surprising. When the functional product of the gene is eliminated, the plants exhibit enhanced disease resistance, but without infection the plants do not exhibit constitutive expression of the pathogenesis-related genes or spontaneous necrotic lesions. Additionally, the induction of the pathogenesis-related genes and the halting of pathogen growth occurs later after infection than observed with other inducible disease defense responses .
  • the EDRl gene was first isolated in a mutated form ⁇ edrl ) from libraries of mutagenized Arabidopsis seed.
  • the edrl mutant was isolated firstly because of its enhanced resistance to the pathogenic bacterium
  • the edrl mutant was found to have reduced susceptibility to Pseudomonas syringae maculicola M4 and the powdery mildew fungus Erysiphe ci chora cearum .
  • the expression of the SAR-related genes, PR-1 , PR-5 and BGL2 was not detectable prior to inoculation with E . ci chora cearum, indicating that the edrl mutant does not function by way of constitutive stimulation of the SAR pathway.
  • the PR-1 message was four times higher in the mutant than wildtype plants, indicating that the mutant displays stronger induction of the SAR pathway in response to normally virulent pathogens than does the wild-type plant.
  • the enhanced disease resistance of the edrl mutant segregates as a recessive trait.
  • Genetic linkage analysis establishes that the EDRl gene is located on chromosome 1, 3.2 centiMorgans centromeric from the SSLP (simple sequence length polymorphism) marker ATEAT1 and 0.85 centiMorgans telomeric from the CAPS (co-dominant amplified polymorphic sequence) marker NCC1 .
  • the EDRl gene was isolated using a positional cloning approach. Two overlapping bacterial artificial chromosome (BAC) clones containing Arabidopsis genomic DNA were identified that contained closely-linked molecular markers.
  • SSLP simple sequence length polymorphism
  • CAPS co-dominant amplified polymorphic sequence
  • Sequence information from the Arabidopsis Genome Project was used to create additional molecular markers and allowed the EDRl gene to be mapped to a 100 kb interval. Sequence analysis of this 100 kb region revealed 25 potential protein coding genes.
  • Candidate genes were sequenced from the edrl mutant and compared to the known wildtype sequence. Upon sequencing a gene with similarity to MAP3 kinases (also known as "mitogen activated protein kinase kinase kinase”) , a single nucleotide difference was found that produces a premature stop codon, thereby truncating the C-terminal half of the protein containing the kinase domain.
  • EDRl gene a cDNA is SEQ ID NO:l and the genomic clone is found on BAC F22013, Genbank AC003981, and in Genbank at No. AAC14047.1.
  • the EDRl predicted protein (SEQ ID NO: 2) is 933 amino acids long and contains a "kinase domain" from residues 668 to 933 (Hanks et al . , 1988, Science 241:42-52).
  • EDRl genomic clone and cDNA from Arabidopsis thaliana are described and exemplified herein, this invention is intended to encompass nucleic acid sequences and proteins from other plants that are sufficiently similar to be used instead of the • Arabidopsis EDRl nucleic acid and proteins for the purposes described below. These include, but are not limited to, allelic variants and natural mutants of SEQ ID NO:l, which are likely to be found in different species of plants or varieties of Arabidopsis .
  • this invention provides an isolated EDRl nucleic acid molecule having at least about 50% (preferably 60%, more preferably 70% and even more preferably over 80%) sequence identity in the coding regions with the nucleotide sequence set forth as SEQ ID NO:l (and, most preferably, specifically comprising the coding region of SEQ ID NO:l) .
  • This invention also provides isolated polypeptide products of SEQ ID NO:l, having at least about 50% (preferably 60%, 70%, 80% or greater) sequence identity with the amino acid sequences of SEQ ID NO: 2.
  • the EDRl protein sequence contains regions of high similarity to a large number of protein kinases.
  • the functional specialization that allows these kinases to operate in specific signal transduction pathways lies both in the kinase domain and non-kinase domains.
  • the Arabidopsis CTR1 kinase protein (SEQ ID NO: 3) displays moderate similarity to EDRl (40% identity, 49% similarity). While the similarity is highest in the putative kinase domain (residues 668-933 of SEQ ID NO:2), it extends to the rest of the protein as well (Fig.
  • CTR1 is a negative regulator of the ethylene response pathway (Kieber, 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:277-296).
  • EDRl may function as a negative regulator of specific defense response pathways.
  • EDRl genes have been identified in crop plants by their extensive homology to EDRl outside the kinase region.
  • the tomato TCTR2 predicted protein sequence (SEQ ID NO: 4) shows extensive homology throughout the protein sequence (55% identity, 61% similarity) (Fig. 3).
  • TCTR2 is more similar to EDRl than CTR1 is to EDRl, both inside and outside the kinase region (Table 1), but the high similarity outside the kinase region in particular indicates that they are homologous proteins.
  • EDR1 also shows a high similarity to a gene product from rice (SEQ ID NOS : 5 and 6 for the cDNA and predicted amino acid sequences, respectively) .
  • the amino acid sequence encoded by this rice gene is 85% identical to EDRl in the kinase domain. This high degree of identity is comparable to the TCTR2-EDR1 identity of 86% in the kinase region, indicating that the rice gene is also part of an EDRl gene family.
  • EDRl homolog from barley also has been identified.
  • a cDNA sequence corresponding to the barley EDRl homolog and its predicted amino acid sequence are set forth herein as SEQ ID NO: 9 and SEQ ID NO: 10, respectively.
  • the present invention encompasses these EDRl genes from tomato, rice and barley, as well as TCTR2.
  • EDRl genes encompass both monocots and dicots and therefore are excellent candidates for determining conserved amino acid sequences that can be used as probes to identify new EDRl genes from the entire plant kingdom.
  • the sequences on which to base such probes are indicated by arrows in Fig. 4, however many other sequences are present that would be equally as appropriate.
