US20040016029A1 - Hypersensitive response elicitor-induced stress resistance - Google Patents

Hypersensitive response elicitor-induced stress resistance Download PDF

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US20040016029A1
US20040016029A1 US10/441,736 US44173603A US2004016029A1 US 20040016029 A1 US20040016029 A1 US 20040016029A1 US 44173603 A US44173603 A US 44173603A US 2004016029 A1 US2004016029 A1 US 2004016029A1
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Zhong-Min Wei
Richard Schading
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Plant Health Care Inc
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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
    • 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
    • C12N15/8273—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 drought, cold, salt resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/27—Erwinia (G)
    • 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
    • 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
    • C12N15/8274—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 herbicide resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/847—Erwinia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/874—Pseudomonas
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/91—Xanthomonas

Definitions

  • the present invention relates to imparting stress resistance to plants with a hypersensitive response elicitor.
  • environmental stress effecting plants can be in the form of climate related stress, air pollution stress, chemical stress, and nutritional stress.
  • climate related stress include drought, water, frost, cold temperature, high temperature, excessive light, and insufficient light.
  • Air pollution stress can be in the form of carbon dioxide, carbon monoxide, sulfur dioxide, NO x , hydrocarbons, ozone, ultraviolet radiation, and acidic rain.
  • Chemical stress can result from application of insecticides, fungicides, herbicides, and heavy metals.
  • Nutritional stress can be caused by fertilizers, micronutrients, and macronutrients.
  • Chilling injury occurs in sensitive species at temperatures that are too low for normal growth but not sufficiently low to form ice. Such injury typically occurs in species of tropical or subtropical origin. When chilling occurs, discoloration or lesions appear on leaves giving them a water-soaked appearance. If roots are chilled, the plants may wilt. On the other hand, freezing temperatures and the accompanying formation of ice crystals in plants can be lethal if ice crystals extend into protoplasts or remain for long periods.
  • High salt concentrations are another form of environmental stress which can afflict plants.
  • high concentrations of salt are found close to seashores and estuaries.
  • natural salt may seep from geological deposits adjoining agricultural areas.
  • salt can accumulate in irrigation water when pure water is evaporated or transpired from soil. About 1 ⁇ 3 of all irrigated farmland is effected by high salt concentrations.
  • High salt content not only injures plants but degrades soil structure by decreasing porosity and water permeability.
  • the present invention is directed to overcoming various forms of environmental stress and imparting resistance in plants to such stress.
  • the present invention relates to the use of a hypersensitive response elicitor protein or polypeptide to impart stress resistance to plants.
  • the hypersensitive response elicitor protein or polypeptide is applied to plants or plant seeds under conditions effective to impart stress resistance.
  • stress resistance is imparted by providing a transgenic plant or plant seed transformed with a DNA molecule which encodes for a hypersensitive response elicitor protein or polypeptide and growing the transgenic plant or plants produced from the transgenic plant seeds under conditions effective to impart stress resistance.
  • Stress encompasses any environmental factor having an adverse effect on plant physiology and development.
  • environmental stress include climate-related stress (e.g., drought, water, frost, cold temperature, high temperature, excessive light, and insufficient light), air pollution stress (e.g., carbon dioxide, carbon monoxide, sulfur dioxide, NO x , hydrocarbons, ozone, ultraviolet radiation, acidic rain), chemical (e.g., insecticides, fungicides, herbicides, heavy metals), and nutritional stress (e.g., fertilizer, micronutrients, macronutrients).
  • climate-related stress e.g., drought, water, frost, cold temperature, high temperature, excessive light, and insufficient light
  • air pollution stress e.g., carbon dioxide, carbon monoxide, sulfur dioxide, NO x , hydrocarbons, ozone, ultraviolet radiation, acidic rain
  • chemical e.g., insecticides, fungicides, herbicides, heavy metals
  • nutritional stress e.g., fertilizer, micronutri
  • the present invention relates to the use of a hypersensitive response elicitor protein or polypeptide to impart stress resistance to plants.
  • the hypersensitive response elicitor protein or polypeptide is applied to plants or plant seeds under conditions effective to impart stress resistance.
  • the stress resistance is imparted by providing a transgenic plant or plant seed transformed with a DNA molecule which encodes for a hypersensitive response elicitor protein or polypeptide and growing the transgenic plant or plants produced from the transgenic plant seeds under conditions effective to impart stress resistance.
  • the hypersensitive response elicitor polypeptides or proteins according to the present invention are derived from hypersensitive response elicitor polypeptides or proteins of a wide variety of fungal and bacterial pathogens. Such polypeptides or proteins are able to elicit local necrosis in plant tissue contacted by the elicitor.
  • Suitable bacterial sources of polypeptide or protein elicitors include Erwinia, Pseudomonas, and Xanthamonas species (e.g., the following bacteria: Erwinia amylovora, Erwinia chrysanthemi, Erwinia stewartii, Erwinia carotovora, Pseudomonas syringae, Pseudomonas solancearum, Xanthomonas campestris, and mixtures thereof).
  • elicitors from Gram positive bacteria is Clavibacter michiganensis subsp. sepedonicus.
  • An example of a fungal source of a hypersensitive response elicitor protein or polypeptide is Phytophthora.
  • Suitable species of Phytophthora include Phytophthora parasitica, Phytophthora cryptogea, Phytophthora cinnamomi, Phytophthora capsici, Phytophthora megasperma, and Phytophthora citrophthora.
  • the hypersensitive response elicitor polypeptide or protein from Erwinia chrysanthemi has an amino acid sequence corresponding to SEQ ID NO: 1 as follows: Met Gln Ile Thr Ile Lys Ala His Ile Gly Gly Asp Leu Gly Val Ser 1 5 10 15 Gly Leu Gly Ala Gln Gly Leu Lys Gly Leu Asn Ser Ala Ala Ser Ser 20 25 30 Leu Gly Ser Ser Val Asp Lys Leu Ser Ser Thr Ile Asp Lys Leu Thr 35 40 45 Ser Ala Leu Thr Ser Met Met Phe Gly Gly Ala Leu Ala Gln Gly Leu 50 55 60 Gly Ala Ser Ser Lys Gly Leu Gly Met Ser Asn Gln Leu Gly Gln Ser 65 70 75 80 Phe Gly Asn Gly Ala Gln Gly Ala Ser Asn Leu Leu Ser Val Pro Lys 85 90 95 Ser Gly Gly Asp Ala Leu Ser Lys Met P
  • This hypersensitive response elicitor polypeptide or protein has a molecular weight of 34 kDa, is heat stable, has a glycine content of greater than 16%, and contains substantially no cysteine.
  • the Erwinia chrysanthemi hypersensitive response elicitor polypeptide or protein is encoded by a DNA molecule having a nucleotide sequence corresponding to SEQ ID NO: 2 as follows: CGATTTTACC CGGGTGAACG TGCTATGACC GACAGCATCA CGGTATTCGA CACCGTTACG 60 GCGTTTATGC CCGCGATGAA CCGGCATCAG GCGGCGCGCT GGTCGCCGCA ATCCGGCGTC 120 GATCTGGTAT TTCAGTTTGG GGACACCGGG CGTGAACTCA TGATOCAGAT TCAGCCGGGG 180 CAGCAATATC CCGGCATGTT GCGCACGCTG CTCGCTCGTC GTTATCAGCA GGCGGCAGAG 240 TGCGATGG
  • the hypersensitive response elicitor polypeptide or protein derived from Erwinia amylovora has an amino acid sequence corresponding to SEQ ID NO: 3 as follows: Met Ser Leu Asn Thr Ser Gly Leu Gly Ala Ser Thr Met Gln Ile Ser 1 5 10 15 Ile Gly Gly Ala Gly Gly Asn Asn Gly Leu Leu Gly Thr Ser Arg Gln 20 25 30 Asn Ala Gly Leu Gly Gly Asn Ser Ala Leu Gly Leu Gly Gly Gly Asn 35 40 45 Gln Asn Asp Thr Val Asn Gln Leu Ala Gly Leu Leu Thr Gly Met Met 50 55 60 Met Met Met Ser Met Met Gly Gly Gly Gly Leu Met Gly Gly Gly Leu 65 70 75 80 Gly Gly Gly Leu Gly Asn Gly Leu Gly Gly Ser Gly Gly Leu Gly Glu 85 90 95 Gly Leu Ser Asn Ala Leu Asn Ala
  • This hypersensitive response elicitor polypeptide or protein has a molecular weight of about 39 kDa, has a pI of approximately 4.3, and is heat stable at 100° C. for at least 10 minutes.
  • This hypersensitive response elicitor polypeptide or protein has substantially no cysteine.
  • the hypersensitive response elicitor polypeptide or protein derived from Erwinia amylovora is more fully described in Wei, Z.-M., R. J. Laby, C. H. Zumoff, D. W. Bauer, S.-Y. He, A. Collmer, and S. V.
  • the DNA molecule encoding this polypeptide or protein has a nucleotide sequence corresponding to SEQ ID NO: 4 as follows: AAGCTTCGGC ATGCCACGTT TGACCGTTGG GTCGGCAGGG TACGTTTGAA TTATTCATAA 60 CAGGAATACG TTATGAGTCT GAATACAAGT GGGCTGGGAG CGTCAACGAT GCAAATTTCT 120 ATCGGCGGTG CGGGCGGAAA TAACGGGTTG CTGGGTACCA GTCGCCAGAA TGCTGGGTTG 180 GGTGCCAATT CTGCACTGGG GCTGGGCGGC GGTAATCAAA ATGATACCGT CAATCAGCTG 240 GCTGGCTTAC TCACCGGCAT GATGATGATG ATGAGCATGA TGGGCGGTGG TGGGCTGATG
  • the isolated DNA molecule of the present invention encodes a hypersensitive response elicitor protein or polypeptide having an amino acid sequence of SEQ ID NO: 6 as follows: Met Ser Ile Leu Thr Leu Asn Asn Asn Thr Ser Ser Ser Pro Gly Leu 1 5 10 15 Phe Gln Ser Gly Gly Asp Asn Gly Leu Gly Gly His Asn Ala Asn Ser 20 25 30 Ala Leu Gly Gln Gln Pro Ile Asp Arg Gln Thr Ile Glu Gln Met Ala 35 40 45 Gln Leu Leu Ala Glu Leu Leu Lys Ser Leu Leu Ser Pro Gln Ser Gly 50 55 60 Asn Ala Ala Thr Gly Ala Gly Gly Asn Asp Gln Thr Thr Gly Val Gly 65 70 75 80 Asn Ala Gly Gly Leu Asn Gly Arg Lys Gly Thr Ala Gly Thr Pro
  • This protein or polypeptide is acidic, rich in glycine and serine, and lacks cysteine. It is also heat stable, protease sensitive, and suppressed by inhibitors of plant metabolism.
  • the protein or polypeptide of the present invention has a predicted molecular size of ca. 4.5 kDa.
  • This DNA molecule is known as the dspE gene for Erwinia amylovora.
  • This isolated DNA molecule of the present invention encodes a protein or polypeptide which elicits a plant pathogen's hypersensitive response having an amino acid sequence of SEQ ID NO: 8 as follows: Met Glu Leu Lys Ser Leu Gly Thr Glu His Lys Ala Ala Val His Thr 1 5 10 15 Ala Ala His Asn Pro Val Gly His Gly Val Ala Leu Gln Gln Gly Ser 20 25 30 Ser Ser Ser Ser Pro Gln Asn Ala Ala Ala Ser Leu Ala Ala Glu Gly 35 40 45 Lys Asn Arg Gly Lys Met Pro Arg Ile His Gln Pro Ser Thr Ala Ala 50 55 60 Asp Gly Ile Ser Ala Ala His Gln Gln Lys Lys Ser Phe Ser Leu Ary 65 70 75 80 Gly Cys Leu Gly Thr Lys Lys Phe Ser Arg Ser Al
  • the present invention relates to an isolated DNA molecule having a nucleotide sequence of SEQ ID NO: 9 as follows: ATGACATCGT CACAGCAGCG GGTTGAAAGG TTTTTACAGT ATTTCTCCGC CGGGTGTAAA 60 ACGCCCATAC ATCTGAAAGA CGGGGTGTGC GCCCTGTATA ACGAACAACA TGAGGAGGCG 120 GCGGTGCTGG AAGTACCGCA ACACAGCGAC AGCCTGTTAC TACACTGCCG AATCATTCAG 180 GCTGACCCAC AAACTTCAAT AACCCTGTAT TCGATGCTAT TACAGCTGAA TTTTGAAATG 240 GCGGCCATGC GCGGCTGTTG GCTGGCTG GATGAACTGC ACAACGTGCG TTTATGTTTT 300 CAGCAGTCGC TGGAGCATCT GGATGAAGCA AGTTTTAGCG ATATCGTTAG CGGCTTCATC 360 GAACATGCGG CAGAAGTGCG TGAGTATATA GCGCAATT
  • This isolated DNA molecule of the present invention encodes a hypersensitive response elicitor protein or polypeptide having an amino acid sequence of SEQ ID NO: 10 as follows: Met Thr Ser Ser Gln Glu Arg Val Glu Arg Phe Leu 1 5 10 Gln Tyr Phe Ser Ala Gly Cys Lys Thr Pro Ile His 15 20 Leu Lys Asp Gly Val Cys Ala Leu Tyr Asn Glu Gln 25 30 35 Asp Glu Glu Ala Ala Val Leu Glu Val Pro Gln His 40 45 Ser Asp Ser Leu Leu Leu His Cys Arg Ile Ile Glu 50 55 60 Ala Asp Pro Gln Thr Ser Ile Thr Leu Tyr Ser Met 65 70 Leu Leu Gln Leu Asn Phe Glu Met Ala Ala Met Arg 75 80 Gly Cys Trp Leu Ala Leu Asp Glu Leu His Asn Val 85 90 95 Arg Leu Cys P
  • This protein or polypeptide is about 16 kDa and has a pI of 4.45.
  • the hypersensitive response elicitor polypeptide or protein derived from Pseudomonas syringae has an amino acid sequence corresponding to SEQ ID NO: 11 as follows: Met Gln Ser Leu Ser Leu Asn Ser Ser Ser Leu Gln Thr Pro Ala Met 1 5 10 15 Ala Leu Val Leu Val Arg Pro Glu Ala Glu Thr Thr Gly Ser Thr Ser 20 25 30 Ser Lys Ala Leu Gln Glu Val Val Val Lys Leu Ala Glu Glu Leu Met 35 40 45 Arg Asn Gly Gln Leu Asp Asp Ser Ser Pro Leu Gly Lys Leu Leu Ala 50 55 60 Lys Ser Met Ala Ala Asp Gly Lys Ala Gly Gly Gly Ile Glu Asp Val 65 70 75 80 Ile Ala Ala Leu Asp Lys Leu Ile His Glu Lys Leu Gly Asp Asn Phe 85 90 95 Gly Ala Ser Ala Asp Ser Ala Ser
  • This hypersensitive response elicitor polypeptide or protein has a molecular weight of 34-35 kDa. It is rich in glycine (about 13.5%) and lacks cysteine and tyrosine.
  • the DNA molecule encoding the hypersensitive response elicitor from Pseudomonas syringae has a nucleotide sequence corresponding to SEQ ID NO: 12 as follows: ATGCAGAGTC TCAGTCTTAA CACCAGCTCG CTGCAAACCC CGGCAATGGC CCTTGTCCTG 60 GTACGTCCTG AAGCCGAGAC GACTGGCAGT ACGTCGAGCA AGGCGCTTCA GGAAGTTGTC 120 GTCAAGCTGG CCGAGGAACT GATGCGCAAT GGTCAACTCG ACGACAGCTC GCCATTGGGA 180 AAACTGTTGG CCAAGTCGAT GGCCGCAGAT GGCAAGGCGG GCGCCGGTAT TCAGGATCTC 240 ATCGCTGCGC TGGACAAGCT GATCCATGAA AAGCTCGGTG ACAACTTCGG CGCGTCTGCG 300 GACAGCCT CGGGTACCGG ACAGCAGGAC CTGATGACTC AGGTGCTCAA TGGCCTGGCC 360 AAGTCG
  • This DNA molecule is known as the dspE gene for Pseudomonas syringae.
  • This isolated DNA molecule of the present invention encodes a protein or polypeptide which elicits a plant pathogen's hypersensitive response having an amino acid sequence of SEQ ID NO: 14 as follows: Met Ser Ile Gly Ile Thr Pro Arg Pro Gln Gln Thr Thr Pro Leu 1 5 10 15 Asp Phe Ser Ala Leu Ser Gly Lys Ser Pro Gln Pro Asn Thr Phe Gly 20 25 30 Glu Gln Asn Thr Gln Gln Ala Ile Asp Pro Ser Ala Leu Leu Phe Gly 35 40 45 Ser Asp Thr Gln Lys Asp Val Asn Phe Gly Thr Pro Asp Ser Thr Val 50 55 60 Gln Asn Pro Gln Asp Ala Ser Lys Pro Asn Asp Ser Gln Ser Asn Ile 65 70 75 80 Ala Lys Leu Ile
  • This protein or polypeptide is about 42.9 kDa.
  • the hypersensitive response elicitor polypeptide or protein derived from Pseudomonas solanacearum has an amino acid sequence corresponding to SEQ ID NO: 15 as follows: Met Ser Val Gly Asn Ile Gln Ser Pro Ser Asn Leu Pro Gly Leu Gln 1 5 10 15 Asn Leu Asn Leu Asn Thr Asn Thr Asn Ser Gln Gln Ser Gly Gln Ser 20 25 30 Val Gln Asp Leu Ile Lys Gln Val Glu Lys Asp Ile Leu Asn Ile Ile 35 40 45 Ala Ala Leu Val Gln Lys Ala Ala Gln Ser Ala Gly Gly Asn Thr Gly 50 55 60 Asn Thr Gly Asn Ala Pro Ala Lys Asp Gly Asn Ala Asn Ala Gly Ala 65 70 75 80 Asn Asp Pro Ser Lys Asn Asp Pro Ser Lys Ser Gln Ala Pro Gln Ser 85 90 95 Ala Asn
  • This sequence is an amino terminal sequence having only 26 residues from the hypersensitive response elicitor polypeptide or protein of Xanthomonas campestris pv. glycines. It matches with fimbrial subunit proteins determined in other Xanthomonas campestris pathovars.
  • the hypersensitive response elicitor polypeptide or protein from Xanthomonas campestris pv. pelargonii is heat stable, protease sensitive, and has a molecular weight of 20 kDa. It includes an amino acid sequence corresponding to SEQ ID NO: 18 as follows: Ser Ser Gln Gln Ser Pro Ser Ala Gly Ser Glu Gln 1 5 10 Gln Leu Asp Gln Leu Leu Ala Met 15 20
  • Hypersensitive response elicitor proteins or polypeptides from Phytophthora parasitica, Phytophthora cryptogea, Phytophthora cinnamoni, Phytophthora capsici, Phytophthora megasperma, and Phytophora citrophthora are described in Kaman, et al., “Extracellular Protein Elicitors from Phytophthora: Most Specificity and Induction of Resistance to Bacterial and Fungal Phytopathogens,” Molec.
  • elicitors are exemplary.
  • Other elicitors can be identified by growing fungi or bacteria that elicit a hypersensitive response under conditions which genes encoding an elicitor are expressed.
  • Cell-free preparations from culture supernatants can be tested for elicitor activity (i.e. local necrosis) by using them to infiltrate appropriate plant tissues.
  • Fragments of the above hypersensitive response elicitor polypeptides or proteins as well as fragments of full length elicitors from other pathogens are encompassed by the method of the present invention.
  • Suitable fragments can be produced by several means.
  • subclones of the gene encoding a known elicitor protein are produced by conventional molecular genetic manipulation by subcloning gene fragments.
  • the subclones then are expressed in vitro or in vivo in bacterial cells to yield a smaller protein or peptide that can be tested for elicitor activity according to the procedure described below.
  • fragments of the elicitor protein gene may be synthesized by using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein. These then would be cloned into an appropriate vector for expression of a truncated peptide or protein.
  • the internal fragment of the amino acid sequence of SEQ ID NO: 3 can span the following amino acids of SEQ ID NO: 3: 105 and 179, 137 and 166, 121 and 150, or 137 and 156. Other suitable fragments can be identified in accordance with the present invention.
  • the N-terminal fragment of the amino acid sequence of SEQ ID NO: 3 can span the following amino acids of SEQ ID NO: 3: 1 and 98, 1 and 104, 1 and 122, 1 and 168, 1 and 218, 1 and 266, 1 and 342, 1 and 321, and 1 and 372.
  • the internal fragment of the amino acid sequence of SEQ ID NO: 3 can span the following amino acids of SEQ ID NO: 3: 76 and 209, 105 and 209, 99 and 209, 137 and 204, 137 and 200, 109 and 204, 109 and 200, 137 and 180, and 105 and 180.
  • the coli coli ) carrying a recombinant plasmid is propagated, lysed by sonication, heat, or chemical treatment, and the homogenate is centrifuged to remove bacterial debris. The supernatant is then subjected to heat treatment and the hypersensitive response elicitor is separated by centrifugation. The supernatant fraction containing the hypersensitive response elicitor is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the fragment. If necessary, the protein fraction may be further purified by ion exchange or HPLC.
  • the DNA molecule encoding the hypersensitive response elicitor polypeptide or protein can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • U.S. Pat. No. 4,237,224 to Cohen and Boyer which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture.
  • Recombinant genes may also be introduced into viruses, such as vaccina virus.
  • Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
  • Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK ⁇ or KS ⁇ (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see F.
  • viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC
  • Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation.
  • the DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference.
  • host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used.
  • Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria.
  • the expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
  • mRNA messenger RNA
  • SD Shine-Dalgamo
  • Promotors vary in their “strength” (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E.
  • promotors such as the T7 phage promotor, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promotor or other E. coli promotors produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • tac hybrid trp-lacUV5
  • Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unless specifically induced. In certain operations, the addition of specific inducers is necessary for efficient transcription of the inserted DNA.
  • the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside).
  • IPTG isopropylthio-beta-D-galactoside
  • Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively.
  • the DNA expression vector which contains a promotor, may also contain any combination of various “strong” transcription and/or translation initiation signals.
  • efficient translation in E. coli requires an SD sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site.
  • ATG initiation codon
  • any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes.
  • any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
  • the isolated DNA molecule encoding the hypersensitive response elicitor polypeptide or protein has been cloned into an expression system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system.
  • Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.
  • the present invention's method of imparting stress resistance to plants can involve applying the hypersensitive response elicitor polypeptide or protein in a non-infectious form to all or part of a plant or a plant seed under conditions effective for the elicitor to impart stress resistance.
  • the hypersensitive response elicitor protein or polypeptide can be applied to plants such that seeds recovered from such plants themselves are able to impart stress resistance in plants.
  • transgenic plants or plant seeds can be utilized. When utilizing transgenic plants, this involves providing a transgenic plant transformed with a DNA molecule encoding a hypersensitive response elicitor polypeptide or protein and growing the plant under conditions effective to permit that DNA molecule to impart stress resistance to plants.
  • a transgenic plant seed transformed with a DNA molecule encoding a hypersensitive response elicitor polypeptide or protein can be provided and planted in soil. A plant is then propagated from the planted seed under conditions effective to permit that DNA molecule to impart stress resistance to plants.
  • the methods of the present invention can be utilized to treat a wide variety of plants or their seeds to impart stress resistance.
  • Suitable plants include dicots and monocots. More particularly, useful crop plants can include: alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane.
  • suitable ornamental plants are: Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.
  • stress refers to drought, salt, cold temperatures (e.g., frost), chemical treatment (e.g., insecticides, fungicides, herbicides, fertilizers), water, excessive light, and insufficient light.
  • the hypersensitive response elicitor protein or polypeptide in accordance with present invention, can be applied by low or high pressure spraying, coating, immersion, or injection. Other suitable application procedures can be envisioned by those skilled in the art provided they are able to effect contact of the elicitor with cells of the plant or plant seed.
  • the seeds can be planted in natural or artificial soil and cultivated using conventional procedures to produce plants. After plants have been propagated from seeds treated in accordance with the present invention, the plants may be treated with one or more applications of the hypersensitive response elicitor protein or polypeptide to impart stress resistance to plants.
  • the hypersensitive response elicitor polypeptide or protein in accordance with the present invention, can be applied to plants or plant seeds alone or in a mixture with other materials. Alternatively, the hypersensitive response elicitor polypeptide or protein can be applied separately to plants with other materials being applied at different times.
  • a composition suitable for treating plants or plant seeds in accordance with the application embodiment of the present invention contains a hypersensitive response elicitor polypeptide or protein in a carrier.
  • Suitable carriers include water, aqueous solutions, slurries, or dry powders.
  • the composition contains greater than 500 nM of the elicitor.
  • Suitable additives include buffering agents, wetting agents, coating agents, and abrading agents. These materials can be used to facilitate the process of the present invention.
  • the hypersensitive response elicitor can be applied to plant seeds with other conventional seed formulation and treatment materials, including clays and polysaccharides.
  • transgenic plants and transgenic seeds a hypersensitive response elicitor need not be applied topically to the plants or seeds. Instead, transgenic plants transformed with a DNA molecule encoding such an elicitor are produced according to procedures well known in the art.
  • the vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA. Crossway, Mol. Gen. Genetics, 202:179-85 (1985), which is hereby incorporated by reference.
  • the genetic material may also be transferred into the plant cell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982), which is hereby incorporated by reference.
  • Another approach to transforming plant cells with a gene is particle bombardment (also known as biolistic transformation) of the host cell.
  • particle bombardment also known as biolistic transformation
  • This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which are hereby incorporated by reference.
  • this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof.
  • the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA.
  • the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle.
  • Biologically active particles e.g., dried bacterial cells containing the vector and heterologous DNA
  • the DNA molecule may also be introduced into the plant cells by electroporation. Fromm et al., Proc. Natl. Acad. Sci. USA, 82:5824 (1985), which is hereby incorporated by reference. In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate.
  • Another method of introducing the DNA molecule into plant cells is to infect a plant cell with Agrobacterium tumefaciens or A. rhizogenes previously transformed with the gene. Under appropriate conditions known in the art, the transformed plant cells are grown to form shoots or roots, and develop further into plants. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28° C.
  • Agrobacterium is a representative genus of the Gram-negative family Rhizobiaceae. Its species are responsible for crown gall ( A. tumefaciens ) and hairy root disease ( A. rhizogenes ). The plant cells in crown gall tumors and hairy roots are induced to produce amino acid derivatives known as opines, which are catabolized only by the bacteria.
  • the bacterial genes responsible for expression of opines are a convenient source of control elements for chimeric expression cassettes. In addition, assaying for the presence of opines can be used to identify transformed tissue.
  • Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes.
  • the Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science, 237:1176-83 (1987), which is hereby incorporated by reference.
  • the transformed plant cells After transformation, the transformed plant cells must be regenerated.
  • Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants.
  • the culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
  • the expression cassette After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure with the presence of the gene encoding the hypersensitive response elicitor resulting in stress resistance to the plant.
  • transgenic seeds or propagules are recovered from the transgenic plants.
  • the seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants.
  • the transgenic plants are propagated from the planted transgenic seeds under conditions effective to impart stress resistance to plants. While not wishing to be bound by theory, such stress resistance may be RNA mediated or may result from expression of the elicitor polypeptide or protein.
  • transgenic plants and plant seeds are used in accordance with the present invention, they additionally can be treated with the same materials as are used to treat the plants and seeds to which a hypersensitive response elicitor in accordance with the present invention is applied.
  • these other materials including a hypersensitive response elicitor in accordance with the present invention, can be applied to the transgenic plants and plant seeds by the above-noted procedures, including high or low pressure spraying, injection, coating, and immersion.
  • the plants may be treated with one or more applications of the hypersensitive response elicitor in accordance with the present invention to impart stress resistance.
  • Such plants may also be treated with conventional plant treatment agents (e.g., insecticides, fertilizers, etc.).
  • Aphids ( Aphids gossypii ) infect cotton during the entire growth season.
  • the damage of aphid infection ranges from honeydew deposit that contaminates the lint and reduces crop value to defoliation that reduces or destroys crops.
  • cotton is usually sprayed with insecticides, for example Asana XL when the infection pressure is not very high, and Admire when the infestation pressure is high.
  • insecticides for example Asana XL when the infection pressure is not very high, and Admire when the infestation pressure is high.
  • the effect of a hypersensitive response elicitor on aphids in cotton was studied by a trial involving a randomized complete block design. This involved treatment with Erwinia amylovora hypersensitive response elicitor (i.e.
  • HP-1000TM at 20, 60, and 80 ppm and a chemical insecticide, Asana XL, at 8 oz./ac.
  • Each treatment involved foliar application beginning at cotyledon to three true leaves and thereafter at 14 day intervals using a backpack sprayer. Aphid counts and overall growth of the cotton were made immediately prior to spray application at 14, 28, 35, and 42 days after the first treatment (“DAT 1”). Twenty-five randomly selected leaves per plot were collected at the first three sampling dates and the leaves per plot at the final sampling date.
  • Hypersensitive response elicitor-treated cotton was more resistant to the damage caused by Pravado (Admire) and Asana. After the second chemical spraying, it was observed that cotton plants were stress shocked by the insecticides. The cotton plants previously treated with Asana and untreated control were defoliated. On most of the chemical-treated cotton, there were no leaves, or very few leaves, in the lower portion of plants. However, the hypersensitive response elicitor-treated plants, especially the plot where hypersensitive response elicitor was applied at 80 ppm, had no defoliation and the cotton plants were vigorous and healthy.
  • a cucumber field trial was set up to test the effect of Erwinia amylovora hypersensitive response elicitor on disease control, tolerance to drought stress, and yield. Three different rates were tested, there at 15, 30, and 60 ⁇ g/ml. In addition to hypersensitive response elicitor treatment, there was an untreated control. Each treatment contained three replicate plots. When the first true leaf emerges, hypersensitive response elicitor was sprayed with a back bag sprayer. The second spray was applied ten days after the first spray. The third application was right after the recovery of cucumber seedlings after the transplanting to the field. Individual treatment was randomly assigned in the field.
  • the recovery rate of the transplanted cucumber seedlings was higher for the hypersensitive response elicitor-treated plants than for the untreated control. More than 80% of the hypersensitive response elicitor-treated cucumber seedlings survived, while only 57% untreated plants survived.
  • Hypersensitive response elicitor-treated cucumber started to flower 14 days earlier than untreated control cucumber. The early flowering resulted in an earlier harvest. In the first harvest, more than 0.4 kilograms of cucumber fruits per plant were harvested from the hypersensitive response elicitor-treated cucumbers; however, virtually no fruit was harvested from untreated control. By the end of the season, untreated plants died due to severe drought, but hypersensitive response elicitor-treated plants were still alive and had one more harvest.
  • the increased yield was partially attributed to hypersensitive response elicitor-induced growth enhancement and partially resulted from more tolerance of hypersensitive response elicitor-treated cucumber to drought, because usually the yield increase from hypersensitive response elicitor-induced growth enhancement is between 10-40%.
  • Pepper seedlings were drenched with hypersensitive response elicitor at 20 ppm seven days before transplanting, sprayed seven days after the transplanting, and then, sprayed every fourteen days. Standard chemicals, Brave, Maneb, Kocide, and Admire, were used for the rest of the treatment.
  • hypersensitive response elicitor-treated pepper was more tolerant to herbicide damage.
  • the pepper field was applied with the herbicide SENCOR which is not labeled for pepper. This herbicide is known to cause severe foliar damage to pepper in chemically-treated plants but not with hypersensitive response elicitor-treated plants.
  • a field trial was conducted to test if hypersensitive elicitor treated pepper would be more tolerant to herbicide stress.
  • the trial contains 6 treatments and 4 replicates for each treatment. The treatments are described as follows:
  • LEXONETM contains the same active ingredient as SENCORTM herbicide (Bayer, Kansas City, Mo.) used in Example 3. Pepper seedlings were drenched with MESSENGERTM solution at the concentration of HR elicitor protein of about 20 ppm seven days before transplanting into the field and then sprayed every 14 days after the transplanting. LEXONE was applied at high (0.3 pound/acre) and low levels (0.15 pound/acre). 50 gallon water and 100 mL of the herbicide solution was introduced into the root zone of each plant in the respective treatment five weeks after transplant into the field.
  • a non-irrigated cotton trial experienced 26 consecutive days of drought.
  • the average daily heat index was near or over 100 degrees F., adding to the stress placed on the plants in the field.
  • MESSENGERTM treated plants survived the heat and drought stresses much better than the untreated plants did. Plants treated with MESSENGERTM had 37.6% more root and shoot mass than the check plants (Table 7). The MESSENGERTM treated plants also had significantly more cotton bolls than the check plants (Table 8). The number of cotton bolls from positions 1 and 2 have a significant contribution to the overall yield. Table 8 showed that MESSENGERTM treated plants had 47% more bolls in positions 1 and 2 and 57% more boll from a whole plant in comparison to the yield achieved using a grower standard treatment (i.e. with no MESSENGERTM treatment). A common reaction to stress in cotton is for the plant to abort bolls.
  • Calcium is an important element for plant physiology and development.
  • a deficiency in calcium can cause several plant diseases.
  • blossom-end rot is caused by a localized calcium deficiency in the distal end of the tomato fruit.
  • calcium is not a highly mobile element, a deficiency can occur with a fluctuation in water supply.
  • tomato growers experienced higher level of blossom-end rot during dry weather conditions when infrequent rains storms dumped a lot of water and then return to a hot and dry condition quickly. Lowering or raising the irrigation water table erratically during a dry and hot growing season can also increase the disease.
  • MESSENGERTM the formulated product containing 3% HR elicitor
  • the application rate of the MESSENGERTM was 2.27 oz per care.
  • the first spray of MESSENGERTM was carried out 7 days before the transplanting and then every 14-days after transplanting.
  • MESSENGERTM-treated tomatoes were compared with a standard grower treatment not utilizing MESSENGERTM. Each treatment had 4 replicates.

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Abstract

The present invention is directed to imparting stress resistance to plants. This can be achieved by applying a hypersensitive response elicitor in a non-infectious form to plants or plant seeds under conditions effective to impart stress resistance to plants or plants grown from the plant seeds. Alternatively, transgenic plants or plant seeds transformed with a DNA molecule encoding the elicitor can be provided and the transgenic plants or plants resulting from the transgenic plant seeds are grown under conditions effective to impart stress resistance to plants or plants grown from the plant seeds.

Description

  • This application claims benefit of U.S. Provisional Patent Application Serial No. 60/107,243, filed Nov. 5, 1999.[0001]
    • FIELD OF THE INVENTION
  • The present invention relates to imparting stress resistance to plants with a hypersensitive response elicitor. [0002]
    • BACKGROUND OF THE INVENTION
  • Under both natural and agricultural conditions, plants are exposed to various forms of environmental stress. Stress is mainly measured with respect to growth (i.e. biomass accumulation) or with respect to the primary assimilation processes (i.e. carbon dioxide and mineral intake). Soil water deficits, suboptimal and supraoptimal temperatures, salinity, and poor aeration of soils may each cause some growth restrictions during the growing season, so that the yield of plants at the end of the season expresses only a small fraction of their genetic potential. Indeed, it is estimated that in the United States the yield of field-grown crops is only 22% of genetic potential. The same physicochemical factors can become extreme in some habitats, such as deserts or marshes, and only specially adapted vegetation can complete its life cycle in the unusually hostile conditions. In less extreme environments, individual plants can become acclimated to changes in water potential, temperature, salinity, and oxygen deficiency so that their fitness for those environments improves. Some species are better able to adapt than others, and various anatomical, structural, and biochemical mechanisms account for acclimation. [0003]
  • Under natural and agriculture conditions, plants must constantly endure stress. Some environmental factors can become stressful in a very short period of time (e.g., high or low temperature) or may take long periods of time to stress plants (e.g., soil water content or mineral nutrients). Generally, environmental stress effecting plants can be in the form of climate related stress, air pollution stress, chemical stress, and nutritional stress. Examples of climate related stress include drought, water, frost, cold temperature, high temperature, excessive light, and insufficient light. Air pollution stress can be in the form of carbon dioxide, carbon monoxide, sulfur dioxide, NO[0004] x, hydrocarbons, ozone, ultraviolet radiation, and acidic rain. Chemical stress can result from application of insecticides, fungicides, herbicides, and heavy metals. Nutritional stress can be caused by fertilizers, micronutrients, and macronutrients.
  • For most plants, water is essential for growth. Some plants are able to preserve some water in the soil for later use, while others complete their life cycles during a wet season before the onset of any drought. Other plants are able to aggressively consume water to save themselves while causing water deprivation for other plants in that location. Plants lacking any of these capabilities are severely hampered by the absence of water. [0005]
  • Chilling injury occurs in sensitive species at temperatures that are too low for normal growth but not sufficiently low to form ice. Such injury typically occurs in species of tropical or subtropical origin. When chilling occurs, discoloration or lesions appear on leaves giving them a water-soaked appearance. If roots are chilled, the plants may wilt. On the other hand, freezing temperatures and the accompanying formation of ice crystals in plants can be lethal if ice crystals extend into protoplasts or remain for long periods. [0006]
  • Stress is also caused by the other temperature extremes with few plants being able to survive high temperatures. When higher plant cells or tissues are dehydrated or are not growing, they can survive higher temperatures than cells which are hydrated, vegetative, and growing. Tissues which are actively growing can rarely survive at temperatures above 45° C. [0007]
  • High salt concentrations are another form of environmental stress which can afflict plants. In natural conditions, such high concentrations of salt are found close to seashores and estuaries. Farther inland, natural salt may seep from geological deposits adjoining agricultural areas. In addition, salt can accumulate in irrigation water when pure water is evaporated or transpired from soil. About ⅓ of all irrigated farmland is effected by high salt concentrations. High salt content not only injures plants but degrades soil structure by decreasing porosity and water permeability. [0008]
  • Air pollution in the form of ozone, carbon dioxide, carbon monoxide, sulfur dioxide, NO[0009] x, and hydrocarbons can very adversely effect plant growth by creating smog and environmental warming.
  • The present invention is directed to overcoming various forms of environmental stress and imparting resistance in plants to such stress. [0010]
    • SUMMARY OF THE INVENTION
  • The present invention relates to the use of a hypersensitive response elicitor protein or polypeptide to impart stress resistance to plants. In one embodiment of the present invention, the hypersensitive response elicitor protein or polypeptide is applied to plants or plant seeds under conditions effective to impart stress resistance. Alternatively, stress resistance is imparted by providing a transgenic plant or plant seed transformed with a DNA molecule which encodes for a hypersensitive response elicitor protein or polypeptide and growing the transgenic plant or plants produced from the transgenic plant seeds under conditions effective to impart stress resistance. [0011]
  • Stress encompasses any environmental factor having an adverse effect on plant physiology and development. Examples of such environmental stress include climate-related stress (e.g., drought, water, frost, cold temperature, high temperature, excessive light, and insufficient light), air pollution stress (e.g., carbon dioxide, carbon monoxide, sulfur dioxide, NO[0012] x, hydrocarbons, ozone, ultraviolet radiation, acidic rain), chemical (e.g., insecticides, fungicides, herbicides, heavy metals), and nutritional stress (e.g., fertilizer, micronutrients, macronutrients). Applicants have found that use of hypersensitive response elicitors in accordance with the present invention impart resistance to plants against such forms of environmental stress.
    • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention relates to the use of a hypersensitive response elicitor protein or polypeptide to impart stress resistance to plants. In one embodiment of the present invention, the hypersensitive response elicitor protein or polypeptide is applied to plants or plant seeds under conditions effective to impart stress resistance. Alternatively, the stress resistance is imparted by providing a transgenic plant or plant seed transformed with a DNA molecule which encodes for a hypersensitive response elicitor protein or polypeptide and growing the transgenic plant or plants produced from the transgenic plant seeds under conditions effective to impart stress resistance. [0013]
  • The hypersensitive response elicitor polypeptides or proteins according to the present invention are derived from hypersensitive response elicitor polypeptides or proteins of a wide variety of fungal and bacterial pathogens. Such polypeptides or proteins are able to elicit local necrosis in plant tissue contacted by the elicitor. Examples of suitable bacterial sources of polypeptide or protein elicitors include Erwinia, Pseudomonas, and Xanthamonas species (e.g., the following bacteria: [0014] Erwinia amylovora, Erwinia chrysanthemi, Erwinia stewartii, Erwinia carotovora, Pseudomonas syringae, Pseudomonas solancearum, Xanthomonas campestris, and mixtures thereof). In addition to hypersensitive response elicitors from these Gram negative bacteria, it is possible to use elicitors from Gram positive bacteria. One example is Clavibacter michiganensis subsp. sepedonicus.
  • An example of a fungal source of a hypersensitive response elicitor protein or polypeptide is Phytophthora. Suitable species of Phytophthora include [0015] Phytophthora parasitica, Phytophthora cryptogea, Phytophthora cinnamomi, Phytophthora capsici, Phytophthora megasperma, and Phytophthora citrophthora.