  • the criteria for selecting probe sequence is that they should be well conserved among the EDRl homologs EDRl, TCTR2 and the rice and barley EDRl, but not conserved in the general kinase CTRl, and therefore will only identify EDRl homologous kinases.
  • the edrl mutant from Arabidopsis is also part of the present invention. It exhibits an enhanced disease resistance with characteristics of regulation that have not been previously observed.
  • This mutant is novel in its ability to poise the plant metabolism for a heightened disease-resistance response without the constitutive induction of the pathogenesis-related genes and non-infection related necrotic lesions on leaf tissue .
  • the pathogenesis-related genes are induced much later than currently known disease resistance mutants, resulting in a halting of pathogen growth at a later stage of infection. This distinction is useful because the enhanced disease resistance pathways exhibited by edrl mutants of this invention are not constitutive in the uninfected plant, and so do not cause death of tissue vital for photosynthesis until the plant is actually challenged with a pathogen.
  • the edrl mutants may well be effective against a spectrum of diseases because they are active against a later infection stage. Due to the unique phenotype conferred by the edrl mutation, it is easy to screen populations of mutagenized plants and obtain other edrl mutants. Such edrl mutants from all other species of plants are considered to be within the scope of this invention.
  • the present invention encompasses not only other plant homologs of the EDRl gene, but also using these homologs to engineer enhanced disease resistance in other plant species.
  • the edrl mutant establishes that null mutations in this gene result in plants with enhanced disease resistance.
  • edrl mutants are available from which edrl mutants in other plant species can be isolated. Many of these populations are very likely to contain plants with null mutations in the EDRl gene. Such populations can be made by chemical mutagenesis, radiation mutagenesis, and transposon or T-DNA insertions. The methods to make mutant populations are well known in the art.
  • the nucleic acids of the invention can be used to isolate edrl mutants in other species. In species such as maize where transposon insertion lines are available, oligonucleotide primers can be designed to screen lines for insertions in the EDRl gene. Plants with transposon or T-DNA insertions in the EDRl gene are very likely to have lost the function of the gene product.
  • a plant line may then be developed that is homozygous for the non-functional copy of the EDRl gene.
  • the PCR primers for this purpose are designed so that a large portion of the coding sequence the EDRl gene are specifically amplified using the sequence of the EDRl gene from the species to be probed (see Baumann et al . , 1998, Theor. Appl. Genet. 97:729- 734) .
  • Other e rl-like mutants can easily be isolated from mutant populations using the distinctive phenotype characterized in accordance with the present invention. This approach is particularly appropriate in plants with low ploidy numbers where the phenotype of a recessive mutation is more easily detected.
  • the population of plants would be inoculated with a strain of powdery mildew, for example Erysiphe ci choracearum or E . cruciferarum strain UEA1. After a suitable period of incubation (e.g., 5-10 days), plants would then be screened for phenotype of the edrl mutant: the development of distinct necrotic and collapsed regions on the leaves. That the phenotype is caused by an edrl mutation is then established by molecular means well known in the art.
  • Species contemplated to be screened with this approach include but are not limited to: aster, barley, begonia, beet, cantaloupe, carrot, chrysanthemum, clover, corn, cucumber, delphinium, grape, lawn and turf grasses, lettuce, pea, peppermint, rice, rutabaga, sugar beet, tomatillo, tomato, turnip, wheat, and zinnia.
  • a gene can be defined by its mapped position in the plant genome. Although the chromosomal position of the gene can change dramatically, the position of the gene in relation to its neighbor genes is often highly conserved (Lagercrantz et al . , 1996, Plant J. 9:13-20). This conserved micro-colinearity can be used to isolate the EDRl gene from distantly related plant species.
  • the screening of genes and markers that flank EDRl on the chromosome are known and are further present on the BAC clone of the Arabidopsis genome (BAC F22013, Genbank locus AC003981) .
  • genes and markers can be used to isolate the EDRl gene in their midst, or to confirm the identity of an isolated EDRl nucleic acid (described below) .
  • the various coding sequences can be used to design probes to isolate the EDRl gene on BAC clones or to map the chromosomal location of the EDRl gene using recombination frequencies.
  • genes highly homologous to those on Arabidopsis BAC F22013 are already known in other species, and these homologous genes may be used to locate EDRl in these genomes. There are several versions of these procedures, and all will be well known to those skilled in the art.
  • Nucleic acid molecules encoding the EDRl protein may be isolated from Arabidopsis or any other plant of interest using methods well known in the art. Nucleic acid molecules from Arabidopsis may be isolated by screening Arabidopsis cDNA or genomic libraries with oligonucleotides designed to match the Arabidopsis nucleic acid sequence of EDRl gene (SEQ ID NO:l). In order to isolate EDRl-encoding nucleic acids from plants other than Arabidopsis , oligonucleotides designed to match the nucleic acids encoding the Arabidopsis EDRl protein may be likewise used with cDNA or genomic libraries from the desired species. If the EDRl gene from a species is desired, the genomic library is screened.
  • the cDNA library is screened.
  • all the appropriate nucleic acids residues may be incorporated to create a mixed oligonucleotide population, or a neutral base such as inosine may be used.
  • the strategy of oligonucleotide design is well known in the art (see also Sambrook et al.) .
  • PCR (polymerase chain reaction) primers may be designed by the above method to encode a portion of the Arabidopsis EDRl protein, and these primers used to amplify nucleic acids from isolated cDNA or genomic DNA.
  • the oligonucleotides used to isolate EDRl nucleic acids are designed to encode sequences conserved in Arabidopsis EDRl (SEQ ID NO:2), tomato TCTR2 (SEQ ID NO:4), rice EDRl (SEQ ID NO: 6), and barley EDRl (SEQ ID NO: 10), but not Arabidopsis CTRl (SEQ ID NO: 3).
  • the sequences marked by arrows on Fig. 4, AVKKFLDQD and DPNLRPSFA (SEQ ID NOS : 7 and 8) are used to design oligonucleotides and probes.
  • nucleic acids having the appropriate sequence homology with an Arabidopsis EDRl nucleic acid molecule may be identified by using hybridization and washing conditions of appropriate stringency.
  • hybridizations may be performed, according to the method of Sambrook et al. (1989, supra ) , using a hybridization solution comprising: 5X SSC, 5X Denhardt ' s reagent, 1.0% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide.
  • Hybridization is carried out at 37-42°C for at least six hours.
  • filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature m 2X SSC and 0.1% SDS; (3) 30 mmutes-1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65° m IX SSC and 1% SDS, changing the solution every 30 minutes.
  • T m 81.5°C + 16.6Log [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex
  • the T m is 57°C.
  • the T m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.
  • the hybridization is at 37°C and the final wash is at 42°C, in a more preferred embodiment the hybridization is at 42° and the final wash is at 50°, and in a most preferred embodiment the hybridization is at 42°C and final wash is at 65°C, with the above hybridization and wash solutions.
  • Conditions of high stringency include hybridization at 42°C in the above hybridization solution and a final wash at 65°C in 0. IX SSC and 0.1% SDS for 10 minutes.
  • Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector.
  • clones are maintained in plasmid cloning/expression vector, such as pBluescript
  • Arabidopsis EDRl nucleic acid molecules of the invention include DNA, RNA, and fragments thereof which may be single- or double-stranded.
  • this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule encoding the protein of the present invention. Such oligonucleotides are useful as probes for detecting Arabidopsis EDRl genes or transcripts.
  • edrl null mutant of the present invention is a naturally occurring mutant, any plant may be transgenically engineered to display a similar phenotype. While the natural edrl mutant has lost the functional product of the EDRl gene due to a premature stop codon in its coding sequence, a transgenic plant can be made that also has a similar loss of the EDRl product. This approach is particularly appropriate to plants with high ploidy numbers, including but not limited to wheat.
  • a synthetic null mutant can be created by a expressing a mutant form of the EDRl protein to create a "dominant negative effect". While not limiting the invention to any one mechanism, this mutant EDRl protein will compete with wild-type EDRl protein for interacting proteins in a transgenic plant. By over-producing the mutant form of the protein, the signaling pathway used by the wild-type EDRl protein can be effectively blocked. Examples of this type of "dominant negative" effect are well known for both insect and vertebrate systems (Radke et al, 1997, Genetics 145:163-171; Kolch et al., 1991, Nature 349:426-428).
  • the mutant protein is produced by mutating the coding sequence corresponding to several residues in the kinase domain of EDRl (amino acids 750-1015, Hanks et al . , 1988, Science 241:42-52).
  • the coding sequence corresponding to the lysine residue at position 778 of SEQ ID NO: 2 (which is conserved in all known kinases and required for kinase function) is mutated to code for a different, preferably non-similar, amino acid residue.
  • a second kind of synthetic null mutant can be created by inhibiting the translation of the EDRl mRNA by "post-transcriptional gene silencing".
  • the EDRl gene from the species targeted for down-regulation, or a fragment thereof, may be utilized to control the production of the encoded protein.
  • Full-length antisense molecules can be used for this purpose.
  • antisense oligonucleotides targeted to specific regions of the EDRl-encoded RNA that are critical for translation may be utilized.
  • the use of antisense molecules to decrease expression levels of a pre-deter ined gene is known in the art.
  • Antisense molecules may be provided in si tu by transforming plant cells with a DNA construct which, upon transcription, produces the antisense RNA sequences. Such constructs can be designed to produce full-length or partial antisense sequences.
  • This gene silencing effect can be enhanced by transgenically over-producing both sense and antisense RNA of the gene coding sequence so that a high amount of dsRNA is produced (for example see Waterhouse et al., 1998, PNAS 95:13959-13964).
  • part or all of the EDRl coding sequence antisense strand is expressed by a transgene.
  • hybridizing sense and antisense strands of part or all of the EDRl coding sequence are transgenically expressed.
  • a third type of synthetic null mutant can also be created by the technique of "co-suppression". Plant cells are transformed with a copy of the endogenous gene targeted for repression.
  • the EDRl gene from the plant species of interest is isolated and used to transform cells of that same species.
  • Transgenic plants displaying enhanced EDRl activity can also be created. This is accomplished by transforming plant cells with a transgene that expresses part of all of an EDRl coding sequence, or a sequence that encodes the either the EDRl protein or a protein functionally similar to it.
  • the complete EDRl coding sequence is transgenically over- expressed.
  • the coding sequence corresponding to the kinase domain of EDRl is over-expressed.
  • Transgenic plants with one of the transgenes mentioned above can be generated using standard plant transformation methods known to those skilled in the art. These include, but are not limited to, Agroba cterium vectors, polyethylene glycol treatment of protoplasts, biolistic DNA delivery, UV laser microbeam, gemini virus vectors, calcium phosphate treatment of protoplasts, electroporation of isolated protoplasts, agitation of cell suspensions in solution with microbeads coated with the transforming DNA, agitation of cell suspension in solution with silicon fibers coated with transforming
  • Agroba cteri um vectors are often used to transform dicot species.
  • Agroba cterium binary vectors include, but are not limited to, BIN19 (Bevan, 1984) and derivatives thereof, the pBI vector series (Jefferson et al., 1987), and binary vectors pGA482 and pGA492 (An, 1986)
  • BIN19 Bevan, 1984
  • pBI vector series Jefferson et al., 1987
  • binary vectors pGA482 and pGA492 are often useful for nuclear transformation.
  • DNA constructs for transforming a selected plant comprise a coding sequence of interest operably linked to appropriate 5' (e.g., promoters and translational regulatory sequences) and 3' regulatory sequences (e.g., terminators).
  • the coding region is placed under a powerful constitutive promoter, such as the Cauliflower Mosaic Virus (CaMV) 35S promoter or the figwort mosaic virus 35S promoter.