  • The hypersensitive response elicitor polypeptide or protein from [0016] Erwinia chrysanthemi has an amino acid sequence corresponding to SEQ ID NO: 1 as follows:
    Met Gln Ile Thr Ile Lys Ala His Ile Gly Gly Asp Leu Gly Val Ser
    1               5               10              15             
    Gly Leu Gly Ala Gln Gly Leu Lys Gly Leu Asn Ser Ala Ala Ser Ser
                20                  25                  30         
    Leu Gly Ser Ser Val Asp Lys Leu Ser Ser Thr Ile Asp Lys Leu Thr
            35                  40                  45             
    Ser Ala Leu Thr Ser Met Met Phe Gly Gly Ala Leu Ala Gln Gly Leu
        50                  55                  60                 
    Gly Ala Ser Ser Lys Gly Leu Gly Met Ser Asn Gln Leu Gly Gln Ser
    65                  70                  75                  80
    Phe Gly Asn Gly Ala Gln Gly Ala Ser Asn Leu Leu Ser Val Pro Lys
                    85                  90                  95     
    Ser Gly Gly Asp Ala Leu Ser Lys Met Phe Asp Lys Ala Leu Asp Asp
                100                 105                 110        
    Leu Leu Gly His Asp Thr Val Thr Lys Leu Thr Asn Gln Ser Asn Gln
            115                 120                 125            
    Leu Ala Asn Ser Met Leu Asn Ala Ser Gln Met Thr Gln Gly Asn Met
        130                 135                 140                
    Asn Ala Phe Gly Ser Gly Val Asn Asn Ala Leu Ser Ser Ile Leu Gly
    145                 150                 155                 160
    Asn Gly Leu Gly Gln Ser Met Ser Gly Phe Ser Gln Pro Ser Leu Gly
                    165                 170                 175    
    Ala Gly Gly Leu Gln Gly Leu Ser Gly Ala Gly Ala Phe Asn Gln Leu
                180                 185                 190        
    Gly Asn Ala Ile Gly Met Gly Val Gly Gln Asn Ala Ala Leu Ser Ala
            195                 200                 205            
    Leu Ser Asn Val Ser Thr His Val Asp Gly Asn Asn Arg His Phe Val
        210                 215                 220                
    Asp Lys Glu Asp Arg Gly Met Ala Lys Glu Ile Gly Gln Phe Met Asp
    225                 230                 235                 240
    Gln Tyr Pro Glu Ile Phe Gly Lys Pro Glu Tyr Gln Lys Asp Gly Trp
                    245                 250                 255    
    Ser Ser Pro Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser Lys
                260                 265                 270        
    Pro Asp Asp Asp Gly Met Thr Gly Ala Ser Met Asp Lys Phe Arg Gln
            275                 280                 285            
    Ala Met Gly Met Ile Lys Ser Ala Val Ala Gly Asp Thr Gly Asn Thr
        290                 295                 300                
    Asn Leu Asn Leu Arg Gly Ala Gly Gly Ala Ser Leu Gly Ile Asp Ala
    305                 310                 315                 320
    Ala Val Val Gly Asp Lys Ile Ala Asn Met Ser Leu Gly Lys Leu Ala
                    325                 330                 335    
    Asn Ala
  • This hypersensitive response elicitor polypeptide or protein has a molecular weight of 34 kDa, is heat stable, has a glycine content of greater than 16%, and contains substantially no cysteine. The [0017] Erwinia chrysanthemi hypersensitive response elicitor polypeptide or protein is encoded by a DNA molecule having a nucleotide sequence corresponding to SEQ ID NO: 2 as follows:
    CGATTTTACC CGGGTGAACG TGCTATGACC GACAGCATCA CGGTATTCGA CACCGTTACG 60
    GCGTTTATGC CCGCGATGAA CCGGCATCAG GCGGCGCGCT GGTCGCCGCA ATCCGGCGTC 120
    GATCTGGTAT TTCAGTTTGG GGACACCGGG CGTGAACTCA TGATOCAGAT TCAGCCGGGG 180
    CAGCAATATC CCGGCATGTT GCGCACGCTG CTCGCTCGTC GTTATCAGCA GGCGGCAGAG 240
    TGCGATGGCT GCCATCTGTG CCTGAACGGC AGCGATGTAT TGATCCTCTG GTGGCCGCTG 300
    CCGTCGGATC CCGGCAGTTA TCCGCAGGTG ATCGAACGTT TGTTTGAACT GGCGGGAATG 360
    ACGTTGCCGT CGCTATCCAT AGCACCGACG GCGCGTCCGC AGACAGGGAA CGGACGCGCC 420
    CGATCATTAA GATAAAGGCC GCTTTTTTTA TTGCAAAACG GTAACGGTGA GGAACCGTTT 480
    CACCGTCGGC GTCACTCAGT AACAAGTATC CATCATGATG CCTACATCGG GATCGGCGTG 540
    GGCATCCGTT GCAGATACTT TTGCGAACAC CTGACATGAA TGAGGAAACG AAATTATGCA 600
    AATTACGATC AAAGCGCACA TCGGCGGTGA TTTGGGCGTC TCCGGTCTGG GGCTGGGTGC 660
    TCAGGGACTG AAAGGACTGA ATTCCGCGGC TTCATCGCTG GGTTCCAGCG TGGATAAACT 720
    GAGCAGCACC ATCGATAAGT TGACCTCCGC GCTGACTTCG ATGATGTTTG GCGGCGCGCT 780
    GGCGCAGGGG CTGGGCGCCA GCTCGAAGGG GCTGGGGATG AGCAATCAAC TGGGCCAGTC 840
    TTTCGGCAAT GGCGCGCAGG GTGCGAGCAA CCTGCTATCC GTACCGAAAT CCGGCGGCCA 900
    TGCGTTGTCA AAAATGTTTG ATAAAGCGCT CGACGATCTG CTGGGTCATG ACACCGTGAC 960
    CAAGCTGACT AACCAGAGCA ACCAACTGGC TAATTCAATG CTGAACGCCA GCCAGATGAC 1020
    CCAGGGTAAT ATGAATGCGT TCGGCAGCGG TGTGAACAAC GCACTGTCGT CCATTCTCGG 1080
    CAACGGTCTC GGCCAGTCGA TGAGTGGCTT CTCTCAGCCT TCTCTGGGGG CAGGCGGCTT 1140
    GCAGGGCCTG AGCGGCGCGG GTGCATTCAA CCAGTTGGGT AATGCCATCG GCATGGGCGT 1200
    GGGGCAGAAT GCTGCGCTGA GTGCGTTGAG TAACGTCAGC ACCCACGTAG ACGGTAACAA 1260
    CCGCCACTTT GTAGATAAAG AAGATCGCGG CATGGCGAAA GAGATCGGCC AGTTTATGGA 1320
    TCAGTATCCG GAAATATTCG GTAAACCGGA ATACCAGAAA GATGGCTGGA GTTCGCCGAA 1380
    GACGGACGAC AAATCCTGGG CTAAAGCGCT GAGTAAACCG GATGATGACG GTATGACCGG 1440
    CGCCAGCATG GACAAATTCC GTCAGGCGAT GGGTATGATC AAAAGCGCGG TGGCGGGTGA 1500
    TACCGGCAAT ACCAACCTGA ACCTGCGTGG CGCGGGCGGT GCATCGCTGG GTATCGATGC 1560
    GGCTGTCGTC GGCGATAAAA TAGCCAACAT GTCGCTGGGT AAGCTGGCCA ACGCCTGATA 1620
    ATCTGTGCTG GCCTGATAAA GCGGAAACGA AAAAAGAGAC GGGGAAGCCT GTCTCTTTTC 1680
    TTATTATGCG GTTTATGCGG TTACCTGGAC CGGTTAATCA TCGTCATCGA TCTGGTACAA 1740
    ACGCACATTT TCCCGTTCAT TCGCGTCGTT ACGCGCCACA ATCGCGATGG CATCTTCCTC 1800
    GTCGCTCAGA TTGCGCGGCT GATGGGGAAC GCCGGGTGGA ATATAGAGAA ACTCGCCGCC 1860
    CAGATGGAGA CACGTCTGCG ATAAATCTGT GCCGTAACGT GTTTCTATCC GCCCCTTTAG 1920
    CAGATAGATT GCGGTTTCGT AATCAACATG CTAATGCGGT TCCGCCTGTG CGCCGGCCGG 1980
    GATCACCACA ATATTCATAG AAAGCTGTCT TGCACCTACC GTATCGCGGG AGATACCGAC 2040
    AAAATAGGGC AGTTTTTGCG TCGTATCCGT GGGGTGTTCC GGCCTGACAA TCTTGAGTTG 2100
    GTTCGTCATC ATCTTTCTCC ATCTGGGCGA CCTGATCGGT T  2141
  • The hypersensitive response elicitor polypeptide or protein derived from Erwinia amylovora has an amino acid sequence corresponding to SEQ ID NO: 3 as follows: [0018]
    Met Ser Leu Asn Thr Ser Gly Leu Gly Ala Ser Thr Met Gln Ile Ser
    1               5                   10                  15     
    Ile Gly Gly Ala Gly Gly Asn Asn Gly Leu Leu Gly Thr Ser Arg Gln
                20                  25                  30         
    Asn Ala Gly Leu Gly Gly Asn Ser Ala Leu Gly Leu Gly Gly Gly Asn
            35                  40                  45             
    Gln Asn Asp Thr Val Asn Gln Leu Ala Gly Leu Leu Thr Gly Met Met
        50                  55                  60                 
    Met Met Met Ser Met Met Gly Gly Gly Gly Leu Met Gly Gly Gly Leu
    65                  70                  75                  80 
    Gly Gly Gly Leu Gly Asn Gly Leu Gly Gly Ser Gly Gly Leu Gly Glu
                    85                  90                  95     
    Gly Leu Ser Asn Ala Leu Asn Asp Met Leu Gly Gly Ser Leu Asn Thr
                100                 105                 110        
    Leu Gly Ser Lys Gly Gly Asn Asn Thr Thr Ser Thr Thr Asn Ser Pro
            115                 120                 125            
    Leu Asp Gln Ala Leu Gly Ile Asn Ser Thr Ser Gln Asn Asp Asp Ser
        130                 135                 140                
    Thr Ser Gly Thr Asp Ser Thr Ser Asp Ser Ser Asp Pro Met Gln Gln
    145                 150                 155                 160
    Leu Leu Lys Met Phe Ser Glu Ile Met Gln Ser Leu Phe Gly Asp Gly
                    165                 170                 175    
    Gln Asp Gly Thr Gln Gly Ser Ser Ser Gly Gly Lys Gln Pro Thr Glu
                180                 185                 190        
    Gly Glu Gln Asn Ala Tyr Lys Lys Gly Val Thr Asp Ala Leu Ser Gly
            195                 200                 205            
    Leu Met Gly Asn Gly Leu Ser Gln Leu Leu Gly Asn Gly Gly Leu Gly
        210                 215                 220                
    Gly Gly Gln Gly Gly Asn Ala Gly Thr Gly Leu Asp Gly Ser Ser Leu
    225                 230                 235                 240
    Gly Gly Lys Gly Leu Gln Asn Leu Ser Gly Pro Val Asp Tyr Gln Gln
                    245                 250                 255    
    Leu Gly Asn Ala Val Gly Thr Gly Ile Gly Met Lys Ala Gly Ile Gln
                260                 265                 270        
    Ala Leu Asn Asp Ile Gly Thr His Arg His Ser Ser Thr Arg Ser Phe
            275                 280                 285            
    Val Asn Lys Gly Asp Arg Ala Met Ala Lys Glu Ile Gly Gln Phe Met
        290                 295                 300                
    Asp Gln Tyr Pro Glu Val Phe Gly Lys Pro Gln Tyr Gln Lys Gly Pro
    305                 310                 315                 320
    Gly Gln Glu Val Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser
                    325                 330                 335    
    Lys Pro Asp Asp Asp Gly Met Thr Pro Ala Ser Met Glu Gln Phe Asn
                340                 345                 350        
    Lys Ala Lys Gly Met Ile Lys Arg Pro Met Ala Gly Asp Thr Gly Asn
            355                 360                 365            
    Gly Asn Leu Gln Ala Arg Gly Ala Gly Gly Ser Ser Leu Gly Ile Asp
        370                 375                 380                
    Ala Met Met Ala Gly Asp Ala Ile Asn Asn Met Ala Leu Gly Lys Leu
    385                 390                 395                 400
    Gly Ala Ala
  • This hypersensitive response elicitor polypeptide or protein has a molecular weight of about 39 kDa, has a pI of approximately 4.3, and is heat stable at 100° C. for at least 10 minutes. This hypersensitive response elicitor polypeptide or protein has substantially no cysteine. The hypersensitive response elicitor polypeptide or protein derived from [0019] Erwinia amylovora is more fully described in Wei, Z.-M., R. J. Laby, C. H. Zumoff, D. W. Bauer, S.-Y. He, A. Collmer, and S. V. Beer, “Harpin, Elicitor of the Hypersensitive Response Produced by the Plant Pathogen Erwinia amylovora,” Science 257:85-88 (1992), which is hereby incorporated by reference. The DNA molecule encoding this polypeptide or protein has a nucleotide sequence corresponding to SEQ ID NO: 4 as follows:
    AAGCTTCGGC ATGCCACGTT TGACCGTTGG GTCGGCAGGG TACGTTTGAA TTATTCATAA 60
    CAGGAATACG TTATGAGTCT GAATACAAGT GGGCTGGGAG CGTCAACGAT GCAAATTTCT 120
    ATCGGCGGTG CGGGCGGAAA TAACGGGTTG CTGGGTACCA GTCGCCAGAA TGCTGGGTTG 180
    GGTGCCAATT CTGCACTGGG GCTGGGCGGC GGTAATCAAA ATGATACCGT CAATCAGCTG 240
    GCTGGCTTAC TCACCGGCAT GATGATGATG ATGAGCATGA TGGGCGGTGG TGGGCTGATG 300
    GGCGGTGGCT TAGGCGGTGG CTTAGGTAAT CGCTTGGGTG GCTCAGGTGG CCTGGGCGAA 360
    GGACTGTCGA ACGCGCTGAA CGATATGTTA GGCGGTTCGC TGAACACGCT GGGCTCGAAA 420
    GGCGCCAACA ATACCACTTC AACAACAAAT TCCCCGCTGG ACCAGGCGCT GGGTATTAAC 480
    TCAACGTCCC AAAACGACGA TTCCACCTCC GGCACAGATT CCACCTCAGA CTCCAGCGAC 540
    CCGATGCAGC AGCTGCTGAA GATGTTCAGC GAGATAATGC AAAGCCTGTT TGCTGATGGG 600
    CAAGATGGCA CCCAGGGCAG TTCCTCTGGG GGCAAGCAGC CGACCGAAGG CGAGCAGAAC 660
    GCCTATAAAA AAGGAGTCAC TGATGCGCTG TCGGGCCTGA TGGGTAATGG TCTGAGCCAG 720
    CTCCTTGGCA ACGGGGGACT GGGAGCTGGT CAGGGCGGTA ATGCTGGCAC GGGTCTTGAC 780
    GGTTCGTCGC TGGGCGGCAA AGGGCTGCAA AACCTGAGCG GGCCGGTGGA CTACCAGCAG 840
    TTAGGTAACG CCGTGGGTAC CGGTATCGGT ATGAAAGCGG GCATTCAGGC GCTGAATGAT 900
    ATCGGTACGC ACAGCCACAG TTCAACCCGT TCTTTCGTCA ATAAAGGCGA TCGGGCGATG 960
    GCGAAGGAAA TCGGTCAGTT CATGGACCAG TATCCTGAGG TGTTTGGCAA GCCGCAGTAC 1020
    CAGAAAGGCC CGGGTCAGGA GGTGAAAACC GATGACAAAT CATGGGCAAA AGCACTGAGC 1080
    AAGCCAGATG ACGACGGAAT GACACCAGCC AGTATGGAGC AGTTCAACAA AGCCAAGGGC 1140
    ATGATCAAAA CGCCCATGGC GGCTGATACC GGCAACGGCA ACCTGCAGGC ACGCGGTGCC 1200
    GGTGGTTCTT CGCTGGGTAT TGATGCCATG ATGGCCGGTG ATGCCATTAA CAATATGGCA 1260
    CTTGGCAAGC TGGGCGCGGC TTAAGCTT  1288
  • Another potentially suitable hypersensitive response elicitor from [0020] Erwinia amylovora is disclosed in U.S. patent application Ser. No. 09/120,927, which is hereby incorporated by reference. The protein is encoded by a DNA molecule having a nucleic acid sequence of SEQ ID NO: 5 as follows:
    ATGTCAATTC TTACGCTTAA CAACAATACC TCGTCCTCGC CGGGTCTGTT CCAGTCCGGG 60
    GGGGACAACG GGCTTGGTGG TCATAATGCA AATTCTGCGT TGGGGCAACA ACCCATCGAT 120
    CGGCAAACCA TTGAGCAAAT GGCTCAATTA TTGGCGGAAC TGTTAAAGTC ACTGCTATCG 180
    CCACAATCAG GTAATGCGGC AACCGGAGCC GGTGGCAATG ACCAGACTAC AGGAGTTGGT 240
    AACGCTGGCG GCCTGAACGG ACGAAAAGGC ACAGCAGGAA CCACTCCGCA GTCTGACAGT 300
    CAGAACATGC TGAGTGAGAT GGGCAACAAC GGGCTGGATC AGGCCATCAC GCCCGATGGC 360
    CAGGGCGGCG GGCAGATCGG CGATAATCCT TTACTGAAAG CCATGCTGAA GCTTATTGCA 420
    CGCATGATGG ACGGCCAAAG CGATCAGTTT GGCCAACCTG GTACGGGCAA CAACAGTGCC 480
    TCTTCCGGTA CTTCTTCATC TGGCGGTTCC CCTTTTAACG ATCTATCAGG GGGGAAGGCC 540
    CCTTCCGGCA ACTCCCCTTC CGGCAACTAC TCTCCCGTCA GTACCTTCTC ACCCCCATCC 600
    ACGCCAACGT CCCCTACCTC ACCGCTTGAT TTCCCTTCTT CTCCCACCAA AGCAGCCGGG 660
    GGCAGCACGC CGGTAACCGA TCATCCTGAC CCTGTTGGTA GCGCCGGCAT CGGGGCCGGA 720
    AATTCGGTGG CCTTCACCAG CGCCGGCGCT AATCAGACCG TGCTGCATGA CACCATTACC 780
    GTGAAAGCGG GTCAGGTGTT TGATGGCAAA GGACAAACCT TCACCGCCGG TTCAGAATTA 840
    GGCCATGGCG GCCAGTCTGA AAACCAGAAA CCGCTGTTTA TACTGGAAGA CGGTGCCAGC 900
    CTCAAAAACG TCACCATGGG CGACGACGGG GCGGATGGTA TTCATCTTTA CGGTGATGCC 960
    AAAATAGACA ATCTGCACGT CACCAACGTG GGTGAGGACG CGATTACCGT TAAGCCAAAC 1020
    AGCGCGGGCA AAAAATCCCA CGTTGAAATC ACTAACAGTT CCTTCGAGCA CGCCTCTGAC 1080
    AAGATCCTGC AGCTGAATGC CGATACTAAC CTGAGCGTTG ACAACGTGAA GGCCAAAGAC 1140
    TTTGGTACTT TTGTACGCAC TAACGGCGGT CAACAGGGTA ACTGGGATCT GAATCTGAGC 1200
    CATATCAGCG CAGAAGACGG TAAGTTCTCG TTCGTTAAAA GCGATAGCGA GGGGCTAAAC 1260
    GTCAATACCA GTGATATCTC ACTGGGTGAT GTTGAAAACC ACTACAAAGT GCCGATGTCC 1320
    GCCAACCTGA AGGTGGCTGA ATGA  1344
  • See GenBank Accession No. U94513. The isolated DNA molecule of the present invention encodes a hypersensitive response elicitor protein or polypeptide having an amino acid sequence of SEQ ID NO: 6 as follows: [0021]
    Met Ser Ile Leu Thr Leu Asn Asn Asn Thr Ser Ser Ser Pro Gly Leu
    1               5                   10                  15     
    Phe Gln Ser Gly Gly Asp Asn Gly Leu Gly Gly His Asn Ala Asn Ser
                20                  25                  30         
    Ala Leu Gly Gln Gln Pro Ile Asp Arg Gln Thr Ile Glu Gln Met Ala
            35                  40                  45             
    Gln Leu Leu Ala Glu Leu Leu Lys Ser Leu Leu Ser Pro Gln Ser Gly
        50                  55                  60                 
    Asn Ala Ala Thr Gly Ala Gly Gly Asn Asp Gln Thr Thr Gly Val Gly
    65                  70                  75                  80 
    Asn Ala Gly Gly Leu Asn Gly Arg Lys Gly Thr Ala Gly Thr Thr Pro
                    85                  90                  95     
    Gln Ser Asp Ser Gln Asn Met Leu Ser Glu Met Gly Asn Asn Gly Leu
                100                 105                 110        
    Asp Gln Ala Ile Thr Pro Asp Gly Gln Gly Gly Gly Gln Ile Gly Asp
            115                 120                 125            
    Asn Pro Leu Leu Lys Ala Met Leu Lys Leu Ile Ala Arg Met Met Asp
        130                 135                 140                
    Gly Gln Ser Asp Gln Phe Gly Gln Pro Gly Thr Gly Asn Asn Ser Ala
    145                 150                 155                 160
    Ser Ser Gly Thr Ser Ser Ser Gly Gly Ser Pro Phe Asn Asp Leu Ser
                    165                 170                 175    
    Gly Gly Lys Ala Pro Ser Gly Asn Ser Pro Ser Gly Asn Tyr Ser Pro
                180                 185                 190        
    Val Ser Thr Phe Ser Pro Pro Ser Thr Pro Thr Ser Pro Thr Ser Pro
            195                 200                 205            
    Leu Asp Phe Pro Ser Ser Pro Thr Lys Ala Ala Gly Gly Ser Thr Pro
        210                 215                 220                
    Val Thr Asp His Pro Asp Pro Val Gly Ser Ala Gly Ile Gly Ala Gly
    225                 230                 235                 240
    Asn Ser Val Ala Phe Thr Ser Ala Gly Ala Asn Gln Thr Val Leu His
                    245                 250                 255    
    Asp Thr Ile Thr Val Lys Ala Gly Gln Val Phe Asp Gly Lys Gly Gln
                260                 265                 270        
    Thr Phe Thr Ala Gly Ser Glu Leu Gly Asp Gly Gly Gln Ser Glu Asn
            275                 280                 285            
    Gln Lys Pro Leu Phe Ile Leu Glu Asp Gly Ala Ser Leu Lys Asn Val
        290                 295                 300                
    Thr Met Gly Asp Asp Gly Ala Asp Gly Ile His Leu Tyr Gly Asp Ala
    305                 310                 315                 320
    Lys Ile Asp Asn Leu His Val Thr Asn Val Gly Glu Asp Ala Ile Thr
                    325                 330                 335    
    Val Lys Pro Asn Ser Ala Gly Lys Lys Ser His Val Glu Ile Thr Asn
                340                 345                 350        
    Ser Ser Phe Glu His Ala Ser Asp Lys Ile Leu Gln Leu Asn Ala Asp
            355                 360                 365            
    Thr Asn Leu Ser Val Asp Asn Val Lys Ala Lys Asp Phe Gly Thr Phe