  • CaMV Cauliflower Mosaic Virus
  • Other constitutive promoters contemplated for use in the present invention include, but are not limited to: T-DNA mannopine synthetase, nopaline synthase (NOS) and octopine synthase (OCS) promoters.
  • Transgenic plants expressing a sense or antisense EDRl coding sequence under an inducible promoter are also contemplated to be within the scope of the present invention.
  • Inducible plant promoters include the tetracycline repressor/operator controlled promoter, the heat shock gene promoters, stress (e.g., wounding) - induced promoters, defense responsive gene promoters (e.g. phenylalanine ammonia lyase genes), wound induced gene promoters (e.g.
  • hydroxyproline rich cell wall protein genes hydroxyproline rich cell wall protein genes
  • chemically-inducible gene promoters e.g., nitrate reductase genes, glucanase genes, chitinase genes, etc.
  • dark-inducible gene promoters e.g., asparagine synthetase gene
  • Tissue specific and development-specific promoters are also contemplated for use in the present invention. Examples of these included, but are not limited to: the ribulose bisphosphate carboxylase (RuBisCo) small subunit gene promoters or chlorophyll a/b binding protein (CAB) gene promoters for expression in photosynthetic tissue; the various seed storage protein gene promoters for expression in seeds; and the root- specific glutamine synthetase gene promoters where expression in roots is desired.
  • RuBisCo ribulose bisphosphate carboxylase
  • CAB chlorophyll a/b binding protein
  • the coding region is also operably linked to an appropriate 3 1 regulatory sequence.
  • the nopaline synthetase polyadenylation region NOS
  • Other useful 3' regulatory regions include, but are not limited to the octopine (OCS) polyadenylation region.
  • the selected coding region under control of a constitutive or inducible promoter as described above, is linked to a nuclear drug resistance marker, such as kanamycin resistance.
  • a nuclear drug resistance marker such as kanamycin resistance.
  • Other useful selectable marker systems include, but are not limited to: other genes that confer antibiotic resistances (e.g., resistance to hygromycin or bialaphos) or herbicide resistance (e.g., resistance to sulfonylurea, phosphinothricin, or glyphosate) .
  • Plants are transformed and thereafter screened for one or more properties, including the lack of EDRl protein, EDRl mRNA, or enhanced resistance to plant pathogens, in particular powdery mildew fungi, the Erysiphe spp . , and most particularly E . ci chora cearum and E . cruciferarum .
  • EDRl protein EDRl mRNA
  • EDRl mRNA enhanced resistance to plant pathogens
  • E ci chora cearum E . cruciferarum
  • Transgenic plants that exhibit one or more of the aforementioned desirable phenotypes can be used for plant breeding, or directly in agricultural or horticultural applications. Plants containing one transgene may also be crossed with plants containing a complementary transgene in order to produce plants with enhanced or combined phenotypes . E. In Vivo Synthesis of the EDRl Protein
  • amino acid sequence information such as the full length sequence in SEQ ID NO: 2
  • the sequence encoding Arabidopsis EDRl from isolated native nucleic acid molecules can be utilized.
  • an isolated nucleic acid that encodes the amino acid sequences of the invention can be prepared by oligonucleotide synthesis.
  • Codon usage tables can be used to design a synthetic sequence that encodes the protein of the invention. In a preferred embodiment, the codon usage table has been derived from the organism in which the synthetic nucleic acid will be expressed.
  • the codon usage for pea ⁇ Pisum sa tivum would be used to design an expression DNA construct to produce the Arabidopsis EDRl in pea.
  • Synthetic nucleic acid molecules may be prepared by the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices, and thereafter may be cloned and amplified in an appropriate vector.
  • the availability of nucleic acids molecules encoding the Arabidopsis EDRl enables production of the protein using in vivo expression methods known in the art.
  • the protein may be produced by expression in a suitable expression system.
  • the EDRl protein of the present invention may also be prepared by in vi tro transcription and translation of either native or synthetic nucleic acid sequences that encode the proteins of the present invention. While in vi tro transcription/translation is not the method of choice for preparing large quantities of the protein, it is ideal for preparing small amounts of native or mutant proteins for research purposes, particularly since in vi tro methods allow the incorporation of radioactive nucleotides such as 35 S- labeled methionine.
  • the EDRl proteins of the present invention may be prepared by various synthetic methods of peptide synthesis via condensation of one or more amino acid residues, in accordance with conventional peptide synthesis methods.
  • the EDRl produced by native cells or by gene expression in a recombinant procaryotic or eukaryotic system may be purified according to methods known in the art.
  • the present invention also provides antibodies that are immunologically specific to the Arabidopsis EDRl of the invention.
  • Polyclonal antibodies may be prepared according to standard methods.
  • monoclonal antibodies are prepared, which are specific to various epitopes of the protein.
  • Monoclonal antibodies may be prepared according to general methods of K ⁇ hler and Milstein, following standard protocols.
  • Polyclonal or monoclonal antibodies that are immunologically specific for the Arabidopsis EDRl can be utilized for identifying and purifying EDRl from Arabidopsis and other species. For example, antibodies may be utilized for affinity separation of proteins for which they are specific or to quantify the protein.
  • Antibodies may also be used to immunoprecipitate proteins from a sample containing a mixture of proteins and other biological molecules.
  • EDRl nucleic acids may be used for a variety of purposes in accordance with the present invention.
  • DNA, RNA, or fragments thereof may be used as probes to detect the presence and/or expression of EDRl genes.
  • Methods in which EDRl nucleic acids may be utilized as probes for such assays include, but are not limited to: (1) in si tu hybridization; (2) Southern hybridization (3) Northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR) .
  • the EDRl nucleic acids of the invention may also be utilized as probes to identify related genes from other plant species.
  • hybridization stringencies may be adjusted to allow hybridization of nucleic acid probes with complementary sequences of varying degrees of homology.
  • EDRl nucleic acids may be used to advantage to produce large quantities of substantially pure EDRl, or selected portions thereof.
  • the EDRl nucleic acids can be used to identify and isolate further members of this novel disease resistance signal transduction pathway in vivo .
  • a yeast two hybrid system can be used to identify proteins that physically interact with the EDRl protein, as well as isolate their nucleic acids. In this system, the sequence encoding the protein of interest is operably linked to the sequence encoding half of a activator protein.
  • This construct is used to transform a yeast cell library which has been transformed with DNA constructs that contain the coding sequence for the other half of the activator protein operably linked to a random coding sequence from the organism of interest.
  • the two halves of the activator protein are physically associated and form a functional unit that activates the reporter gene.
  • all or part of the Arabidopsis EDRl coding sequence may be operably linked to the coding sequence of the first half of the activator, and the library of random coding sequences may be constructed with cDNA from Arabidopsis and operably linked to the coding sequence of the second half of the activator protein.
  • activator protein/reporter genes are customarily used in the yeast two hybrid system.
  • the bacterial repressor LexA DNA-binding domain and the Gal4 transcription activation domain fusion proteins associate to activate the La cZ reporter gene (see Clark et al., 1998, PNAS 95:5401-5406). Kits for the two hybrid system are also commercially available from Clontech, Palo Alto CA, among others.
  • the EDRl proteins of the present invention can be used to identify molecules with binding affinity for EDRl, which are likely to be novel participants in this resistance pathway.
  • the known protein is allowed to form a physical interaction with the unknown binding molecule (s), often in a heterogenous solution of proteins.
  • the known protein in complex with associated molecules is then isolated, and the nature of the associated protein (s) and/or other molecules is determined.
  • Antibodies that are immunologically specific for EDRl may be utilized in affinity chromatography to isolate the EDRl protein, to quantify the EDRl protein utilizing techniques such as western blotting and ELISA, or to immuno-precipitate EDRl from a sample containing a mixture of proteins and other biological materials.
  • the immuno-precipitation of EDRl is particularly advantageous when utilized to isolate affinity binding complexes of EDRl, as described above.
  • the edrl mutants of the invention display enhanced disease resistance to plant pathogens of both fungal and bacterial origin, and therefore can be used to improve crop and horticultural plant species.
  • the edrl mutants are particularly resistant to powdery mildew. Such mutants will therefore be particularly useful when isolated from crop and horticultural varieties in which an infection by powdery mildew-inducing fungi, such as Erisyphe spp . , Oidium spp . and Uncinula necator, results in loss.
  • Plants species contemplated in regard to this invention include, but are not limited to: aster, barley, begonia, beet, cantaloupe, carrot, chrysanthemum, clover, corn, cucumber, delphinium, grape, lawn and turf grasses, lettuce, pea, peppermint, rice, rutabaga, sugar beet, tomatillo, tomato, turnip, wheat, and zinnia.
  • the edrl mutants have a broad-based resistance to Erysiphe, also encompassing the species E . cruciferarum . It is therefore contemplated that the edrl mutants will exhibit broad-spectrum resistance against a wide range of Erysiphe species and other members of
  • Erysiphales Such species include, but are not limited to, E . graminis , E. polygon! , E . betae, E. heraclei , E. pisi , E. trifolii , the Oidium species, particularly 0. lycopersicon, and the Uncinula species, particularly U. neca tor .
  • the mechanism of resistance involves the repression of fruiting body formation, and since pathogenic fungi in general have very similar methods infection, it is contemplated that these mutants will be more resistant to many forms of pathogenic fungi.
  • Other fungal species considered in connection with the invention include, but are not limited to, biotrophic fungal species, such as those causing downy mildew and rust diseases.
  • the enhanced resistance exhibited by the mutants of the invention extends also to bacterial plant pathogens.
  • the edrl mutants exhibit increased resistance to Pseudomonas syringae and Pseudomonas syringae pv maculi cola M2. This broad spectrum resistance to Pseudomonas strains is very likely to extend to all Pseudomonas species.
  • the edrl mutants are contemplated for use in providing resistance to all Pseudomonas species, including but not limited to, P. cichorii , P. coronafaciens, P. la crymans , P. margina ta and numerous P. syringae pathovars .
  • edrl mutations will be selected from plant species that are susceptible to Pseudomonas infection: blueberry, english laurel, lilac, willow, apple, pear, raspberry, lettuce, oats, cucumber, gladiolus, bean, pea, cherry, maple, prune, plum, tomato, soybean, wheat, barley, rice and corn, among others.
  • the edrl mutants exhibit a heightened induction of normal resistance pathways. It is therefore contemplated that these mutants will have enhanced resistance to a myriad of plant pathogens in addition to the specific fungal and bacterial species mentioned heretofore .
  • the edrl mutants of the invention can be used to identify and isolate additional members of this disease resistance pathway. Mutations that, when combined with edrl , suppress the edrl phenotype, are likely to interact directly with EDRl, or to compensate in some other way for the loss of EDRl function.
  • transgenic plants of the invention are particularly useful in conferring the edrl phenotype to many different plant species. In this manner, a host of plant species with enhanced disease resistance can be easily made, to be used as breeding lines or directly in commercial operations. Such plants can have uses as crop species, or for ornamental use.
  • transgenic edrl-phenotype plants will be used with the same aforementioned pathogens and plant species as the edrl mutants.
  • a transgenic approach is advantageous because it allows edrl-phenotype plants to be created quickly, without time-consuming mutant generation, selection, and back-crossing.
  • Transgenically created edrl-phenotype plants have special utility in polyploid plants, such as wheat, where recessive mutations are difficult to detect.
  • a plant that has had functional EDRl increased may have delayed senescence compared to wild-type plants. Plants with delayed senescence will be extremely valuable to agriculture and horticulture by allowing plants to flower, leaves to remain productive, and harvested fruits, vegetables and flowers to remain fresh for longer periods of time. It may also allow fruit with abscission zones such as tomato to accumulate sugars from a longer period of time.
  • TSA Trypticase soy agar
  • Wild-type Arabidopsis thaliana ecotype Col-0 seed was obtained from B.J. Staskawicz (University of California, Berkeley) .