        370                 375                 380                
    Val Arg Thr Asn Gly Gly Gln Gln Gly Asn Trp Asp Leu Asn Leu Ser
    385                 390                 395                 400
    His Ile Ser Ala Glu Asp Gly Lys Phe Ser Phe Val Lys Ser Asp Ser
                    405                 410                 415    
    Glu Gly Leu Asn Val Asn Thr Ser Asp Ile Ser Leu Gly Asp Val Glu
                420                 425                 430        
    Asn His Tyr Lys Val Pro Met Ser Ala Asn Leu Lys Val Ala Glu
            435                 440                 445        
  • This protein or polypeptide is acidic, rich in glycine and serine, and lacks cysteine. It is also heat stable, protease sensitive, and suppressed by inhibitors of plant metabolism. The protein or polypeptide of the present invention has a predicted molecular size of ca. 4.5 kDa. [0022]
  • Another potentially suitable hypersensitive response elicitor from [0023] Erwinia amylovora is disclosed in U.S. patent application Ser. No. 09/120,663, which is hereby incorporated by reference. The protein is encoded by a DNA molecule having a nucleic acid sequence of SEQ ID NO: 7 as follows:
    ATGGAATTAA AATCACTGGG AACTGAACAC AAGGCGGCAG TACACACAGC GGCGCACAAC 60
    CCTGTGGGGC ATGGTGTTGC CTTACAGCAG GGCAGCAGCA GCAGCAGCCC GCAAAATGCC 120
    GCTGCATCAT TGGCGGCAGA AGCCAAAAAT CGTGGGAAAA TGCCGAGAAT TCACCAGCCA 180
    TCTACTGCGG CTGATGGTAT CAGCGCTGCT CACCAGCAAA AGAAATCCTT CAGTCTCAGG 240
    GGCTGTTTCG GGACGAAAAA ATTTTCCAGA TCGGCACCGC AGGGCCAGCC AGGTACCACC 300
    CACAGCAAAG GGGCAACATT GCGCGATCTG CTGCGCGGG ACGACGGCGA AACGCAGCAT 360
    GAGGCGGCCG CGCCAGATGC GGCGCGTTTG ACCCGTTCGG GCGGCGTCAA ACGCCGCAAT 420
    ATGGACGACA TGGCCGGGCC GCCAATGGTG AAAGGTGGCA GCGGCGAAGA TAAGGTACCA 480
    ACGCAGCAAA AACGGCATCA GCTGAACAAT TTTGGCCAGA TGCGCCAAAC GATGTTGAGC 540
    AAAATGGCTC ACCCGGCTTC AGCCAACGCC GGCGATCGCC TGCAGCATTC ACCGCCGCAC 600
    ATCCCGGGTA GCCACCACGA AATCAAGGAA GAACCGGTTG GCTCCACCAG CAAGGCAACA 660
    ACGGCCCACG CAGACAGAGT GGAAATCGCT CAGGAAGATG ACGACAGCGA ATTCCAGCAA 720
    CTGCATCAAC AGCGGCTGGC GCGCGAACGG GAAAATCCAC CGCAGCCGCC CAAACTCGGC 780
    GTTGCCACAC CGATTAGCGC CAGGTTTCAG CCCAAACTGA CTGCGGTTGC GGAAACCGTC 840
    CTTGAGGGGA CAGATACCAC GCAGTCACCC CTTAAGCCGC AATCAATGCT GAAAGGAAGT 900
    GGAGCCGGGG TAACGCCGCT GGCGGTAACG CTGGATAAAG GCAAGTTGCA GCTGGCACCG 960
    GATAATCCAC CCGCGCTCAA TACGTTGTTG AAGCAGACAT TGGGTAAAGA CACCCAGCAC 1020
    TATCTGGCGC ACCATGCCAG CAGCCACGGT AGCCAGCATC TGCTGCTGGA CAACAAAGGC 1080
    CACCTGTTTG ATATCAAAAG CACCGCCACC AGCTATAGCG TGCTGCACAA CAGCCACCCC 1140
    GGTGAGATAA AGGGCAAGCT GGCGCAGGCG GGTACTGGCT CCGTCAGCGT AGACGGTAAA 1200
    AGCGGCAAGA TCTCGCTGGG GAGCGGTACG CAAAGTCACA ACAAAACAAT GCTAAGCCAA 1260
    CCGGGGGAAG CGCACCGTTC CTTATTAACC GGCATTTGGC AGCATCCTGC TGGCGCAGCG 1320
    CGGCCGCAGG GCGAGTCAAT CCGCCTGCAT GACGACAAAA TTCATATCCT GCATCCGGAG 1380
    CTGGGCGTAT GGCAATCTGC GGATAAAGAT ACCCACAGCC AGCTGTCTCG CCAGGCAGAC 1440
    GGTAAGCTCT ATGCGCTGAA AGACAACCGT ACCCTGCAAA ACCTCTCCGA TAATAAATCC 1500
    TCAGAAAAGC TGGTCGATAA AATCAAATCG TATTCCGTTG ATCAGCGGGG GCAGGTGGCG 1560
    ATCCTGACGG ATACTCCCGG CCGCCATAAG ATGAGTATTA TGCCCTCGCT GGATGCTTCC 1620
    CCGGAGAGCC ATATTTCCCT CAGCCTGCAT TTTGCCGATG CCCACCAGGG GTTATTGCAC 1680
    GGGAAGTCGG AGCTTGAGGC ACAATCTGTC GCGATCAGCC ATGGGCGACT GGTTGTGGCC 1740
    GATAGCGAAG GCAAGCTGTT TAGCGCCGCC ATTCCGAAGC AACGGGATGG AAACGAACTG 1800
    AAAATGAAAG CCATGCCTCA GCATGCGCTC GATGAACATT TTGGTCATGA CCACCAGATT 1860
    TCTGGATTTT TCCATGACGA CCACGGCCAG CTTAATGCGC TGGTGAAAAA TAACTTCAGG 1920
    CAGCAGCATG CCTGCCCGTT GGGTAACGAT CATCAGTTTC ACCCCGGCTG GAACCTGACT 1980
    GATGCGCTGG TTATCGACAA TCAGCTGGGG CTGCATCATA CCAATCCTGA ACCGCATGAG 2040
    ATTCTTGATA TGGGCATTT AGGCAGCCTG GCGTTACAGG AGGGCAAGCT TCACTATTTT 2100
    GACCAGCTGA CCAAAGGGTG GACTGGCGCG GAGTCAGATT GTAAGCAGCT GAAAAAAGGC 2160
    CTGGATGGAG CAGCTTATCT ACTGAAAGAC GGTGAAGTGA AACGCCTGAA TATTAATCAG 2220
    AGCACCTCCT CTATCAAGCA CGGAACGGAA AACGTTTTTT CGCTGCCGCA TGTGCGCAAT 2280
    AAACCGGAGC CGGGAGATGC CCTGCAAGGG CTGAATAAAG ACGATAAGGC CCAGGCCATC 2340
    GCGGTGATTG GGGTAAATAA ATACCTGGCG CTGACGGAAA AAGGGGACAT TCGCTCCTTC 2400
    CAGATAAAAC CCGGCACCCA GCAGTTGGAG CGGCCGGCAC AAACTCTCAG CCGCGAAGGT 2460
    ATCAGCGGCG AACTGAAAGA CATTCATGTC GACCACAAGC AGAACCTGTA TGCCTTGACC 2520
    CACGAGGGAG AGGTGTTTCA TCAGCCGCGT GAAGCCTGGC AGAATGGTGC CGAAAGCAGC 2580
    AGCTGGCACA AACTGGCGTT GCCACAGAGT GAAAGTAAGC TAAAAAGTCT GCACATGAGC 2640
    CATGAGCACA AACCGATTGC CACCTTTGAA GACGGTAGCC AGCATCAGCT GAAGGCTGGC 2700
    GGCTGGCACG CCTATGCGGC ACCTGAACGC GGGCCGCTGG CGGTGGGTAC CAGCGGTTCA 2760
    CAAACCGTCT TTAACCGACT AATGCAGCGG GTGAAAGGCA AGGTGATCCC AGGCACCGGG 2820
    TTGACGGTTA AGCTCTCGGC TCAGACGGGG GGAATGACCG GCGCCGAAGG GCGCAAGGTC 2880
    AGCAGTAAAT TTTCCGAAAG GATCCGCGCC TATGCGTTCA ACCCAACAAT GTCCACGCCG 2940
    CGACCGATTA AAAATGCTGC TTATGCCACA CAGCACGGCT GGCAGGGGCG TGAGGGGTTG 3000
    AAGCCGTTGT ACGAGATGCA GGGAGCGCTG ATTAAACAAC TGGATGCGCA TAACGTTCGT 3060
    CATAACGCGC CACAGCCAGA TTTGCAGAGC AAACTGGAAA CTCTGGATTT AGGCGAACAT 3120
    GCCGCAGAAT TGCTTAACGA CATGAAGCGC TTCCGCGACG AACTGGAGCA GAGTGCAACC 3180
    CGTTCGGTGA CCGTTTTAGG TCAACATCAG GGAGTGCTAA AAAGCAACGG TGAAATCAAT 3240
    AGCGAATTTA AGCCATCGCC CGGCAAGGCG TTGGTCCAGA GCTTTAACGT CAATCGCTCT 3300
    GGTCAGGATC TAAGCAAGTC ACTGCAACAG GCAGTACATG CCACGCCGCC ATCCGCAGAG 3360
    AGTAAACTGC AATCCATGCT GGGGCACTTT GTCAGTGCCG GGGTGGATAT GAGTCATCAG 3420
    AAGGGCGAGA TCCCGCTGGG CCCCCACCGC GATCCGAATG ATAAAACCGC ACTGACCAAA 3480
    TCGCGTTTAA TTTTAGATAC CGTGACCATC GGTCAACTGC ATGAACTGGC CGATAAGCCG 3540
    AAACTGGTAT CTGACCATAA ACCCGATGCC GATCAGATAA AACAGCTGCG CCAGCAGTTC 3600
    GATACGCTGC GTGAAAAGCG GTATGAGAGC AATCCGGTGA AGCATTACAC CGATATGGGC 3660
    TTCACCCATA ATAAGGCGCT GGAAGCAAAC TATGATGCGG TCAAAGCCTT TATCAATGCC 3720
    TTTAAGAAAG AGCACCACGG CGTCAATCTG ACCACCCGTA CCGTACTGCA ATCACAGGGC 3780
    AGTGCGGAGC TGGCGAAGAA GCTCAAGAAT ACGCTGTTGT CCCTGGACAG TGGTGAAAGT 3840
    ATGAGCTTCA GCCGGTCATA TGGCGGGGGC GTCAGCACTG TCTTTGTGCC TACCCTTAGC 3900
    AAGAAGGTGC CAGTTCCGGT GATCCCCGGA GCCCGCATCA CGCTGGATCG CGCCTATAAC 3960
    CTGAGCTTCA GTCGTACCAG CGGCGGATTG AACGTCAGTT TTGGCCGCGA CGGCGGGGTG 4020
    AGTGGTAACA TCATGGTCGC TACCGGCCAT GATGTGATGC CCTATATGAC CGGTAAGAAA 4080
    ACCAGTGCAG CTAACGCCAG TGACTGGTTG AGCGCAAAAC ATAAAATCAG CCCGGACTTG 4140
    CGTATCGGCG CTGCTGTGAG TGGCACCCTG CAACGAACGC TACAAAACAG CCTGAAGTTT 4200
    AAGCTGACAG AGGATGAGCT GCCTGGCTTT ATCCATGGCT TGACGCATGG CACGTTGACC 4260
    CCGCCAGAAC TGTTGCAAAA GGGGATCGAA CATCAGATGA AGCAGGGCAG CAAACTGACG 4320
    TTTAGCGTCG ATACCTCGGC AAATCTGGAT CTGCGTGCCG GTATCAATCT GAACGAAGAC 4380
    GGCAGTAAAC CAAATGGTGT CACTGCCCGT GTTTCTGCCG GGCTAAGTGC ATCGGCAAAC 4440
    CTGGCCGCCG GCTCGCGTGA ACGCAGCACC ACCTCTGGCC AGTTTGGCAG CACGACTTCG 4500
    GCCAGCAATA ACCGCCCAAC CTTCCTCAAC GGGGTCGGCG CGGGTGCTAA CCTGACGGCT 4560
    GCTTTAGGGG TTGCCCATTC ATCTACGCAT GAAGGGAAAC CGGTCGGGAT CTTCCCGGCA 4620
    TTTACCTCGA CCAATGTTTC GGCAGCGCTG GCGCTGGATA ACCGTACCTC ACAGAGTATC 4680
    AGCCTGGAAT TGAAGCGCGC GGACCCGGTG ACCAGCAACG ATATCAGCGA GTTGACCTCC 4740
    ACGCTGGGAA AACACTTTAA GGATAGCGCC ACAACGAAGA TGCTTGCCGC TCTCAAAGAG 4800
    TTAGATGACG CTAAGCCCCC TGAACAACTG CATATTTTAC AGCAGCATTT CAGTGCAAAA 4860
    GATGTCGTCG GTGATGAACG CTACGAGGCG GTGCGCAACC TGAAAAAACT GGTGATACGT 4920
    CAACAGGCTG CGGACAGCCA CAGCATGGAA TTAGGATCTG CCAGTCACAG CACGACCTAC 4980
    AATAATCTGT CGAGAATAAA TAATGACGGC ATTGTCGAGC TGCTACACAA ACATTTCGAT 5040
    GCGGCATTAC CAGCAAGCAC TGCCAAACGT CTTGGTCAAA TGATGAATAA CGATCCGGCA 5100
    CTGAAAGATA TTATTAACCA GCTGCAAAGT ACGCCGTTCA GCAGCGCCAG CGTGTCGATG 5160
    GAGCTGAAAG ATGGTCTGCG TGAGCAGACG GAAAAAGCAA TACTGGACGG TAAGGTCGGT 5220
    CGTGAAGAAG TGGGAGTACT TTTCCAGGAT CGTAACAACT TGCGTGTTAA ATCGGTCAGC 5280
    GTCAGTCAGT CCGTCAGCAA AAGCGAAGGC TTCAATACCC CAGCGCTGTT ACTGGGGACG 5340
    AGCAACAGCG CTGCTATGAG CATGGAGCGC AACATCGGAA CCATTAATTT TAAATACGGC 5400
    CAGGATCAGA ACACCCCACG GCGATTTACC CTGGAGGGTG GAATAGCTCA GGCTAATCCG 5460
    CAGGTCGCAT CTGCGCTTAC TCATTTGAAG AAGGAAGGGC TGGAAATGAA GAGCTAA 5517
  • This DNA molecule is known as the dspE gene for [0024] Erwinia amylovora. This isolated DNA molecule of the present invention encodes a protein or polypeptide which elicits a plant pathogen's hypersensitive response having an amino acid sequence of SEQ ID NO: 8 as follows:
    Met Glu Leu Lys Ser Leu Gly Thr Glu His Lys Ala Ala Val His Thr
    1               5                   10                  15     
    Ala Ala His Asn Pro Val Gly His Gly Val Ala Leu Gln Gln Gly Ser
                20                  25                  30         
    Ser Ser Ser Ser Pro Gln Asn Ala Ala Ala Ser Leu Ala Ala Glu Gly
            35                  40                  45             
    Lys Asn Arg Gly Lys Met Pro Arg Ile His Gln Pro Ser Thr Ala Ala
        50                  55                  60                 
    Asp Gly Ile Ser Ala Ala His Gln Gln Lys Lys Ser Phe Ser Leu Ary
    65                  70                  75                  80 
    Gly Cys Leu Gly Thr Lys Lys Phe Ser Arg Ser Ala Pro Gln Gly Gln
                    85                  90                  95     
    Pro Gly Thr Thr His Ser Lys Gly Ala Thr Leu Arg Asp Leu Leu Ala
                100                 105                 110        
    Arg Asp Asp Gly Glu Thr Gln His Glu Ala Ala Ala Pro Asp Ala Ala
            115                 120                 125            
    Arg Leu Thr Ary Ser Gly Gly Val Lys Arg Arg Asn Met Asp Asp Met
        130                 135                 140                
    Ala Gly Arg Pro Met Val Lys Gly Gly Ser Gly Glu Asp Lys Val Pro
    145                 150                 155                 160
    Thr Gln Gln Lys Arg His Gln Leu Asn Asn Phe Gly Gln Met Arg Gln
                    165                 170                 175    
    Thr Met Leu Ser Lys Met Ala His Pro Ala Ser Ala Asn Ala Gly Asp
                180                 185                 190        
    Arg Leu Gln His Ser Pro Pro His Ile Pro Gly Ser His His Glu Ile
            195                 200                 205            
    Lys Glu Glu Pro Val Gly Ser Thr Ser Lys Ala Thr Thr Ala His Ala
        210                 215                 220                
    Asp Arg Val Glu Ile Ala Gln Glu Asp Asp Asp Ser Glu Phe Gln Gln
    225                 230                 235                 240
    Leu His Gln Gln Arg Leu Ala Arg Glu Arg Glu Asn Pro Pro Gln Pro
                    245                 250                 255    
    Pro Lys Leu Gly Val Ala Thr Pro Ile Ser Ala Arg Phe Gln Pro Lys
                260                 265                 270        
    Leu Thr Ala Val Ala Glu Ser Val Leu Glu Gly Thr Asp Thr Thr Gln
            275                 280                 285            
    Ser Pro Leu Lys Pro Gln Ser Met Leu Lys Gly Ser Gly Ala Gly Val
        290                 295                 300                
    Thr Pro Leu Ala Val Thr Leu Asp Lys Gly Lys Leu Gln Leu Ala Pro
    305                 310                 315                 320
    Asp Asn Pro Pro Ala Leu Asn Thr Leu Leu Lys Gln Thr Leu Gly Lys
                    325                 330                 335    
    Asp Thr Gln His Tyr Leu Ala His His Ala Ser Ser Asp Gly Ser Gln
                340                 345                 350        
    His Leu Leu Leu Asp Asn Lys Gly His Leu Phe Asp Ile Lys Ser Thr
            355                 360                 365            
    Ala Thr Ser Tyr Ser Val Leu His Asn Ser His Pro Gly Glu Ile Lys
        370                 375                 380                
    Gly Lys Leu Ala Gln Ala Gly Thr Gly Ser Val Ser Val Asp Gly Lys
    385                 390                 395                 400
    Ser Gly Lys Ile Ser Leu Gly Ser Gly Thr Gln Ser His Asn Lys Thr
                    405                 410                 415    
    Met Leu Ser Gln Pro Gly Glu Ala His Arg Ser Leu Leu Thr Gly Ile
                420                 425                 430        
    Trp Gln His Pro Ala Gly Ala Ala Arg Pro Gln Gly Glu Ser Ile Arg
            435                 440                 445            
    Leu His Asp Asp Lys Ile His Ile Leu His Pro Glu Leu Gly Val Trp
        450                 455                 460                
    Gln Ser Ala Asp Lys Asp Thr His Ser Gln Leu Ser Arg Gln Ala Asp
    465                 470                 475                 480
    Gly Lys Leu Tyr Ala Leu Lys Asp Asn Arg Thr Leu Gln Asn Leu Ser
                    485                 490                 495    
    Asp Asn Lys Ser Ser Glu Lys Leu Val Asp Lys Ile Lys Ser Tyr Ser
                500                 505                 510        
    Val Asp Gln Arg Gly Gln Val Ala Ile Leu Thr Asp Thr Pro Gly Arg
            515                 520                 525            
    His Lys Met Ser Ile Met Pro Ser Leu Asp Ala Ser Pro Glu Ser His
        530                 535                 540                
    Ile Ser Len Ser Leu His Phe Ala Asp Ala His Gln Gly Leu Leu His
    545                 550                 555                 560
    Gly Lys Ser Glu Leu Glu Ala Gln Ser Val Ala Ile Ser His Gly Arg
                    565                 570                 575    
    Leu Val Val Ala Asp Ser Glu Gly Lys Leu Phe Ser Ala Ala Ile Pro
                580                 585                 590        
    Lys Gln Gly Asp Gly Asn Glu Leu Lys Met Lys Ala Met Pro Gln His
            595                 600                 605            
    Ala Leu Asp Glu His Phe Gly His Asp His Gln Ile Ser Gly Phe Phe
        610                 615                 620                
    His Asp Asp His Gly Gln Leu Asn Ala Leu Val Lys Asn Asn Phe Arg
    625                 630                 635                 640
    Gln Gln His Ala Cys Pro Leu Gly Asn Asp His Gln Phe His Pro Gly
                    645                 650                 655    
    Trp Asn Leu Thr Asp Ala Leu Val Ile Asp Asn Gln Leu Gly Leu His
                660                 665                 670        
    His Thr Asn Pro Glu Pro His Glu Ile Leu Asp Met Gly His Leu Gly
            675                 680                 685            
    Ser Leu Ala Leu Gln Glu Gly Lys Leu His Tyr Phe Asp Gln Leu Thr
        690                 695                 700                
    Lys Gly Trp Thr Gly Ala Glu Ser Asp Cys Lys Gln Leu Lys Lys Gly
    705                 710                 715                 720
    Leu Asp Gly Ala Ala Tyr Leu Leu Lys Asp Gly Glu Val Lys Arg Leu
                    725                 730                 735    
    Asn Ile Asn Gln Ser Thr Ser Ser Ile Lys His Gly Thr Glu Asn Val
                740                 745                 750        
    Phe Ser Leu Pro His Val Arg Asn Lys Pro Glu Pro Gly Asp Ala Leu
            755                 760                 765            
    Gln Gly Len Asn Lys Asp Asp Lys Ala Gln Ala Met Ala Val Ile Gly
        770                 775                 780                
    Val Asn Lys Tyr Leu Ala Leu Thr Glu Lys Gly Asp Ile Arg Ser Phe
    785                 790                 795                 800
    Gln Ile Lys Pro Gly Thr Gln Gln Leu Glu Arg Pro Ala Gln Thr Leu
                    805                 810                 815    
    Ser Arg Glu Gly Ile Ser Gly Glu Leu Lys Asp Ile His Val Asp His
                820                 825                 830        
    Lys Gln Asn Leu Tyr Ala Leu Thr His Glu Gly Glu Val Phe His Gln
            835                 840                 845            
    Pro Arg Glu Ala Trp Gln Asn Gly Ala Glu Ser Ser Ser Trp His Lys
        850                 855                 860                
    Leu Ala Leu Pro Gln Ser Glu Ser Lys Leu Lys Ser Leu Asp Met Ser
    865                 870                 875                 880
    His Glu His Lys Pro Ile Ala Thr Phe Glu Asp Gly Ser Gln His Gln
                    885                 890                 895    
    Leu Lys Ala Gly Gly Trp His Ala Tyr Ala Ala Pro Glu Arg Gly Pro
                900                 905                 910        
    Leu Ala Val Gly Thr Ser Gly Ser Gln Thr Val Phe Asn Arg Leu Met
            915                 920                 925            
    Gln Gly Val Lys Gly Lys Val Ile Pro Gly Ser Gly Leu Thr Val Lys
        930                 935                 940                
    Leu Ser Ala Gln Thr Gly Gly Met Thr Gly Ala Glu Gly Arg Lys Val
    945                 950                 955                 960
    Ser Ser Lys Phe Ser Glu Arg Ile Arg Ala Tyr Ala Phe Asn Pro Thr
                    965                 970                 975    
    Met Ser Thr Pro Arg Pro Ile Lys Asn Ala Ala Tyr Ala Thr Gln His
                980                 985                 990        
    Gly Trp Gln Gly Arg Glu Gly Leu Lys Pro Leu Tyr Glu Met Gln Gly
            995                 1000                1005           
    Ala Leu Ile Lys Gln Leu Asp Ala His Asn Val Arg His Asn Ala Pro
        1010                1015                1020               
    Gln Pro Asp Leu Gln Ser Lys Leu Glu Thr Leu Asp Leu Gly Glu His
    1025                1030                1035                1040
    Gly Ala Glu Leu Leu Asn Asp Met Lys Arg Phe Arg Asp Glu Leu Glu
                    1045                1050                1055   
    Gln Ser Ala Thr Arg Ser Val Thr Val Leu Gly Gln His Gln Gly Val
                1060                1065                1070       