  • Mutagenized seed was obtained from Lehle Seeds (Round Rock, TX; fast-neutron mutagenized) or M. Estelle (Indiana University, Bloomington; ethyl methanesulfonate mutagenized and ⁇ -irradiated) .
  • M 1 generation seeds were mutagenized, planted and allowed to self-pollinate to generate the M 2 population.
  • M 2 populations were bulked from approximately 500 M x plants.
  • the edrl mutant was isolated from the ⁇ -irradiated population.
  • Third and fourth generations (M 3 and M 4 ) of the edrl mutant were used interchangeably for phenotypic analyses and crosses.
  • Arabidopsis Growth and Bacterial Inoculation Arabidopsis Seed were sown in 4-inch-round pots filled with Perlite Plug Mix (Grace Sierra, Milpitas, CA) . Pots were covered with 1.3-mm nylon mesh (window/door screen), and plants were allowed to grow through the screen. Seeded pots were covered and held at 4°C for 3 days before being placed in growth rooms under a 9-hr day length (100 to 150 microE»m-2» sec "1 of light) at 24°C. Covers were removed after the seeds sprouted and the first true leaves were emerging.
  • Bacterial suspensions contained 0.01% L77 Silwet and 10 mM MgCl 2 . At specific time points, samples were removed from rosette leaves using a number 2 cork borer (three discs per sample) and macerated in 200 ⁇ l of 10 mM MgCl 2 . Dilutions were made in 10 M MgCl 2 and plated on trypticase soy agar containing 100 mg/L rifamycin and incubated at 30°C. Colonies were counted 48 hr later.
  • E. cichoracearum Inoculation and Histology E. cichora cearum actively growing on Col-0 plants (7-10 days post-inoculation) was used as an inoculum. To inoculate plants, diseased plants were used to brush healthy plants, thus passing spores onto the new plants.
  • Hyphal length (3 days and 7 days post inoculation) and conidiophore number (7 days post inoculation) were obtained from a minimum of six trypan blue-stained leaves collected from separate experiments.
  • Microscopic images (described above) were captured and digitized using a ZVS-3C75DE 3 CCD video camera (Carl Zeiss) and PowerTower Pro 180 computer (PowerComputing, Round Rock, TX) . Digitized images were viewed and printed using Adobe Photoshop software (Adobe Systems, San Jose, CA) .
  • Hyphal length at 3 days post inoculation was measured on the printed images and converted to actual measurements by comparing to an image of a slide micrometer.
  • hyphal length was estimated using a 50-mm grid on the printed image as described by Olson (1950, Trans. Am. Microscop. Soc. 69:272-279). Conidiophores on the printed image were counted directly. Fields with approximately equal hyphal density were chosen to assure equal sampling.
  • Probes were generated using a Random Primed DNA labeling kit (Boehringer Mannheim) .
  • DNA templates for probes were generated by polymerase chain reaction amplification of Arabidopsis genomic DNA ⁇ BGL2, PR-5, and ubiquitin [ UBQ5] ) or amplification from a cDNA clone ⁇ PR-1 ; Uknes, et al., 1992, Plant Cell, 4:645-656) using published primers (Glazebrook et al., 1996, Genetics 143:973-982).
  • Hybridization was quantified using a Molecular Dynamics (Sunnyvale, CA) Phosphorlmager . Values for PR-1 and BGL2 hybridization were normalized for unequal loading using values obtained from the UBQ5 hybridization. Images were obtained by exposing the membrane to X-ray film (Fuji Film RX, Fisher Scientific) .
  • Arabidopsis mutant edrl was crossed to accession Landsberg erecta (Ler) .
  • the F l r F 2 , and F 3 plants were scored for the mutant phenotype after dusting with E . cichora cearum spores.
  • Resistant F 2 plants were selected for generation of F 3 families, which were used to confirm F 2 mutant phenotypes.
  • DNA for analysis of molecular markers was collected from one or two inner rosette leaves of resistant F 2 plants using a hexadecyltri-methylammonium bromide (CTAB) extraction procedure (Bisgrove et al., 1994, Plant Cell, 6:927-933).
  • CTAB hexadecyltri-methylammonium bromide
  • SSLP Simple sequence length polymorphism
  • CAPS codominant amplified polymorphic sequence
  • Arabidopsis accession Columbia (Col-0) is susceptible to P. s . toma to DC3000. Disease symptoms develop on rosette leaves 4 to 5 days after inoculation and appear as gray lesions surrounded by chlorosis (Whalen et al., 1991, Plant Cell, 3:49-59).
  • To identify Arabidopsis mutants with reduced susceptibility we inoculated mutagenized Col-0 plants (M 2 generation) with P. s . toma to DC3000 and scored for disease lesion severity 4 to 5 days later. Plants were inoculated with a high dose of P. s .
  • toma to DC3000 (1 x 10 9 colony forming units per ml) , facilitating the identification of living resistant plants because at this concentration susceptible plants frequently died.
  • Living plants displaying a decrease in the severity of disease symptoms were selected for further analysis. Reduced disease symptoms included fewer leaves showing disease, smaller lesions, and a lack of lesions on inner-rosette leaves.
  • Approximately 25,000 mutagenized Col-0 plants were screened, and 78 putative mutants were selected. The mutant phenotype of decreased disease symptoms was found to be heritable in 36 of the 78 plants selected.
  • E. cichoracearum is arrested at a late stage of the infection process in edrl plants.
  • the infection process of E . ci chora cearum on Arabidopsis is known.
  • Spores first produce appressorial germ tubes that penetrate the underlying epidermal cells. Inside the epidermal cells the fungus forms a haustorium, which is a bag-like invagination surrounded by host cell plasma membrane.
  • E . ci chora cearum developed extensive hyphal growth that nearly covered the leaf surface on both edrl and wild-type Col-0 plants.
  • E . cichora cearum developed abundant conidiophores on wild-type Col-0 plants; however, these structures were severely reduced in number on edrl leaves (Table 2) .
  • the conidiophores that were present on edrl leaves often were not septated and appeared to be under-developed compared to those on wild-type Col-0 leaves at the same time point.
  • Plant cells undergoing an HR accumulate callose and autofluorescent compounds in the cell wall.
  • To determine whether the necrosis observed in edrl plants shared the biochemical properties of an HR we assayed infected leaves for deposition of autofluorescent compounds and for callose. Both wild-type Col-0 and edrl plants displayed punctate staining of callose in the cell walls of epidermal cells approximately 3 days post inoculation; however, only edrl plants showed callose staining in large clumps of mesophyll cells.
  • the fungus penetrates through the papillae that subsequently becomes a collar around the penetration peg.
  • Callose deposition in the mesophyll cells of edrl leaves is evident 3 days after inoculation with E . ci chora cearum, which is prior to the appearance of dead cells.
  • the pattern of autofluorescence was similar to that observed for callose.
  • Autofluorescing mesophyll cells accumulated in edrl leaves beginning 3 days post inoculation. Col-0 leaves showed only scattered epidermal cells autofluorescing at the same time point.
  • PR-1 and BGL2 mRNA Little to no PR-1 and BGL2 mRNA was detectable prior to inoculation, or 1 day after infection. By 3 days after infection, significant levels of PR-1 and BGL2 were observed in both wild type and edrl plants. We quantified the levels of mRNA detected by using a Phosphorlmager . The level of PR-1 message in edrl leaves at day 3 was approximately four times higher than in Col- 0 leaves. PR-1 and BGL2 transcript levels increased at days 5 and 7 after infection, but the relative difference between edrl and wild-type plants was less. By day 7, PR-1 levels were higher in wild-type Col-0 than in edrl plan ts .
  • a total of 1223 F 2 plants from the Ler cross were scored for E. cichoracearum resistance and 235 plants displaying resistance to E. cichoracearum were selected for mapping.
  • DNA was isolated from the resistant F 2 plants and analyzed for linkage to SSLP and CAPS markers (Konieczny and Ausubel, 1993, supra ; Bell and Ecker, 1994, supra ) .
  • the edrl mutation mapped 3.2 centiMorgans centromeric from the SSLP marker ATEAT1 (15 recombinant chromosomes) and 0.85 centiMorgans telomeric from the CAPS marker NCC1 (4 recombinant chromosomes) on chromosome 1.
  • the edrl mutant displays enhanced resistance to powdery mildew, but does not constitutively express pathogenesis related genes, such as PR-1 and BGL2.
  • pathogenesis related genes such as PR-1 and BGL2.
  • Our data show that multiple defense responses are induced more rapidly in edrl plants than in wild-type plants when infected with a virulent strain of powdery mildew.
  • P. syringae infection compared to E. cichoracearum might be related to the different modes of infection of these pathogens.
  • P. syringae colonizes the intercellular spaces of the leaf mesophyll, reaching maximum population levels within 2 to 3 days after infection.
  • E. cichoracearum remains on the leaf surface and does not produce spores until 6 to 7 days after infection.
  • the mutant was challenged with additional pathogens.
  • the edrl mutant was found to be resistant to a second species of Erysiphe, E . cruciferarum strain UEA1.
  • This pathogen induced a resistance phenotype essentially the same as that induced by E. ci choracearum, including mesophyll cell death associated with a dramatic decrease in conidiophore production.
  • This result suggests the edrl mutation confers a broad-spectrum rather than race-specific resistance against powdery mildew.
  • ci chora cearum The earliest response detected was deposition of callose and autofluorescent compounds - in epidermal cells and underlying mesophyll cells. We also observed a slightly enhanced expression of PR genes at day 3; however, cell death was not observed until 5 days after inoculation.
  • cichora cearum is similar to the phenotypes conferred by the Pm2 and pm5 genes of wheat, and the Mla3 and Mla l genes of barley. These resistance genes affect powdery mildew growth after the development of secondary hyphae, but before conidiophore production, and are associated with the accumulation of large masses of dead mesophyll cells (Hyde and Colhoun, 1975, Phytopath. Z. 82:185-206; Boyd, et al., 1995, supra ) . In addition, plants with rapidly acting resistance genes occasionally allow fungal germlings to form secondary hyphae. Growth of such escapees is usually halted prior to conidiophore formation and is associated with mesophyll cell death (Hyde and Colhoun, 1975, supra ) .
  • the edrl resistance phenotype shares some attributes with resistance mediated by the mlo mutation of barley.
  • the mlo mutation is recessive and confers resistance to multiple races of E . g. hordei , but has no apparent effect on other pathogen species tested, such as Puccinia hordei , the causative agent of rust on barley (Jorgensen, 1992, supra ; Wolter, et al., 1993, supra ) .
  • Puccinia hordei the causative agent of rust on barley
  • edrl plants showed clear resistance to two species of powdery mildew, but variable resistance to bacterial pathogens.
  • mlo resistance is associated with the formation of callose rich papillae that are thought to block penetration of the fungus into the initially infected epidermal cell (Aist et al., 1988, Physiol. Molec. Plant Pathol . 33:17- 32; Wolter, et al . , 1993, supra ) ; secondary hyphae very rarely form on mlo plants.
  • the edrl mutation does not map to the same location as the Mlo-like Arabidopsis genes identified previously (Buschges, et al., 1997, Cell, 88:695-705) .
  • EXAMPLE 2 Isolation and Analysis of the EDRl Gene
  • EDRl Isolation of the EDRl gene.
  • the edrl mutation was located on the genetic map of Arabidopsis by genetic linkage analysis (see Example 1) .
  • two overlapping bacterial artificial chromosome (BAC) clones were identified that genetically span the region containing edrl .
  • EDRl was therefore contained on one of these two BAC clones, F22013 and F7G19, which are publicly available clones generated as part of the ongoing Arabidopsis genome project.