    Leu Lys Ser Asn Gly Glu Ile Asn Ser Glu Phe Lys Pro Ser Pro Gly
            1075                1080                1085           
    Lys Ala Leu Val Gln Ser Phe Asn Val Asn Arg Ser Gly Gln Asp Leu
        1090                1095                1100               
    Ser Lys Ser Leu Gln Gln Ala Val His Ala Thr Pro Pro Ser Ala Glu
    1105                1110                1115                1120
    Ser Lys Leu Gln Ser Met Leu Gly His Phe Val Ser Ala Gly Val Asp
                    1125                1130                1135   
    Met Ser His Gln Lys Gly Glu Ile Pro Leu Gly Arg Gln Arg Asp Pro
                1140                1145                1150       
    Asn Asp Lys Thr Ala Leu Thr Lys Ser Arg Leu Ile Leu Asp Thr Val
            1155                1160                1165           
    Thr Ile Gly Glu Leu His Glu Leu Ala Asp Lys Ala Lys Leu Val Ser
        1170                1175                1180               
    Asp His Lys Pro Asp Ala Asp Gln Ile Lys Gln Leu Arg Gln Gln Phe
    1185                1190                1195                1200
    Asp Thr Leu Arg Glu Lys Arg Tyr Glu Ser Asn Pro Val Lys His Tyr
                    1205                1210                1215   
    Thr Asp Met Gly Phe Thr His Asn Lys Ala Leu Glu Ala Asn Tyr Asp
                1220                1225                1230       
    Ala Val Lys Ala Phe Ile Asn Ala Phe Lys Lys Glu His His Gly Val
            1235                1240                1245           
    Asn Leu Thr Thr Arg Thr Val Leu Glu Ser Gln Gly Ser Ala Glu Leu
        1250                1255                1260               
    Ala Lys Lys Leu Lys Asn Thr Leu Leu Ser Leu Asp Ser Gly Glu Ser
    1265                1270                1275                1280
    Met Ser Phe Ser Arg Ser Tyr Gly Gly Gly Val Ser Thr Val Phe Val
                    1285                1290                1295   
    Pro Thr Leu Ser Lys Lys Val Pro Val Pro Val Ile Pro Gly Ala Gly
                1300                1305                1310       
    Ile Thr Leu Asp Arg Ala Tyr Asn Leu Ser Phe Ser Arg Thr Ser Gly
            1315                1320                1325           
    Gly Leu Asn Val Ser Phe Gly Arg Asp Gly Gly Val Ser Gly Asn Ile
        1330                1335                1340               
    Met Val Ala Thr Gly His Asp Val Met Pro Tyr Met Thr Gly Lys Lys
    1345                1350                1355                1360
    Thr Ser Ala Gly Asn Ala Ser Asp Trp Leu Ser Ala Lys His Lys Ile
                    1365                1370                1375   
    Ser Pro Asp Leu Arg Ile Gly Ala Ala Val Ser Gly Thr Leu Gln Gly
                1380                1385                1390       
    Thr Leu Gln Asn Ser Leu Lys Phe Lys Leu Thr Glu Asp Gln Leu Pro
            1395                1400                1405           
    Gly Phe Ile His Gly Leu Thr His Gly Thr Leu Thr Pro Ala Glu Leu
        1410                1415                1420               
    Leu Gln Lys Gly Ile Glu His Glu Met Lys Gln Gly Ser Lys Leu Thr
    1425                1430                1435                1440
    Phe Ser Val Asp Thr Ser Ala Asn Leu Asp Leu Arg Ala Gly Ile Asn
                    1445                1450                1455   
    Leu Asn Glu Asp Gly Ser Lys Pro Asn Gly Val Thr Ala Arg Val Ser
                1460                1465                1470       
    Ala Gly Leu Ser Ala Ser Ala Asn Leu Ala Ala Gly Ser Arg Glu Arg
            1475                1480                1485           
    Ser Thr Thr Ser Gly Gln Phe Gly Ser Thr Thr Ser Ala Ser Asn Asn
        1490                1495                1500               
    Arg Pro Thr Phe Leu Asn Gly Val Gly Ala Gly Ala Asn Leu Thr Ala
    1505                1510                1515                1520
    Ala Leu Gly Val Ala His Ser Ser Thr His Glu Gly Lys Pro Val Gly
                    1525                1530                1535   
    Ile Phe Pro Ala Phe Thr Ser Thr Asn Val Ser Ala Ala Leu Ala Leu
                1540                1545                1550       
    Asp Asn Arg Thr Ser Gln Ser Ile Ser Leu Glu Leu Lys Arg Ala Glu
            1555                1560                1565           
    Pro Val Thr Ser Asn Asp Ile Ser Glu Leu Thr Ser Thr Leu Gly Lys
        1570                1575                1580               
    His Phe Lys Asp Ser Ala Thr Thr Lys Met Leu Ala Ala Leu Lys Glu
    1585                1590                1595                1600
    Leu Asp Asp Ala Lys Pro Ala Glu Gln Leu His Ile Leu Gln Gln His
                    1605                1610                1615   
    Phe Ser Ala Lys Asp Val Val Gly Asp Glu Ary Tyr Glu Ala Val Arg
                1620                1625                1630       
    Asn Leu Lys Lys Leu Val Ile Arg Gln Gln Ala Ala Asp Ser His Ser
            1635                1640                1645           
    Met Glu Leu Gly Ser Ala Ser His Ser Thr Thr Tyr Asn Asn Leu Ser
        1650                1655                1660               
    Arg Ile Asn Asn Asp Gly Ile Val Glu Leu Leu His Lys His Phe Asp
    1665                1670                1675                1680
    Ala Ala Leu Pro Ala Ser Ser Ala Lys Arg Leu Gly Glu Met Met Asn
                    1685                1690                1695   
    Asn Asp Pro Ala Leu Lys Asp Ile Ile Lys Gln Leu Gln Ser Thr Pro
                1700                1705                1710       
    Phe Ser Ser Ala Ser Val Ser Met Glu Leu Lys Asp Gly Leu Arg Glu
            1715                1720                1725           
    Gln Thr Glu Lys Ala Ile Leu Asp Gly Lys Val Gly Arg Glu Glu Val
        1730                1735                1740               
    Gly Val Leu Phe Gln Asp Arg Asn Asn Leu Arg Val Lys Ser Val Ser
    1745                1750                1755                1760
    Val Ser Gln Ser Val Ser Lys Ser Glu Gly Phe Asn Thr Pro Ala Leu
                    1765                1770                1775   
    Leu Leu Gly Thr Ser Asn Ser Ala Ala Met Ser Met Glu Arg Asn Ile
                1780                1785                1790       
    Gly Thr Ile Asn Phe Lys Tyr Gly Gln Asp Gln Asn Thr Pro Arg Arg
            1795                1800                1805           
    Phe Thr Leu Glu Gly Gly Ile Ala Gln Ala Asn Pro Gln Val Ala Ser
        1810                1815                1820               
    Ala Leu Thr Asp Leu Lys Lys Glu Gly Leu Glu Met Lys Ser
    1825                1830                1835           
  • This protein or polypeptide is about 198 kDa and has a pI of 8.98. [0025]
  • The present invention relates to an isolated DNA molecule having a nucleotide sequence of SEQ ID NO: 9 as follows: [0026]
    ATGACATCGT CACAGCAGCG GGTTGAAAGG TTTTTACAGT ATTTCTCCGC CGGGTGTAAA 60
    ACGCCCATAC ATCTGAAAGA CGGGGTGTGC GCCCTGTATA ACGAACAACA TGAGGAGGCG 120
    GCGGTGCTGG AAGTACCGCA ACACAGCGAC AGCCTGTTAC TACACTGCCG AATCATTCAG 180
    GCTGACCCAC AAACTTCAAT AACCCTGTAT TCGATGCTAT TACAGCTGAA TTTTGAAATG 240
    GCGGCCATGC GCGGCTGTTG GCTGGCGCTG GATGAACTGC ACAACGTGCG TTTATGTTTT 300
    CAGCAGTCGC TGGAGCATCT GGATGAAGCA AGTTTTAGCG ATATCGTTAG CGGCTTCATC 360
    GAACATGCGG CAGAAGTGCG TGAGTATATA GCGCAATTAG ACGAGAGTAG CGCGGCATAA 420
  • This is known as the dspF gene. This isolated DNA molecule of the present invention encodes a hypersensitive response elicitor protein or polypeptide having an amino acid sequence of SEQ ID NO: 10 as follows: [0027]
    Met Thr Ser Ser Gln Glu Arg Val Glu Arg Phe Leu
    1               5                   10         
    Gln Tyr Phe Ser Ala Gly Cys Lys Thr Pro Ile His
            15                  20                 
    Leu Lys Asp Gly Val Cys Ala Leu Tyr Asn Glu Gln
    25                  30                  35     
    Asp Glu Glu Ala Ala Val Leu Glu Val Pro Gln His
                40                  45             
    Ser Asp Ser Leu Leu Leu His Cys Arg Ile Ile Glu
        50                  55                  60 
    Ala Asp Pro Gln Thr Ser Ile Thr Leu Tyr Ser Met
                    65                  70         
    Leu Leu Gln Leu Asn Phe Glu Met Ala Ala Met Arg
            75                  80                 
    Gly Cys Trp Leu Ala Leu Asp Glu Leu His Asn Val
    85                  90                  95     
    Arg Leu Cys Phe Gln Gln Ser Leu Glu His Leu Asp
                100                 105            
    Glu Ala Ser Phe Ser Asp Ile Val Ser Gly Phe Ile
        110                 115                 120
    Glu His Ala Ala Glu Val Arg Glu Tyr Ile Ala Gln
                    125                 130        
    Leu Asp Glu Ser Ser Ala Ala
            135
  • This protein or polypeptide is about 16 kDa and has a pI of 4.45. [0028]
  • The hypersensitive response elicitor polypeptide or protein derived from Pseudomonas syringae has an amino acid sequence corresponding to SEQ ID NO: 11 as follows: [0029]
    Met Gln Ser Leu Ser Leu Asn Ser Ser Ser Leu Gln Thr Pro Ala Met
    1               5                   10                  15     
    Ala Leu Val Leu Val Arg Pro Glu Ala Glu Thr Thr Gly Ser Thr Ser
                20                  25                  30         
    Ser Lys Ala Leu Gln Glu Val Val Val Lys Leu Ala Glu Glu Leu Met
            35                  40                  45             
    Arg Asn Gly Gln Leu Asp Asp Ser Ser Pro Leu Gly Lys Leu Leu Ala
        50                  55                  60                 
    Lys Ser Met Ala Ala Asp Gly Lys Ala Gly Gly Gly Ile Glu Asp Val
    65                  70                  75                  80
    Ile Ala Ala Leu Asp Lys Leu Ile His Glu Lys Leu Gly Asp Asn Phe
                    85                  90                  95     
    Gly Ala Ser Ala Asp Ser Ala Ser Gly Thr Gly Gln Gln Asp Leu Met
                100                 105                 110        
    Thr Gln Val Leu Asn Gly Leu Ala Lys Ser Met Leu Asp Asp Leu Leu
            115                 120                 125            
    Thr Lys Gln Asp Gly Gly Thr Ser Phe Ser Glu Asp Asp Met Pro Met
        130                 135                 140                
    Leu Asn Lys Ile Ala Gln Phe Met Asp Asp Asn Pro Ala Gln Phe Pro
    145                 150                 155                 160
    Lys Pro Asp Ser Gly Ser Trp Val Asn Glu Leu Lys Glu Asp Asn Phe
                    165                 170                 175    
    Leu Asp Gly Asp Glu Thr Ala Ala Phe Arg Her Ala Leu Asp Ile Ile
                180                 185                 190        
    Gly Gln Gln Leu Gly Asn Gln Gln Ser Asp Ala Gly Ser Leu Ala Gly
            195                 200                 205            
    Thr Gly Gly Gly Leu Gly Thr Pro Ser Ser Phe Ser Asn Asn Ser Ser
        210                 215                 220                
    Val Met Gly Asp Pro Leu Ile Asp Ala Asn Thr Gly Pro Gly Asp Ser
    225                 230                 235                 240
    Gly Asn Thr Arg Gly Glu Ala Gly Gln Leu Ile Gly Glu Leu Ile Asp
                    245                 250                 255    
    Arg Gly Leu Gln Ser Val Leu Ala Gly Gly Gly Leu Gly Thr Pro Val
                260                 265                 270        
    Asn Thr Pro Gln Thr Gly Thr Ser Ala Asn Gly Gly Gln Her Ala Gln
            275                 280                 285            
    Asp Leu Asp Gln Leu Leu Gly Gly Leu Leu Leu Lys Gly Leu Glu Ala
        290                 295                 300                
    Thr Leu Lys Asp Ala Gly Gln Thr Gly Thr Asp Val Gln Ser Ser Ala
    305                 310                 315                 320
    Ala Gln Ile Ala Thr Leu Leu Val Ser Thr Leu Leu Gln Gly Thr Arg
                    325                 330                 335   
    Asn Gln Ala Ala Ala
                340    
  • This hypersensitive response elicitor polypeptide or protein has a molecular weight of 34-35 kDa. It is rich in glycine (about 13.5%) and lacks cysteine and tyrosine. [0030]
  • Further information about the hypersensitive response elicitor derived from [0031] Pseudomonas syringae is found in He, S. Y., H. C. Huang, and A. Collmer, “Pseudomonas syringae pv. syringae HarpinPss: a Protein that is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants,” Cell 73:1255-1266 (1993), which is hereby incorporated by reference. The DNA molecule encoding the hypersensitive response elicitor from Pseudomonas syringae has a nucleotide sequence corresponding to SEQ ID NO: 12 as follows:
    ATGCAGAGTC TCAGTCTTAA CACCAGCTCG CTGCAAACCC CGGCAATGGC CCTTGTCCTG 60
    GTACGTCCTG AAGCCGAGAC GACTGGCAGT ACGTCGAGCA AGGCGCTTCA GGAAGTTGTC 120
    GTCAAGCTGG CCGAGGAACT GATGCGCAAT GGTCAACTCG ACGACAGCTC GCCATTGGGA 180
    AAACTGTTGG CCAAGTCGAT GGCCGCAGAT GGCAAGGCGG GCGCCGGTAT TCAGGATCTC 240
    ATCGCTGCGC TGGACAAGCT GATCCATGAA AAGCTCGGTG ACAACTTCGG CGCGTCTGCG 300
    GACAGCGCCT CGGGTACCGG ACAGCAGGAC CTGATGACTC AGGTGCTCAA TGGCCTGGCC 360
    AAGTCGATGC TCGATGATCT TCTGACCAAG CAGGATGGCG GGACAAGCTT CTCCGAAGAC 420
    GATATGCCGA TGCTGAACAA GATCGCGCAG TTCATGGATG ACAATCCCGC ACAGTTTCCC 480
    AAGCCGGACT CGCGCTCCTG GGTGAACGAA CTCAAGGAAG ACAACTTCCT TGATGGCGAC 540
    GAAACGGCTG CGTTCCGTTC GGCACTCGAC ATCATTGGCC AGCAACTGGG TAATCAGCAG 600
    AGTGACGCTC GCAGTCTGGC AGGGACGGGT GGAGGTCTGG GCACTCCGAG CAGTTTTTCC 660
    AACAACTCGT CCGTGATGGG TGATCCGCTG ATCGACGCCA ATACCGGTCC CGGTGACAGC 720
    GGCAATACCC GTGGTGAAGC GGGCAACTG ATCGGCGAGC TTATCGACCG TGGCCTGCAA 780
    TCGGTATTGG CCGGTGGTGG ACTGGCCACA CCCGTAAACA CCCCGCAGAC CGCTACGTCG 840
    GCGAATGGCG GACAGTCCGC TCAGGATCTT GATCAGTTGC TGGGCGGCTT GCTGCTCAAG 900
    GGCCTGGAGG CAACGCTCAA GGATGCCGGG CAAACAGGCA CCGACGTGCA GTCGAGCGCT 960
    GCGCAAATCG CCACCTTGCT GGTCAGTACG CTGCTCCAAG GCACCCGCAA TCAGGCTGCA 1020
    GCCTGA  1026
  • Another potentially suitable hypersensitive response elicitor from [0032] Pseudomonas syringae is disclosed in U.S. patent application Ser. No. 09/120,817, which is hereby incorporated by reference. The protein has a nucleotide sequence of SEQ ID NO: 13 as follows:
    TCCACTTCGC TGATTTTGAA ATTGGCAGAT TCATAGAAAC GTTCAGGTGT GGAAATCAGG 60
    CTGAGTGCGC AGATTTCGTT GATAAGGGTG TGGTACTGGT CATTGTTGGT CATTTCAAGG 120
    CCTCTGAGTG CGGTGCGGAG CAATACCAGT CTTCCTGCTG GCGTGTGCAC ACTGAGTCGC 180
    AGGCATAGGC ATTTCAGTTC CTTGCGTTGG TTGGGCATAT AAAAAAAGGA ACTTTTAAAA 240
    ACAGTGCAAT GAGATGCCGG CAAAACGGGA ACCGGTCGCT GCGCTTTGCC ACTCACTTCG 300
    AGCAAGCTCA ACCCCAAACA TCCACATCCC TATCGAACGG ACAGCGATAC GGCCACTTGC 360
    TCTGGTAAAC CCTGGAGCTG GCGTCGGTCC AATTGCCCAC TTAGCGAGGT AACGCAGCAT 420
    GAGCATCGGC ATCACACCCC GGCCGCAACA GACCACCACG CCACTCGATT TTTCGGCGCT 480
    AAGCGGCAAG AGTCCTCAAC CAAACACGTT CGGCGAGCAG AACACTCAGC AAGCGATCGA 540
    CCCGAGTGCA CTGTTGTTCG GCAGCGACAC ACAGAAAGAC GTCAACTTCG GCACGCCCGA 600
    CAGCACCGTC CAGAATCCGC AGGACOCCAC CAAGCCCAAC GACAGCCACT CCAACATCGC 660
    TAAATTCATC AGTGCATTGA TCATCTCGTT CCTGCAGATG CTCACCAACT CCAATAAAAA 720
    GCAGGACACC AATCAGGAAC AGCCTGATAG CCAGGCTCCT TTCCAGAACA ACGGCGGGCT 780
    CGGTACACCG TCGGCCGATA GCGGGGGCGG CGCTACACCG GATGCGACAG GTGGCGGCGG 840
    CGGTGATACG CCAAGCGCAA CAGGCGGTGG CGGCGGTGAT ACTCCGACCG CAACAGGCGG 900
    TGGCGGCAGC GGTGGCGGCG GCACACCCAC TGCAACAGGT GGCGGCAGCG GTGGCACACC 960
    CACTGCAACA GGCGCTGCCC AGCGTCGCCT AACACCGCAA ATCACTCCCC AGTTCGCCAA 1020
    CCCTAACCGT ACCTCAGGTA CTGGCTCGGT GTCGGACACC CCAGGTTCTA CCGAGCAAGC 1080
    CGGCAAGATC AATGTGGTGA AAGACACCAT CAACGTCGGC GCTGGCGAAG TCTTTGACGG 1140
    CCACGGCGCA ACCTTCACTG CCGACAAATC TATGGGTAAC GGAGACCAGG GCGAAAATCA 1200
    GAAGCCCATC TTCGACCTGG CTCAACCCGC TACGTTGAAC AATGTGAACC TGGGTGAGAA 1260
    CGAGGTCGAT GGCATCCACG TGAAACCCAA AAACGCTCAG GAAGTCACCA TTGACAACGT 1320
    GCATGCCCAG AACGTCGGTG AAGACCTGAT TACGGTCAAA GGCGAGGGAG GCGCAGCGGT 1380
    CACTAATCTG AACATCAAGA ACAGCAGTGC CAAAGGTGCA GACGACAACG TTGTCCAGCT 1440
    CAACGCCAAC ACTCACTTGA AAATCCACAA CTTCAAGCCC GACGATTTCG GCACGATGGT 1500
    TCGCACCAAC CCTGGCAAGC ACTTTGATGA CATGAGCATC GAGCTGAACG GCATCCAACC 1560
    TAACCACGGC AAGTTCGCCC TGGTGAAAAG CGACAGTGAC GATCTGAAGC TGGCAACGGG 1620
    CAACATCGCC ATGACCGACG TCAAACACGC CTACGATAAA ACCCAGGCAT CGACCCAACA 1680
    CACCGAGCTT TGAATCCAGA CAAGTACCTT GAAAAAAGCG GGTGGACTC  1729
  • This DNA molecule is known as the dspE gene for [0033] Pseudomonas syringae. This isolated DNA molecule of the present invention encodes a protein or polypeptide which elicits a plant pathogen's hypersensitive response having an amino acid sequence of SEQ ID NO: 14 as follows:
    Met Ser Ile Gly Ile Thr Pro Arg Pro Gln Gln Thr Thr Thr Pro Leu
    1               5                   10                  15     
    Asp Phe Ser Ala Leu Ser Gly Lys Ser Pro Gln Pro Asn Thr Phe Gly
                20                  25                  30         
    Glu Gln Asn Thr Gln Gln Ala Ile Asp Pro Ser Ala Leu Leu Phe Gly
            35                  40                  45             
    Ser Asp Thr Gln Lys Asp Val Asn Phe Gly Thr Pro Asp Ser Thr Val
        50                  55                  60                 
    Gln Asn Pro Gln Asp Ala Ser Lys Pro Asn Asp Ser Gln Ser Asn Ile
    65                  70                  75                  80
    Ala Lys Leu Ile Ser Ala Leu Ile Met Ser Leu Leu Gln Met Leu Thr
                    85                  90                  95     
    Asn Ser Asn Lys Lys Gln Asp Thr Asn Gln Glu Gln Pro Asp Ser Gln
                100                 105                 110        
    Ala Pro Phe Gln Asn Asn Gly Gly Leu Gly Thr Pro Ser Ala Asp Ser