  • the complete DNA sequence of these two clones is available from the Genbank DNA database.
  • PCR DNA sequences of PCR products were determined using an ABI Dye Terminator FS kit protocol (Perkm-Elmer, Foster City, CA) on an ABI Prism 377 DNA sequencer . Candidates were chosen based on the probability that mutations m such genes could lead to the observed edrl phenotype. In particular, genes known to play a role m stress responses (e.g. superoxide dismutase) or signal transduction (e.g. protein kinases and transcription factors) were focused on.
  • stress responses e.g. superoxide dismutase
  • signal transduction e.g. protein kinases and transcription factors
  • the EDRl protein sequence (SEQ ID NO: 2) contains high similarity to a large number of protein kinases, which are proteins that can catalyze the addition of phosphate groups to other proteins. Many protein kinases are known to participate in signal relays, passing information from outside the cell to other sites within the cell ("signal transduction") .
  • One of the proteins most similar to EDRl is another Arabidopsis protein called CTRl (SEQ ID NO: 3).
  • CTRl Arabidopsis protein
  • the similarity is highest in the putative kinase domain, but extends to other regions of the protein as well (Fig. 2) . This similarity is informative, as a considerable amount is known about CTRl . Loss of function mutations in CTRl result in induction of ethylene-induced responses.
  • plants with null mutations in CTRl behave as if they are being exposed to high levels of ethylene all the time.
  • the CTRl protein functions to repress (i.e. keep off) these responses in the absence of ethylene.
  • the CTRl protein is inactivated, leading to the induction of these responses.
  • the CTRl protein is thus described as a negative regulator of the ethylene response pathway.
  • the EDRl protein may by analogy function as a negative regulator of specific defense response pathways. In the absence of functional EDRl protein, these pathways are more easily induced by pathogens, leading to enhanced disease resistance .
  • the CTRl protein has been shown to interact with a second protein known as ETR1, which is a receptor for the ethylene molecule.
  • CTRl functions near the beginning of the signal relay between perception of ethylene and the activation of ethylene responses.
  • EDRl may also function near the beginning of a signal relay, perhaps activated by a pathogen molecule.
  • EDRl Homologs of EDRl exist in Crop Species.
  • the Arabidopsis EDRl sequence has been used to identify homologs in rice and barley, and can be used to identify homologs in other crop plants . Once these homologous genes are identified they can be inactivated, thus creating edr -like mutants that have enhanced disease resistance.
  • a search of the Genbank DNA, protein, and expressed sequence tag (EST) databases reveals several strongly homologous genes. Although many genes show similarity to the kinase domain encoding portion of EDRl , one gene from tomato called TCTR2 (SEQ ID NO: 4) shows extensive homology throughout the protein-coding region (Fig. 3), indicating that this gene encodes the tomato version of EDRl . Alignment of EDRl and TCTR2 reveals numerous regions where the amino acid sequences are identical. Such regions may be used to design PCR primers to enable amplification of EDRl homologs from other plant species.
  • EDRl showed high similarity to an EST sequence from rice. Because EST sequences represent only partial sequences of the corresponding cDNA clones, this cDNA clone was obtained and the complete DNA sequence determined (SEQ ID NO: 5) .
  • the rice EDRl protein sequence (SEQ ID NO: 6) is aligned with EDRl, TCTR2, and CTRl in Fig. 4.
  • the high degree of similarity between EDRl and the protein encoded by the rice cDNA indicates that this cDNA corresponds to the rice version of EDRl .
  • the protein sequence encoded by the rice cDNA is more similar to EDRl and TCTR2 than any other sequences in the public databases, including CTRl (Table 1) .

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Abstract

La présente invention concerne un nouveau gène, l'EDR1, dont la disruption est associée à une résistance accrue aux maladies chez les plantes. L'invention se rapporte en outre à des plantes transgéniques et des mutants possédant une résistance augmentée aux agents pathogènes des plantes et d'autres propriétés agronomiques désirables. La résistance aux maladies conférée par l'invention diffère des mécanismes de résistance aux maladies des plantes décrits jusqu'à présent.
PCT/US2000/014718 1999-05-26 2000-05-26 Gene permettant de reguler la resistance aux maladies chez les plantes Ceased WO2000071696A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN104830872A (zh) * 2015-05-18 2015-08-12 山西省农业科学院棉花研究所 棉花GhEDR2基因及其编码蛋白与应用
WO2025103148A1 (fr) * 2023-11-17 2025-05-22 中国科学院遗传与发育生物学研究所 Clonage du gène pm26 résistant au mildiou pulvérulent à large spectre de blé et son utilisation

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WO1998004586A2 (fr) * 1996-07-29 1998-02-05 Plant Bioscience Limited Polynucleotide et son utilisation pour moduler une reponse de defense dans des plantes
US6057490A (en) * 1993-01-08 2000-05-02 Novartis Finance Corporation Method for selecting disease resistant mutant plants

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US6057490A (en) * 1993-01-08 2000-05-02 Novartis Finance Corporation Method for selecting disease resistant mutant plants
WO1998004586A2 (fr) * 1996-07-29 1998-02-05 Plant Bioscience Limited Polynucleotide et son utilisation pour moduler une reponse de defense dans des plantes

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CAO ET AL.: "Characterization of an arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance", THE PLANT CELL,, vol. 6, November 1994 (1994-11-01), pages 1583 - 1592, XP002931554 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104830872A (zh) * 2015-05-18 2015-08-12 山西省农业科学院棉花研究所 棉花GhEDR2基因及其编码蛋白与应用
CN104830872B (zh) * 2015-05-18 2017-10-24 山西省农业科学院棉花研究所 棉花GhEDR2基因及其编码蛋白与应用
WO2025103148A1 (fr) * 2023-11-17 2025-05-22 中国科学院遗传与发育生物学研究所 Clonage du gène pm26 résistant au mildiou pulvérulent à large spectre de blé et son utilisation

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