            115                 120                 125            
    Gly Gly Gly Gly Thr Pro Asp Ala Thr Gly Gly Gly Gly Gly Asp Thr
        130                 135                 140                
    Pro Ser Ala Thr Gly Gly Gly Gly Gly Asp Thr Pro Thr Ala Thr Gly
    145                 150                 155                 160
    Gly Gly Gly Ser Gly Gly Gly Gly Thr Pro Thr Ala Thr Gly Gly Gly
                    165                 170                 175    
    Ser Gly Gly Thr Pro Thr Ala Thr Gly Gly Gly Glu Gly Gly Val Thr
                180                 185                 190        
    Pro Gln Ile Thr Pro Gln Leu Ala Asn Pro Asn Arg Thr Ser Gly Thr
            195                 200                 205            
    Gly Ser Val Ser Asp Thr Ala Gly Ser Thr Glu Gln Ala Gly Lys Ile
        210                 215                 220                
    Asn Val Val Lys Asp Thr Ile Lys Val Gly Ala Gly Glu Val Phe Asp
    225                 230                 235                 240
    Gly His Gly Ala Thr Phe Thr Ala Asp Lys Ser Met Gly Asn Gly Asp
                    245                 250                 255    
    Gln Gly Glu Asn Gln Lys Pro Met Phe Gln Len Ala Glu Gly Ala Thr
                260                 265                 270        
    Leu Lys Asn Val Asn Leu Gly Glu Asn Glu Val Asp Gly Ile His Val
            275                 280                 285            
    Lys Ala Lys Asn Ala Gln Glu Val Thr Ile Asp Asn Val His Ala Gln
        290                 295                 300                
    Asn Val Gly Glu Asp Leu Ile Thr Val Lys Gly Glu Gly Gly Ala Ala
    305                 310                 315                 320
    Val Thr Asn Leu Asn Ile Lys Asn Ser Ser Ala Lys Gly Ala Asp Asp
                    325                 330                 335    
    Lys Val Val Gln Leu Asn Ala Asn Thr His Leu Lys Ile Asp Asn Phe
                340                 345                 350        
    Lys Ala Asp Asp Phe Gly Thr Met Val Arg Thr Asn Gly Gly Lys Gln
            355                 360                 365            
    Phe Asp Asp Met Ser Ile Glu Leu Asn Gly Ile Glu Ala Asn His Gly
        370                 375                 380                
    Lys Phe Ala Leu Val Lys Ser Asp Ser Asp Asp Leu Lys Leu Ala Thr
    385                 390                 395                 400
    Gly Asn Ile Ala Met Thr Asp Val Lys His Ala Tyr Asp Lys Thr Gln
                    405                 410                 415    
    Ala Ser Thr Gln His Thr Glu Leu
                420                
  • This protein or polypeptide is about 42.9 kDa. [0034]
  • The hypersensitive response elicitor polypeptide or protein derived from [0035] Pseudomonas solanacearum has an amino acid sequence corresponding to SEQ ID NO: 15 as follows:
    Met Ser Val Gly Asn Ile Gln Ser Pro Ser Asn Leu Pro Gly Leu Gln
    1               5                   10                  15     
    Asn Leu Asn Leu Asn Thr Asn Thr Asn Ser Gln Gln Ser Gly Gln Ser
                20                  25                  30         
    Val Gln Asp Leu Ile Lys Gln Val Glu Lys Asp Ile Leu Asn Ile Ile
            35                  40                  45             
    Ala Ala Leu Val Gln Lys Ala Ala Gln Ser Ala Gly Gly Asn Thr Gly
        50                  55                  60                 
    Asn Thr Gly Asn Ala Pro Ala Lys Asp Gly Asn Ala Asn Ala Gly Ala
    65                  70                  75                  80
    Asn Asp Pro Ser Lys Asn Asp Pro Ser Lys Ser Gln Ala Pro Gln Ser
                    85                  90                  95     
    Ala Asn Lys Thr Gly Asn Val Asp Asp Ala Asn Asn Gln Asp Pro Met
                100                 105                 110        
    Gln Ala Leu Met Gln Leu Leu Glu Asp Leu Val Lys Leu Leu Lys Ala
            115                 120                 125            
    Ala Leu His Met Gln Gln Pro Gly Gly Asn Asp Lys Gly Asn Gly Val
        130                 135                 140                
    Gly Gly Ala Asn Gly Ala Lys Gly Ala Gly Gly Gln Gly Gly Leu Ala
    145                 150                 155                 160
    Glu Ala Leu Gln Glu Ile Glu Gln Ile Leu Ala Gln Leu Gly Gly Gly
                    165                 170                 175    
    Gly Ala Gly Ala Gly Gly Ala Gly Gly Gly Val Gly Gly Ala Gly Gly
                180                 185                 190        
    Ala Asp Gly Gly Ser Gly Ala Gly Gly Ala Gly Gly Ala Asn Gly Ala
            195                 200                 205            
    Asp Gly Gly Asn Gly Val Asn Gly Asn Gln Ala Asn Gly Pro Gln Asn
        210                 215                 220                
    Ala Gly Asp Val Asn Gly Ala Asn Gly Ala Asp Asp Gly Ser Glu Asp
    225                 230                 235                 240
    Gln Gly Gly Leu Thr Gly Val Leu Gln Lys Leu Met Lys Ile Leu Asn
                    245                 250                 255    
    Ala Leu Val Gln Met Met Gln Gln Gly Gly Leu Gly Gly Gly Asn Gln
                260                 265                 270        
    Ala Gln Gly Gly Ser Lys Gly Ala Gly Asn Ala Ser Pro Ala Ser Gly
            275                 280                 285            
    Ala Asn Pro Gly Ala Asn Gln Pro Gly Ser Ala Asp Asp Gln Ser Ser
        290                 295                 300                
    Gly Gln Asn Asn Leu Gln Ser Gln Ile Met Asp Val Val Lys Glu Val
    305                 310                 315                 320
    Val Gln Ile Leu Gln Gln Met Leu Ala Ala Gln Asn Gly Gly Ser Gln
                    325                 330                 335    
    Gln Ser Thr Ser Thr Gln Pro Met
                340                
  • It is encoded by a DNA molecule having a nucleotide sequence corresponding SEQ ID NO: 16 as follows: [0036]
    ATGTCAGTCG GAAACATCCA GAGCCCGTCG AACCTCCCGG GTCTGCAGAA CCTGAACCTC 60
    AACACCAACA CCAACAGCCA GCAATCGCGC CAGTCCGTGC AAGACCTGAT CAAGCAGGTC 120
    GAGAAGGACA TCCTCAACAT CATCGCAGCC CTCGTGCACA AGGCCCCACA GTCGGCGGGC 180
    GGCAACACCG GTAACACCGG CAACGCGCCG GCGAAGGACG GCAATGCCAA CGCGGGCGCC 240
    AACGACCCGA GCAAGAACGA CCCGAGCAAG AGCCAGGCTC CGCAGTCGGC CAACAAGACC 300
    GGCAACGTCG ACGACGCCAA CAACCAGGAT CCGATGCAAG CGCTGATGCA GCTGCTGGAA 360
    GACCTGGTGA AGCTGCTGAA GGCGGCCCTG CACATGCAGC AGCCCGGCGG CAATGACAAG 420
    GGCAACGGCG TGGGCGGTGC CAACGCCGCC AAGGGTGCCG GCGGCCAGGG CCGCCTCGCC 480
    GAAGCGCTGC AGGAGATCGA GCAGATCCTC GCCCAGCTCG GCGGCGGCGG TGCTGGCGCC 540
    GGCGGCGCGG GTGGCGGTGT CGGCGGTGCT GGTGGCGCGG ATGGCGCCTC CGGTCCGGGT 600
    GGCGCAGGCG GTGCGAACGG CGCCGACGGC GGCAATGGCG TGAACGGCAA CCAGGCGAAC 660
    GGCCCGCAGA ACGCAGGCCA TGTCAACGGT GCCAACGGCG CGGATGACGG CAGCGAAGAC 720
    CAGGGCCGCC TCACCGGCGT GCTGCAAAAG CTGATGAAGA TCCTGAACGC GCTGGTGCAG 780
    ATGATGCAGC AAGGCGGCCT CGGCGGCGGC AACCAGGCGC AGGGCGGCTC GAAGGGTGCC 840
    GGCAACGCCT CGCCGGCTTC CGGCGCGAAC CCGGGCGCGA ACCAGCCCGG TTCGGCGGAT 900
    GATCAATCGT CCCGCCAGAA CAATCTGCAA TCCCAGATCA TGCATGTGGT GAAGGAGGTC 960
    GTCCAGATCC TGCAGCAGAT GCTGGCGGCG CAGAACGGCG GCAGCCAGCA GTCCACCTCG 1020
    ACGCAGCCGA TGTAA  1035
  • Further information regarding the hypersensitive response elicitor polypeptide or protein derived from [0037] Pseudomonas solanacearum is set forth in Arlat, M., F. Van Gijsegem, J. C. Huet, J. C. Pemollet, and C. A. Boucher, “PopA1, a Protein which Induces a Hypersensitive-like Response in Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum,” EMBO J. 13:543-533 (1994), which is hereby incorporated by reference.
  • The hypersensitive response elicitor polypeptide or protein from [0038] Xanthomonas campestris pv. glycines has an amino acid sequence corresponding to SEQ ID NO: 17 as follows:
    Thr Leu Ile Glu Leu Met Ile Val Val Ala Ile Ile
    1               5                   10
    Ala Ile Leu Ala Ala Ile Ala Leu Pro Ala Tyr Gln
            15                  20
    Asp Tyr
    25
  • This sequence is an amino terminal sequence having only 26 residues from the hypersensitive response elicitor polypeptide or protein of [0039] Xanthomonas campestris pv. glycines. It matches with fimbrial subunit proteins determined in other Xanthomonas campestris pathovars.
  • The hypersensitive response elicitor polypeptide or protein from [0040] Xanthomonas campestris pv. pelargonii is heat stable, protease sensitive, and has a molecular weight of 20 kDa. It includes an amino acid sequence corresponding to SEQ ID NO: 18 as follows:
    Ser Ser Gln Gln Ser Pro Ser Ala Gly Ser Glu Gln
    1               5                   10
    Gln Leu Asp Gln Leu Leu Ala Met
            15                  20
  • Isolation of [0041] Erwinia carotovora hypersensitive response elictor protein or polypeptide is described in Cui et al., “The RsmA Mutants of Erwinia carotovora subsp. carotovora Strain Ecc71 Overexpress hrp NEcc and Elicit a Hypersensitive Reaction-like Response in Tobacco Leaves,” MPMI, 9(7):565-73 (1996), which is hereby incorporated by reference. The hypersensitive response elicitor protein or polypeptide of Erwinia stewartii is set forth in Ahmad et al., “Harpin is Not Necessary for the Pathogenicity of Erwinia stewartii on Maize,” 8th Int'l. Cong. Molec. Plant-Microbe Interact., Jul. 14-19, 1996 and Ahmad, et al., “Harpin is Not Necessary for the Pathogenicity of Erwinia stewartii on Maize,” Ann. Mtg. Am. Phytopath. Soc., Jul. 27-31, 1996, which are hereby incorporated by reference.
  • Hypersensitive response elicitor proteins or polypeptides from [0042] Phytophthora parasitica, Phytophthora cryptogea, Phytophthora cinnamoni, Phytophthora capsici, Phytophthora megasperma, and Phytophora citrophthora are described in Kaman, et al., “Extracellular Protein Elicitors from Phytophthora: Most Specificity and Induction of Resistance to Bacterial and Fungal Phytopathogens,” Molec. Plant-Microbe Interact., 6(1):15-25 (1993), Ricci et al., “Structure and Activity of Proteins from Pathogenic Fungi Phytophthora Eliciting Necrosis and Acquired Resistance in Tobacco,” Eur. J. Biochem., 183:555-63 (1989), Ricci et al., “Differential Production of Parasiticein, and Elicitor of Necrosis and Resistance in Tobacco, by Isolates of Phytophthora parasitica,” Plant Path. 41:298-307 (1992), Baillreul et al, “A New Elicitor of the Hypersensitive Response in Tobacco: A Fungal Glycoprotein Elicits Cell Death, Expression of Defence Genes, Production of Salicylic Acid, and Induction of Systemic Acquired Resistance,” Plant J., 8(4):551-60 (1995), and Bonnet et al., “Acquired Resistance Triggered by Elicitors in Tobacco and Other Plants,” Eur. J. Plant Path. 102:181-92 (1996), which are hereby incorporated by reference.
  • Another hypersensitive response elicitor in accordance with the present invention is from [0043] Clavibacter michiganensis subsp. sepedonicus which is fully described in U.S. patent application Ser. No. 09/136,625, which is hereby incorporated by reference.
  • The above elicitors are exemplary. Other elicitors can be identified by growing fungi or bacteria that elicit a hypersensitive response under conditions which genes encoding an elicitor are expressed. Cell-free preparations from culture supernatants can be tested for elicitor activity (i.e. local necrosis) by using them to infiltrate appropriate plant tissues. [0044]
  • Fragments of the above hypersensitive response elicitor polypeptides or proteins as well as fragments of full length elicitors from other pathogens are encompassed by the method of the present invention. [0045]
  • Suitable fragments can be produced by several means. In the first, subclones of the gene encoding a known elicitor protein are produced by conventional molecular genetic manipulation by subcloning gene fragments. The subclones then are expressed in vitro or in vivo in bacterial cells to yield a smaller protein or peptide that can be tested for elicitor activity according to the procedure described below. [0046]
  • As an alternative, fragments of an elicitor protein can be produced by digestion of a full-length elicitor protein with proteolytic enzymes like chymotrypsin or Staphylococcus proteinase A, or trypsin. Different proteolytic enzymes are likely to cleave elicitor proteins at different sites based on the amino acid sequence of the elicitor protein. Some of the fragments that result from proteolysis may be active elicitors of resistance. [0047]
  • In another approach, based on knowledge of the primary structure of the protein, fragments of the elicitor protein gene may be synthesized by using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein. These then would be cloned into an appropriate vector for expression of a truncated peptide or protein. [0048]
  • Chemical synthesis can also be used to make suitable fragments. Such a synthesis is carried out using known amino acid sequences for the elicitor being produced. Alternatively, subjecting a full length elicitor to high temperatures and pressures will produce fragments. These fragments can then be separated by conventional procedures (e.g., chromatography, SDS-PAGE). [0049]
  • An example of suitable fragments of a hypersensitive response elicitor which do not elicit a hypersensitive response include fragments of the Erwinia. Suitable fragments include a C-terminal fragment of the amino acid sequence of SEQ ID NO: 3, an N-terminal fragment of the amino acid sequence of SEQ ID NO: 3, or an internal fragment of the amino acid sequence of SEQ ID NO: 3. The C-terminal fragment of the amino acid sequence of SEQ ID NO: 3 can span the following amino acids of SEQ ID NO: 3: 169 and 403, 210 and 403, 267 and 403, or 343 and 403. The internal fragment of the amino acid sequence of SEQ ID NO: 3 can span the following amino acids of SEQ ID NO: 3: 105 and 179, 137 and 166, 121 and 150, or 137 and 156. Other suitable fragments can be identified in accordance with the present invention. [0050]
  • Another example of suitable fragments of a hypersensitive response elicitor which do elicit a hypersensitive response are [0051] Erwinia amylovora fragments including a C-terminal fragment of the amino acid sequence of SEQ ID NO: 3, an N-terminal fragment of the amino acid sequence of SEQ ID NO: 3, or an internal fragment of the amino acid sequence of SEQ ID NO: 3. The C-terminal fragment of the amino acid sequence of SEQ ID NO: 3 can span amino acids 105 and 403 of SEQ ID NO: 3. The N-terminal fragment of the amino acid sequence of SEQ ID NO: 3 can span the following amino acids of SEQ ID NO: 3: 1 and 98, 1 and 104, 1 and 122, 1 and 168, 1 and 218, 1 and 266, 1 and 342, 1 and 321, and 1 and 372. The internal fragment of the amino acid sequence of SEQ ID NO: 3 can span the following amino acids of SEQ ID NO: 3: 76 and 209, 105 and 209, 99 and 209, 137 and 204, 137 and 200, 109 and 204, 109 and 200, 137 and 180, and 105 and 180.
  • Suitable DNA molecules are those that hybridize to the DNA molecule comprising a nucleotide sequence of SEQ ID NOS: 2, 4, 5, 7, 9, 12, 13, and 16 under stringent conditions. An example of suitable high stringency conditions is when hybridization is carried out at 65° C. for 20 hours in a medium containing 1M NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.1% sodium dodecyl sulfate, 0.2% ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, 50 μm g/ml [0052] E. coli DNA.
  • Variants may be made by, for example, the deletion or addition of amino acids that have minimal influence on the properties, secondary structure and hydropathic nature of the polypeptide. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide. [0053]
  • The hypersensitive response elicitor of the present invention is preferably in isolated form (i.e. separated from its host organism) and more preferably produced in purified form (preferably at least about 60%, more preferably 80%, pure) by conventional techniques. Typically, the hypersensitive response elicitor of the present invention is produced but not secreted into the growth medium of recombinant host cells. Alternatively, the protein or polypeptide of the present invention is secreted into growth medium. In the case of unsecreted protein, to isolate the protein, the host cell (e.g., [0054] E. coli) carrying a recombinant plasmid is propagated, lysed by sonication, heat, or chemical treatment, and the homogenate is centrifuged to remove bacterial debris. The supernatant is then subjected to heat treatment and the hypersensitive response elicitor is separated by centrifugation. The supernatant fraction containing the hypersensitive response elicitor is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the fragment. If necessary, the protein fraction may be further purified by ion exchange or HPLC.
  • The DNA molecule encoding the hypersensitive response elicitor polypeptide or protein can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences. [0055]
  • U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture. [0056]
  • Recombinant genes may also be introduced into viruses, such as vaccina virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus. [0057]
  • Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK ± or KS ± (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see F. W. Studier et. al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” [0058] Gene Expression Technology vol. 185 (1990), which is hereby incorporated by reference), and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference.
  • A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used. [0059]
  • Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation). [0060]
  • Transcription of DNA is dependent upon the presence of a promotor which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promotors differ from those of procaryotic promotors. Furthermore, eucaryotic promotors and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promotors are not recognized and do not function in eucaryotic cells. [0061]
  • Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgamo (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, [0062] Methods in Enzymology, 68:473 (1979), which is hereby incorporated by reference.
  • Promotors vary in their “strength” (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in [0063] E. coli, its bacteriophages, or plasmids, promotors such as the T7 phage promotor, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promotor or other E. coli promotors produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unless specifically induced. In certain operations, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls. [0064]
  • Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promotor, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in [0065] E. coli requires an SD sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
  • Once the isolated DNA molecule encoding the hypersensitive response elicitor polypeptide or protein has been cloned into an expression system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like. [0066]
  • The present invention's method of imparting stress resistance to plants can involve applying the hypersensitive response elicitor polypeptide or protein in a non-infectious form to all or part of a plant or a plant seed under conditions effective for the elicitor to impart stress resistance. Alternatively, the hypersensitive response elicitor protein or polypeptide can be applied to plants such that seeds recovered from such plants themselves are able to impart stress resistance in plants. [0067]
  • As an alternative to applying a hypersensitive response elicitor polypeptide or protein to plants or plant seeds in order to impart stress resistance in plants or plants grown from the seeds, transgenic plants or plant seeds can be utilized. When utilizing transgenic plants, this involves providing a transgenic plant transformed with a DNA molecule encoding a hypersensitive response elicitor polypeptide or protein and growing the plant under conditions effective to permit that DNA molecule to impart stress resistance to plants. Alternatively, a transgenic plant seed transformed with a DNA molecule encoding a hypersensitive response elicitor polypeptide or protein can be provided and planted in soil. A plant is then propagated from the planted seed under conditions effective to permit that DNA molecule to impart stress resistance to plants. [0068]
  • The embodiment of the present invention where the hypersensitive response elicitor polypeptide or protein is applied to the plant or plant seed can be carried out in a number of ways, including: 1) application of an isolated hypersensitive response elicitor or 2) application of bacteria which do not cause disease and are transformed with a genes encoding the elicitor. In the latter embodiment, the elicitor can be applied to plants or plant seeds by applying bacteria containing the DNA molecule encoding a hypersensitive response elicitor polypeptide or protein. Such bacteria must be capable of secreting or exporting the elicitor so that the elicitor can contact plant or plant seed cells. In these embodiments, the elicitor is produced by the bacteria in planta or on seeds or just prior to introduction of the bacteria to the plants or plant seeds. [0069]
  • The methods of the present invention can be utilized to treat a wide variety of plants or their seeds to impart stress resistance. Suitable plants include dicots and monocots. More particularly, useful crop plants can include: alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane. Examples of suitable ornamental plants are: [0070] Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.
  • In accordance with the present invention, the term “stress” refers to drought, salt, cold temperatures (e.g., frost), chemical treatment (e.g., insecticides, fungicides, herbicides, fertilizers), water, excessive light, and insufficient light. [0071]
  • The method of the present invention involving application of the hypersensitive response elicitor polypeptide or protein can be carried out through a variety of procedures when all or part of the plant is treated, including leaves, stems, roots, propagules (e.g., cuttings), etc. This may (but need not) involve infiltration of the hypersensitive response elicitor polypeptide or protein into the plant. Suitable application methods include high or low pressure spraying, injection, and leaf abrasion proximate to when elicitor application takes place. When treating plant seeds or propagules (e.g., cuttings), in accordance with the application embodiment of the present invention, the hypersensitive response elicitor protein or polypeptide, in accordance with present invention, can be applied by low or high pressure spraying, coating, immersion, or injection. Other suitable application procedures can be envisioned by those skilled in the art provided they are able to effect contact of the elicitor with cells of the plant or plant seed. Once treated with the hypersensitive response elicitor of the present invention, the seeds can be planted in natural or artificial soil and cultivated using conventional procedures to produce plants. After plants have been propagated from seeds treated in accordance with the present invention, the plants may be treated with one or more applications of the hypersensitive response elicitor protein or polypeptide to impart stress resistance to plants. [0072]
  • The hypersensitive response elicitor polypeptide or protein, in accordance with the present invention, can be applied to plants or plant seeds alone or in a mixture with other materials. Alternatively, the hypersensitive response elicitor polypeptide or protein can be applied separately to plants with other materials being applied at different times. [0073]
  • A composition suitable for treating plants or plant seeds in accordance with the application embodiment of the present invention contains a hypersensitive response elicitor polypeptide or protein in a carrier. Suitable carriers include water, aqueous solutions, slurries, or dry powders. In this embodiment, the composition contains greater than 500 nM of the elicitor. [0074]
  • Although not required, this composition may contain additional additives including fertilizer, insecticide, fungicide, nematacide, and mixtures thereof. Suitable fertilizers include (NH[0075] 4)2NO3. An example of a suitable insecticide is Malathion. Useful fungicides include Captan.
  • Other suitable additives include buffering agents, wetting agents, coating agents, and abrading agents. These materials can be used to facilitate the process of the present invention. In addition, the hypersensitive response elicitor can be applied to plant seeds with other conventional seed formulation and treatment materials, including clays and polysaccharides. [0076]
  • In the alternative embodiment of the present invention involving the use of transgenic plants and transgenic seeds, a hypersensitive response elicitor need not be applied topically to the plants or seeds. Instead, transgenic plants transformed with a DNA molecule encoding such an elicitor are produced according to procedures well known in the art. [0077]
  • The vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA. Crossway, [0078] Mol. Gen. Genetics, 202:179-85 (1985), which is hereby incorporated by reference. The genetic material may also be transferred into the plant cell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982), which is hereby incorporated by reference.
  • Another approach to transforming plant cells with a gene is particle bombardment (also known as biolistic transformation) of the host cell. This can be accomplished in one of several ways. The first involves propelling inert or biologically active particles at cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which are hereby incorporated by reference. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells. [0079]
  • Yet another method of introduction is fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies. Fraley, et al., [0080] Proc. Natl. Acad. Sci. USA, 79:1859-63 (1982), which is hereby incorporated by reference.
  • The DNA molecule may also be introduced into the plant cells by electroporation. Fromm et al., [0081] Proc. Natl. Acad. Sci. USA, 82:5824 (1985), which is hereby incorporated by reference. In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate.
  • Another method of introducing the DNA molecule into plant cells is to infect a plant cell with [0082] Agrobacterium tumefaciens or A. rhizogenes previously transformed with the gene. Under appropriate conditions known in the art, the transformed plant cells are grown to form shoots or roots, and develop further into plants. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28° C.
  • Agrobacterium is a representative genus of the Gram-negative family Rhizobiaceae. Its species are responsible for crown gall ([0083] A. tumefaciens) and hairy root disease (A. rhizogenes). The plant cells in crown gall tumors and hairy roots are induced to produce amino acid derivatives known as opines, which are catabolized only by the bacteria. The bacterial genes responsible for expression of opines are a convenient source of control elements for chimeric expression cassettes. In addition, assaying for the presence of opines can be used to identify transformed tissue.
  • Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of [0084] A. tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science, 237:1176-83 (1987), which is hereby incorporated by reference.
  • After transformation, the transformed plant cells must be regenerated. [0085]
  • Plant regeneration from cultured protoplasts is described in Evans et al., [0086] Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. 1, 1984, and Vol. III (1986), which are hereby incorporated by reference.
  • It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugarcane, sugar beets, cotton, fruit trees, and legumes. [0087]
  • Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable. [0088]
  • After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. [0089]
  • Once transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure with the presence of the gene encoding the hypersensitive response elicitor resulting in stress resistance to the plant. Alternatively, transgenic seeds or propagules (e.g., cuttings) are recovered from the transgenic plants. The seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants. The transgenic plants are propagated from the planted transgenic seeds under conditions effective to impart stress resistance to plants. While not wishing to be bound by theory, such stress resistance may be RNA mediated or may result from expression of the elicitor polypeptide or protein. [0090]
  • When transgenic plants and plant seeds are used in accordance with the present invention, they additionally can be treated with the same materials as are used to treat the plants and seeds to which a hypersensitive response elicitor in accordance with the present invention is applied. These other materials, including a hypersensitive response elicitor in accordance with the present invention, can be applied to the transgenic plants and plant seeds by the above-noted procedures, including high or low pressure spraying, injection, coating, and immersion. Similarly, after plants have been propagated from the transgenic plant seeds, the plants may be treated with one or more applications of the hypersensitive response elicitor in accordance with the present invention to impart stress resistance. Such plants may also be treated with conventional plant treatment agents (e.g., insecticides, fertilizers, etc.).[0091]
    • EXAMPLES Example 1
  • Hypersensitive Response Elicitor-Treated Cotton is More Resistant to the Damage Caused by Insecticide Stress [0092]
  • Aphids ([0093] Aphids gossypii) infect cotton during the entire growth season. The damage of aphid infection ranges from honeydew deposit that contaminates the lint and reduces crop value to defoliation that reduces or destroys crops. To protect plants from aphid infection, cotton is usually sprayed with insecticides, for example Asana XL when the infection pressure is not very high, and Admire when the infestation pressure is high. The effect of a hypersensitive response elicitor on aphids in cotton was studied by a trial involving a randomized complete block design. This involved treatment with Erwinia amylovora hypersensitive response elicitor (i.e. HP-1000™) at 20, 60, and 80 ppm and a chemical insecticide, Asana XL, at 8 oz./ac. Each treatment involved foliar application beginning at cotyledon to three true leaves and thereafter at 14 day intervals using a backpack sprayer. Aphid counts and overall growth of the cotton were made immediately prior to spray application at 14, 28, 35, and 42 days after the first treatment (“DAT 1”). Twenty-five randomly selected leaves per plot were collected at the first three sampling dates and the leaves per plot at the final sampling date.
  • Results [0094]
  • 1. Aphid control: The number of aphids in the hypersensitive response elicitor-treated cotton were significantly reduced in comparison to the chemical treated cotton (see Table 1). [0095]
    TABLE 1
    Aphid count per leaf on cotton after treatment with Asana XL ®
    or HP-1000 ™
    Number of aphids per leaf1
    No. sprays applied/days after treatment
    Treatment Rate2 1/14DAT1 2/28DAT1 3/35DAT1 4/42DAT1
    Asana XL ®  8 oz/ac 0.2 a 32.2 a 110.0 a 546.9 a
    HP-1000 ™ 20 μg/ml 0.2 a  7.8 b  22.9 b 322.1 a
    HP-1000 ™ 60 μg/ml 0.1 a  4.9 b  34.6 b 168.3 a
    HP-1000 ™ 80 μg/ml 0.0 a  2.7 b  25.8 b 510.2 a
  • At 14 days after DAT 1, aphid counts were relatively low across all of the treatments, but by 28 days after DAT 1 (by which time two sprayings had been applied), the number of aphids per leaf were significantly greater in Asana XL-treated plants compared to the hypersensitive response elicitor-treated cottons. By 35 days after DAT 1 (by which time three sprayings had been applied), aphid counts had risen for all treatments, yet aphid counts per leaf were still significantly lower for hypersensitive response elicitor-treated cotton compared to the Asana XL treatment. Finally, at 42 days after DAT 1 (by which time four sprayings had been applied), the number of aphids per leaf had increased to a level that threatened to overwhelm the plants even when treated with the standard chemical insecticide. To save the trial, another chemical, Pravado (Admire), was applied to all plots to eradicate aphids from the field. [0096]
  • 2. Hypersensitive response elicitor-treated cotton was more resistant to the damage caused by Pravado (Admire) and Asana. After the second chemical spraying, it was observed that cotton plants were stress shocked by the insecticides. The cotton plants previously treated with Asana and untreated control were defoliated. On most of the chemical-treated cotton, there were no leaves, or very few leaves, in the lower portion of plants. However, the hypersensitive response elicitor-treated plants, especially the plot where hypersensitive response elicitor was applied at 80 ppm, had no defoliation and the cotton plants were vigorous and healthy. By counting the number of mature balls, it clearly showed that hypersensitive response elicitor-treated plants (at 80 ppm) had more ball setting than chemical and untreated control (Table 2), indicating that hypersensitive response elicitor-treated plants were more tolerant to the stress caused by insecticide. [0097]
    TABLE 2
    Number of Formed Cotton Balls Counted on Ten Plants
    in Each of Four Replicates Per Treatment.
    Treatment No. balls/10 plants/replicate
    UTC 28
    Chemical standard 6
    Hypersensitive Response Elicitor 35
    • Example 2
  • Hypersensitive Response Elicitor-Treated Cucumbers are More Resistant to Drought [0098]
  • A cucumber field trial was set up to test the effect of [0099] Erwinia amylovora hypersensitive response elicitor on disease control, tolerance to drought stress, and yield. Three different rates were tested, there at 15, 30, and 60 μg/ml. In addition to hypersensitive response elicitor treatment, there was an untreated control. Each treatment contained three replicate plots. When the first true leaf emerges, hypersensitive response elicitor was sprayed with a back bag sprayer. The second spray was applied ten days after the first spray. The third application was right after the recovery of cucumber seedlings after the transplanting to the field. Individual treatment was randomly assigned in the field.
  • When the first true leaf emerged (Day 0), a first application was sprayed. Usually cucumber seedlings are transplanted when seedlings show two true leaves. It has been known that the recovery rate after the transplanting is closely related to the size of the seedlings. Because of the drought, the seedlings were maintained in the nursery for an extra ten days and the second spray was applied on Day 10. Two days after the second spray, the plants were transplanted into fields and covered with plastic sheets. The plants had 4-5 true leaves. [0100]
  • Result [0101]
  • The recovery rate of the transplanted cucumber seedlings was higher for the hypersensitive response elicitor-treated plants than for the untreated control. More than 80% of the hypersensitive response elicitor-treated cucumber seedlings survived, while only 57% untreated plants survived. [0102]
  • Throughout the growth season, there was a serious drought problem. Early field visits indicated that hypersensitive response elicitor-treated plants had more root mass and better over-all growth. Hypersensitive response elicitor-treated cucumber started to flower 14 days earlier than untreated control cucumber. The early flowering resulted in an earlier harvest. In the first harvest, more than 0.4 kilograms of cucumber fruits per plant were harvested from the hypersensitive response elicitor-treated cucumbers; however, virtually no fruit was harvested from untreated control. By the end of the season, untreated plants died due to severe drought, but hypersensitive response elicitor-treated plants were still alive and had one more harvest. [0103]
  • The final yield was significantly different between hypersensitive response elicitor-treated and untreated plants. Hypersensitive response elicitor administered at the rate of 30 ppm produced three times greater yield than the control plants (Table 3). [0104]
    TABLE 3
    Yield Increase of Cucumber Fruit from Hypersensitive
    Response Elicitor Treated Plants
    % of the Yield
    Treatment Replicate kg/plant Yield/Replicate Increase
    HP 15 I 1.25 37.5
    II 1.00 30.0 103.8 241
    III 1.21 36.3
    HP 30 I 1.54 46.2
    II 1.43 42.9 133.2 339
    III 1.47 44.1
    Control I 0.43 12.9
    II 0.41 12.3 39.3
    III 0.47 14.1
  • The increased yield was partially attributed to hypersensitive response elicitor-induced growth enhancement and partially resulted from more tolerance of hypersensitive response elicitor-treated cucumber to drought, because usually the yield increase from hypersensitive response elicitor-induced growth enhancement is between 10-40%. [0105]
    • Example 3
  • Hypersensitive Response Elicitor-Treated Pepper is More Tolerant to Herbicide Stress [0106]
  • Pepper seedlings were drenched with hypersensitive response elicitor at 20 ppm seven days before transplanting, sprayed seven days after the transplanting, and then, sprayed every fourteen days. Standard chemicals, Brave, Maneb, Kocide, and Admire, were used for the rest of the treatment. In addition to early growth enhancement, which resulted in a higher yield, larger fruit, and resistance to several diseases, hypersensitive response elicitor-treated pepper was more tolerant to herbicide damage. The pepper field was applied with the herbicide SENCOR which is not labeled for pepper. This herbicide is known to cause severe foliar damage to pepper in chemically-treated plants but not with hypersensitive response elicitor-treated plants. [0107]
  • The difference between the adverse effect of the herbicide on the hypersensitive response elicitor and non-hypersensitive response elicitor treated plants is dramatic. See Table 4 below. Thirty-nine of the 60 elicitor-treated plants showed only minor damage by the herbicide, the damaged leaves were less than 20%. In contrast, 53 out of the 60 chemically-treated pepper plants had severe damage, 40-57% of the leaves were damaged, and 20 plants were dead. The ability of hypersensitive response elicitors to help crops withstand the phytotoxic effects of a herbicide is very important benefit to in agricultural industry. [0108]
    TABLE 4
    Hypersensitive Response Elicitor-Treated Peppers are More
    Tolerant to Herbicide Damage.
    Treatment Damage Rating Damage Index %
    1 2 3 4 5 6 41
    Hypersensitive 1 38 17 3 1 0
    Response Elicitor
    Chemicals 0 1 6 16 19 18 87
    • Example 4
  • Hypersensitive Response Elicitor-Treated Pepper is More Tolerant to Herbicide Stress under Controlled Experimental Conditions [0109]
  • A field trial was conducted to test if hypersensitive elicitor treated pepper would be more tolerant to herbicide stress. The trial contains 6 treatments and 4 replicates for each treatment. The treatments are described as follows: [0110]
  • 1. Control, the peppers were neither treated by a hypersensitive response (“HR”) elicitor nor by LEXONE™ herbicide (DuPont Agricultural Products, Wilmington, Del.). [0111]
  • 2. Control pepper with application of 0.15 pound LEXONE™ herbicide/acre. [0112]
  • 3. Control pepper with application of 0.3 pound LEXONE™ herbicide/acre. [0113]
  • 4. HR elicitor treatment with no application of LEXONE™ herbicide using a formulated product known as MESSENGER™ biopesticide (Eden Bioscience Corporation, Bothell, Wash.) containing 3% HR elicitor protein was used. [0114]
  • 5. HR elicitor treatment with application of 0.15 pound LEXONE™ herbicide/acre. [0115]
  • 6. HR elicitor treatment with application of 0.3 pound LEXONE™ herbicide/acre. [0116]
  • LEXONE™ contains the same active ingredient as SENCOR™ herbicide (Bayer, Kansas City, Mo.) used in Example 3. Pepper seedlings were drenched with MESSENGER™ solution at the concentration of HR elicitor protein of about 20 ppm seven days before transplanting into the field and then sprayed every 14 days after the transplanting. LEXONE was applied at high (0.3 pound/acre) and low levels (0.15 pound/acre). 50 gallon water and 100 mL of the herbicide solution was introduced into the root zone of each plant in the respective treatment five weeks after transplant into the field. [0117]
  • The treatments were evaluated for the percent of chlorosis caused by the LEXONE™ herbicide application and for the pepper yield. HR elicitor-treated plants exposed to the high rate of herbicide had significantly less chlorosis and produced 108% more fruit in comparison to the non-hypersensitive response elicitor treated plants exposed to the same amount of herbicide. See Tables 5 and 6 below. There was no significant difference in the reduction of chlorosis at the low rate of herbicide between the HR elicitor treated and non-HR elicitor treated peppers. However, the HR elicitor treated plants produced 15% more fruit than the corresponding control plants exposed to the same amount of herbicide. There was no chlorosis in either the check or HR elicitor-treated plants that did not receive LEXONE™ herbicide treatment. [0118]
  • The HR elicitor treated plants were much less severely affected by the herbicide application than the respective control plants at the high rate of herbicide. However, the amount of visual chlorosis was similar at the low rate for both the check and HR elicitor-treated plants. More importantly, the yields from both the high and low rate herbicide treatments of HR elicitor treated plants were less severely effected by the herbicide than the checks. These findings further confirm that HR elicitors can help crops withstand the phytotoxic effects of herbicides and are very beneficial to the agricultural industry. [0119]
    TABLE 5
    Reduction of Foliar Chlorosis and Increase in Yield in Hypersensitive
    Response Elicitor Treated Plants after Exposure to LEXONE ™Herbicide
    Percent foliar chlorosis and yield of pepper
    % difference
    from the
    Yield respective
    Treatment A B C D E (pound) control
    6 (MESSENGER ™ + 13.75 30.00 37.50 36.25 40.00 8.31 108%
    High rate
    LEXONE ™)
    3 (High rate 26.25 43.75 51.25 50.00 51.25 4.00 —
    LEXONE ™)
    5 (MESSENGER ™ + 16.25 22.50 28.75 23.75 27.50 8.00  15%
    low rate
    LENOXE ™)
    2 (LENOXE ™) 12.50 20.00 25.00 25.00 23.75 6.81 —
  • [0120]
    TABLE 6
    Weight of Harvested Peppers Increased in Hypersensitive
    Response Elicitor Treated Plants after Exposure to LEXONE ™
    Herbicide Compared to Check Plants.
    Weight of peppers
    Treatment harvested Dec. 1, 1998 in pounds
    HP20 + high rate LEXONE ™ 8.31
    Check + high rate LEXONE ™ 4.00
    HP20 + low rate LEXONE ™ 8.00
    Check + low rate LEXONE ™ 6.81
    • Example 5
  • Hypersensitive Response Elicitor-Treated Cotton is More Tolerant to Drought Stress [0121]
  • A non-irrigated cotton trial experienced 26 consecutive days of drought. The average daily heat index was near or over 100 degrees F., adding to the stress placed on the plants in the field. [0122]
  • Observations in the field indicated that plants treated with HR elicitor at the concentration of 15 ppm (2.2 oz formulated product, MESSENGER™ containing 3% active ingredient HR elicitor protein) were more vigorous and had less defoliation than the check plants as a result of the heat and drought stress. Equal numbers of plants from the MESSENGER™-treated and the non-MESSENGER™ treated plots were carefully removed from the field and mapped for the number of nodes and bolls by position. The plants were also weighed on a Metler analytical scale to determine whole plant, root and shoot weights. [0123]
  • MESSENGER™ treated plants survived the heat and drought stresses much better than the untreated plants did. Plants treated with MESSENGER™ had 37.6% more root and shoot mass than the check plants (Table 7). The MESSENGER™ treated plants also had significantly more cotton bolls than the check plants (Table 8). The number of cotton bolls from positions 1 and 2 have a significant contribution to the overall yield. Table 8 showed that MESSENGER™ treated plants had 47% more bolls in positions 1 and 2 and 57% more boll from a whole plant in comparison to the yield achieved using a grower standard treatment (i.e. with no MESSENGER™ treatment). A common reaction to stress in cotton is for the plant to abort bolls. The results indicate that MESSENGER™-treated plants are more tolerant to the drought stress. [0124]
    TABLE 7
    Weight per Plant of Non-Irrigated Cotton Following 26 Days of Drought.
    Whole plant
    Root weight Shoot weight % weight %
    Treatment (pond/plant) % Difference (pond/plant) difference (pond/plant) difference
    MESSENGER ™  0.041 a* 37.6% 0.505 a 37.5% 0.546 37.5%
    2.2 oz/acre
    Control (Grower 0.0298 b 0.367 b 0.397
    standard)
    Level of P = 0.119 P = 0.034 P = 0.033
    statistically
    significant
  • [0125]
    TABLE 8
    Number of Bolls per 5 Plants at the Number 1 & 2 positions, and
    Total Number of Bolls from Whole Plants in Non-irrigated Cotton
    Following 26 days of drought.
    Avg. # bolls in Avg. # of
    the #1 & 2 Percent total bolls Percent
    Treatment position difference per 5 plant difference
    MESSENGER ™ 18.4 a +46.0% 21.4 a +57.0%
    2.2 OZ.
    Check 12.6 b 13.6 b —
    Statistically P = 0.032 P = 0.01
    significant level
    • Example 6
  • Hypersensitive Response Elicitor-Treated Tomato is More Tolerant to Calcium Deficiency [0126]
  • Calcium is an important element for plant physiology and development. A deficiency in calcium can cause several plant diseases. For example, blossom-end rot is caused by a localized calcium deficiency in the distal end of the tomato fruit. Because calcium is not a highly mobile element, a deficiency can occur with a fluctuation in water supply. In the past, tomato growers experienced higher level of blossom-end rot during dry weather conditions when infrequent rains storms dumped a lot of water and then return to a hot and dry condition quickly. Lowering or raising the irrigation water table erratically during a dry and hot growing season can also increase the disease. [0127]
  • A field trial was designed to test if HR elicitor protein-treated tomato can be more tolerant to the calcium deficiency under a dry hot growing season. MESSENGER™, the formulated product containing 3% HR elicitor, was used for the trial. The application rate of the MESSENGER™ was 2.27 oz per care. The first spray of MESSENGER™ was carried out 7 days before the transplanting and then every 14-days after transplanting. MESSENGER™-treated tomatoes were compared with a standard grower treatment not utilizing MESSENGER™. Each treatment had 4 replicates. [0128]
  • The number of infected fruit was counted from a 100 square foot field. The rot typically begins with light tan water soaked lesions, which then enlarge, and then turn black. In a survey, about 20% of the fruits were infected. Severe end-rot symptoms occurred in the standard treatment; however, an average of only 2.5% of the fruit was infected in the MESSENGER™-treated plants. The harvest data showed that MESSENGER™-treated plants had 8% more marketable fruit (Table 9). The test results demonstrated that MESSENGER™-treatment can reduce the stress resulting from calcium deficiency and increase plant resistance to blossom-end rot. [0129]
    TABLE 9
    Hypersensitive Response Elicitor Treatment Reduced Blossom-End
    Rot Infection, Increased Yield of Tomato Fruit
    Blossom-End Infected Fruit* Tomato Fruit Yield
    Treatment Rep I Rep II Rep III Rep IV Bin/Acre % Difference
    MESSENGER ™ 0 9 0 1 35 8
    Standard Treatment) 24 22 16 17 31.5 —
  • Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims. [0130]
  • 1 18 1 338 PRT Erwinia chrysanthemi 1 Met Gln Ile Thr Ile Lys Ala His Ile Gly Gly Asp Leu Gly Val Ser 1 5 10 15 Gly Leu Gly Ala Gln Gly Leu Lys Gly Leu Asn Ser Ala Ala Ser Ser 20 25 30 Leu Gly Ser Ser Val Asp Lys Leu Ser Ser Thr Ile Asp Lys Leu Thr 35 40 45 Ser Ala Leu Thr Ser Met Met Phe Gly Gly Ala Leu Ala Gln Gly Leu 50 55 60 Gly Ala Ser Ser Lys Gly Leu Gly Met Ser Asn Gln Leu Gly Gln Ser 65 70 75 80 Phe Gly Asn Gly Ala Gln Gly Ala Ser Asn Leu Leu Ser Val Pro Lys 85 90 95 Ser Gly Gly Asp Ala Leu Ser Lys Met Phe Asp Lys Ala Leu Asp Asp 100 105 110 Leu Leu Gly His Asp Thr Val Thr Lys Leu Thr Asn Gln Ser Asn Gln 115 120 125 Leu Ala Asn Ser Met Leu Asn Ala Ser Gln Met Thr Gln Gly Asn Met 130 135 140 Asn Ala Phe Gly Ser Gly Val Asn Asn Ala Leu Ser Ser Ile Leu Gly 145 150 155 160 Asn Gly Leu Gly Gln Ser Met Ser Gly Phe Ser Gln Pro Ser Leu Gly 165 170 175 Ala Gly Gly Leu Gln Gly Leu Ser Gly Ala Gly Ala Phe Asn Gln Leu 180 185 190 Gly Asn Ala Ile Gly Met Gly Val Gly Gln Asn Ala Ala Leu Ser Ala 195 200 205 Leu Ser Asn Val Ser Thr His Val Asp Gly Asn Asn Arg His Phe Val 210 215 220 Asp Lys Glu Asp Arg Gly Met Ala Lys Glu Ile Gly Gln Phe Met Asp 225 230 235 240 Gln Tyr Pro Glu Ile Phe Gly Lys Pro Glu Tyr Gln Lys Asp Gly Trp 245 250 255 Ser Ser Pro Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser Lys 260 265 270 Pro Asp Asp Asp Gly Met Thr Gly Ala Ser Met Asp Lys Phe Arg Gln 275 280 285 Ala Met Gly Met Ile Lys Ser Ala Val Ala Gly Asp Thr Gly Asn Thr 290 295 300 Asn Leu Asn Leu Arg Gly Ala Gly Gly Ala Ser Leu Gly Ile Asp Ala 305 310 315 320 Ala Val Val Gly Asp Lys Ile Ala Asn Met Ser Leu Gly Lys Leu Ala 325 330 335 Asn Ala 2 2141 DNA Erwinia chrysanthemi 2 cgattttacc cgggtgaacg tgctatgacc gacagcatca cggtattcga caccgttacg 60 gcgtttatgg ccgcgatgaa ccggcatcag gcggcgcgct ggtcgccgca atccggcgtc 120 gatctggtat ttcagtttgg ggacaccggg cgtgaactca tgatgcagat tcagccgggg 180 cagcaatatc ccggcatgtt gcgcacgctg ctcgctcgtc gttatcagca ggcggcagag 240 tgcgatggct gccatctgtg cctgaacggc agcgatgtat tgatcctctg gtggccgctg 300 ccgtcggatc ccggcagtta tccgcaggtg atcgaacgtt tgtttgaact ggcgggaatg 360 acgttgccgt cgctatccat agcaccgacg gcgcgtccgc agacagggaa cggacgcgcc 420 cgatcattaa gataaaggcg gcttttttta ttgcaaaacg gtaacggtga ggaaccgttt 480 caccgtcggc gtcactcagt aacaagtatc catcatgatg cctacatcgg gatcggcgtg 540 ggcatccgtt gcagatactt ttgcgaacac ctgacatgaa tgaggaaacg aaattatgca 600 aattacgatc aaagcgcaca tcggcggtga tttgggcgtc tccggtctgg ggctgggtgc 660 tcagggactg aaaggactga attccgcggc ttcatcgctg ggttccagcg tggataaact 720 gagcagcacc atcgataagt tgacctccgc gctgacttcg atgatgtttg gcggcgcgct 780 ggcgcagggg ctgggcgcca gctcgaaggg gctggggatg agcaatcaac tgggccagtc 840 tttcggcaat ggcgcgcagg gtgcgagcaa cctgctatcc gtaccgaaat ccggcggcga 900 tgcgttgtca aaaatgtttg ataaagcgct ggacgatctg ctgggtcatg acaccgtgac 960 caagctgact aaccagagca accaactggc taattcaatg ctgaacgcca gccagatgac 1020 ccagggtaat atgaatgcgt tcggcagcgg tgtgaacaac gcactgtcgt ccattctcgg 1080 caacggtctc ggccagtcga tgagtggctt ctctcagcct tctctggggg caggcggctt 1140 gcagggcctg agcggcgcgg gtgcattcaa ccagttgggt aatgccatcg gcatgggcgt 1200 ggggcagaat gctgcgctga gtgcgttgag taacgtcagc acccacgtag acggtaacaa 1260 ccgccacttt gtagataaag aagatcgcgg catggcgaaa gagatcggcc agtttatgga 1320 tcagtatccg gaaatattcg gtaaaccgga ataccagaaa gatggctgga gttcgccgaa 1380 gacggacgac aaatcctggg ctaaagcgct gagtaaaccg gatgatgacg gtatgaccgg 1440 cgccagcatg gacaaattcc gtcaggcgat gggtatgatc aaaagcgcgg tggcgggtga 1500 taccggcaat accaacctga acctgcgtgg cgcgggcggt gcatcgctgg gtatcgatgc 1560 ggctgtcgtc ggcgataaaa tagccaacat gtcgctgggt aagctggcca acgcctgata 1620 atctgtgctg gcctgataaa gcggaaacga aaaaagagac ggggaagcct gtctcttttc 1680 ttattatgcg gtttatgcgg ttacctggac cggttaatca tcgtcatcga tctggtacaa 1740 acgcacattt tcccgttcat tcgcgtcgtt acgcgccaca atcgcgatgg catcttcctc 1800 gtcgctcaga ttgcgcggct gatggggaac gccgggtgga atatagagaa actcgccggc 1860 cagatggaga cacgtctgcg ataaatctgt gccgtaacgt gtttctatcc gcccctttag 1920 cagatagatt gcggtttcgt aatcaacatg gtaatgcggt tccgcctgtg cgccggccgg 1980 gatcaccaca atattcatag aaagctgtct tgcacctacc gtatcgcggg agataccgac 2040 aaaatagggc agtttttgcg tggtatccgt ggggtgttcc ggcctgacaa tcttgagttg 2100 gttcgtcatc atctttctcc atctgggcga cctgatcggt t 2141 3 403 PRT Erwinia amylovora 3 Met Ser Leu Asn Thr Ser Gly Leu Gly Ala Ser Thr Met Gln Ile Ser 1 5 10 15 Ile Gly Gly Ala Gly Gly Asn Asn Gly Leu Leu Gly Thr Ser Arg Gln 20 25 30 Asn Ala Gly Leu Gly Gly Asn Ser Ala Leu Gly Leu Gly Gly Gly Asn 35 40 45 Gln Asn Asp Thr Val Asn Gln Leu Ala Gly Leu Leu Thr Gly Met Met 50 55 60 Met Met Met Ser Met Met Gly Gly Gly Gly Leu Met Gly Gly Gly Leu 65 70 75 80 Gly Gly Gly Leu Gly Asn Gly Leu Gly Gly Ser Gly Gly Leu Gly Glu 85 90 95 Gly Leu Ser Asn Ala Leu Asn Asp Met Leu Gly Gly Ser Leu Asn Thr 100 105 110 Leu Gly Ser Lys Gly Gly Asn Asn Thr Thr Ser Thr Thr Asn Ser Pro 115 120 125 Leu Asp Gln Ala Leu Gly Ile Asn Ser Thr Ser Gln Asn Asp Asp Ser 130 135 140 Thr Ser Gly Thr Asp Ser Thr Ser Asp Ser Ser Asp Pro Met Gln Gln 145 150 155 160 Leu Leu Lys Met Phe Ser Glu Ile Met Gln Ser Leu Phe Gly Asp Gly 165 170 175 Gln Asp Gly Thr Gln Gly Ser Ser Ser Gly Gly Lys Gln Pro Thr Glu 180 185 190 Gly Glu Gln Asn Ala Tyr Lys Lys Gly Val Thr Asp Ala Leu Ser Gly 195 200 205 Leu Met Gly Asn Gly Leu Ser Gln Leu Leu Gly Asn Gly Gly Leu Gly 210 215 220 Gly Gly Gln Gly Gly Asn Ala Gly Thr Gly Leu Asp Gly Ser Ser Leu 225 230 235 240 Gly Gly Lys Gly Leu Gln Asn Leu Ser Gly Pro Val Asp Tyr Gln Gln 245 250 255 Leu Gly Asn Ala Val Gly Thr Gly Ile Gly Met Lys Ala Gly Ile Gln 260 265 270 Ala Leu Asn Asp Ile Gly Thr His Arg His Ser Ser Thr Arg Ser Phe 275 280 285 Val Asn Lys Gly Asp Arg Ala Met Ala Lys Glu Ile Gly Gln Phe Met 290 295 300 Asp Gln Tyr Pro Glu Val Phe Gly Lys Pro Gln Tyr Gln Lys Gly Pro 305 310 315 320 Gly Gln Glu Val Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser 325 330 335 Lys Pro Asp Asp Asp Gly Met Thr Pro Ala Ser Met Glu Gln Phe Asn 340 345 350 Lys Ala Lys Gly Met Ile Lys Arg Pro Met Ala Gly Asp Thr Gly Asn 355 360 365 Gly Asn Leu Gln Ala Arg Gly Ala Gly Gly Ser Ser Leu Gly Ile Asp 370 375 380 Ala Met Met Ala Gly Asp Ala Ile Asn Asn Met Ala Leu Gly Lys Leu 385 390 395 400 Gly Ala Ala 4 1288 DNA Erwinia amylovora 4 aagcttcggc atggcacgtt tgaccgttgg gtcggcaggg tacgtttgaa ttattcataa 60 gaggaatacg ttatgagtct gaatacaagt gggctgggag cgtcaacgat gcaaatttct 120 atcggcggtg cgggcggaaa taacgggttg ctgggtacca gtcgccagaa tgctgggttg 180 ggtggcaatt ctgcactggg gctgggcggc ggtaatcaaa atgataccgt caatcagctg 240 gctggcttac tcaccggcat gatgatgatg atgagcatga tgggcggtgg tgggctgatg 300 ggcggtggct taggcggtgg cttaggtaat ggcttgggtg gctcaggtgg cctgggcgaa 360 ggactgtcga acgcgctgaa cgatatgtta ggcggttcgc tgaacacgct gggctcgaaa 420 ggcggcaaca ataccacttc aacaacaaat tccccgctgg accaggcgct gggtattaac 480 tcaacgtccc aaaacgacga ttccacctcc ggcacagatt ccacctcaga ctccagcgac 540 ccgatgcagc agctgctgaa gatgttcagc gagataatgc aaagcctgtt tggtgatggg 600 caagatggca cccagggcag ttcctctggg ggcaagcagc cgaccgaagg cgagcagaac 660 gcctataaaa aaggagtcac tgatgcgctg tcgggcctga tgggtaatgg tctgagccag 720 ctccttggca acgggggact gggaggtggt cagggcggta atgctggcac gggtcttgac 780 ggttcgtcgc tgggcggcaa agggctgcaa aacctgagcg ggccggtgga ctaccagcag 840 ttaggtaacg ccgtgggtac cggtatcggt atgaaagcgg gcattcaggc gctgaatgat 900 atcggtacgc acaggcacag ttcaacccgt tctttcgtca ataaaggcga tcgggcgatg 960 gcgaaggaaa tcggtcagtt catggaccag tatcctgagg tgtttggcaa gccgcagtac 1020 cagaaaggcc cgggtcagga ggtgaaaacc gatgacaaat catgggcaaa agcactgagc 1080 aagccagatg acgacggaat gacaccagcc agtatggagc agttcaacaa agccaagggc 1140 atgatcaaaa ggcccatggc gggtgatacc ggcaacggca acctgcaggc acgcggtgcc 1200 ggtggttctt cgctgggtat tgatgccatg atggccggtg atgccattaa caatatggca 1260 cttggcaagc tgggcgcggc ttaagctt 1288 5 1344 DNA Erwinia amylovora 5 atgtcaattc ttacgcttaa caacaatacc tcgtcctcgc cgggtctgtt ccagtccggg 60 ggggacaacg ggcttggtgg tcataatgca aattctgcgt tggggcaaca acccatcgat 120 cggcaaacca ttgagcaaat ggctcaatta ttggcggaac tgttaaagtc actgctatcg 180 ccacaatcag gtaatgcggc aaccggagcc ggtggcaatg accagactac aggagttggt 240 aacgctggcg gcctgaacgg acgaaaaggc acagcaggaa ccactccgca gtctgacagt 300 cagaacatgc tgagtgagat gggcaacaac gggctggatc aggccatcac gcccgatggc 360 cagggcggcg ggcagatcgg cgataatcct ttactgaaag ccatgctgaa gcttattgca 420 cgcatgatgg acggccaaag cgatcagttt ggccaacctg gtacgggcaa caacagtgcc 480 tcttccggta cttcttcatc tggcggttcc ccttttaacg atctatcagg ggggaaggcc 540 ccttccggca actccccttc cggcaactac tctcccgtca gtaccttctc acccccatcc 600 acgccaacgt cccctacctc accgcttgat ttcccttctt ctcccaccaa agcagccggg 660 ggcagcacgc cggtaaccga tcatcctgac cctgttggta gcgcgggcat cggggccgga 720 aattcggtgg ccttcaccag cgccggcgct aatcagacgg tgctgcatga caccattacc 780 gtgaaagcgg gtcaggtgtt tgatggcaaa ggacaaacct tcaccgccgg ttcagaatta 840 ggcgatggcg gccagtctga aaaccagaaa ccgctgttta tactggaaga cggtgccagc 900 ctgaaaaacg tcaccatggg cgacgacggg gcggatggta ttcatcttta cggtgatgcc 960 aaaatagaca atctgcacgt caccaacgtg ggtgaggacg cgattaccgt taagccaaac 1020 agcgcgggca aaaaatccca cgttgaaatc actaacagtt ccttcgagca cgcctctgac 1080 aagatcctgc agctgaatgc cgatactaac ctgagcgttg acaacgtgaa ggccaaagac 1140 tttggtactt ttgtacgcac taacggcggt caacagggta actgggatct gaatctgagc 1200 catatcagcg cagaagacgg taagttctcg ttcgttaaaa gcgatagcga ggggctaaac 1260 gtcaatacca gtgatatctc actgggtgat gttgaaaacc actacaaagt gccgatgtcc 1320 gccaacctga aggtggctga atga 1344 6 447 PRT Erwinia amylovora 6 Met Ser Ile Leu Thr Leu Asn Asn Asn Thr Ser Ser Ser Pro Gly Leu 1 5 10 15 Phe Gln Ser Gly Gly Asp Asn Gly Leu Gly Gly His Asn Ala Asn Ser 20 25 30 Ala Leu Gly Gln Gln Pro Ile Asp Arg Gln Thr Ile Glu Gln Met Ala 35 40 45 Gln Leu Leu Ala Glu Leu Leu Lys Ser Leu Leu Ser Pro Gln Ser Gly 50 55 60 Asn Ala Ala Thr Gly Ala Gly Gly Asn Asp Gln Thr Thr Gly Val Gly 65 70 75 80 Asn Ala Gly Gly Leu Asn Gly Arg Lys Gly Thr Ala Gly Thr Thr Pro 85 90 95 Gln Ser Asp Ser Gln Asn Met Leu Ser Glu Met Gly Asn Asn Gly Leu 100 105 110 Asp Gln Ala Ile Thr Pro Asp Gly Gln Gly Gly Gly Gln Ile Gly Asp 115 120 125 Asn Pro Leu Leu Lys Ala Met Leu Lys Leu Ile Ala Arg Met Met Asp 130 135 140 Gly Gln Ser Asp Gln Phe Gly Gln Pro Gly Thr Gly Asn Asn Ser Ala 145 150 155 160 Ser Ser Gly Thr Ser Ser Ser Gly Gly Ser Pro Phe Asn Asp Leu Ser 165 170 175 Gly Gly Lys Ala Pro Ser Gly Asn Ser Pro Ser Gly Asn Tyr Ser Pro 180 185 190 Val Ser Thr Phe Ser Pro Pro Ser Thr Pro Thr Ser Pro Thr Ser Pro 195 200 205 Leu Asp Phe Pro Ser Ser Pro Thr Lys Ala Ala Gly Gly Ser Thr Pro 210 215 220 Val Thr Asp His Pro Asp Pro Val Gly Ser Ala Gly Ile Gly Ala Gly 225 230 235 240 Asn Ser Val Ala Phe Thr Ser Ala Gly Ala Asn Gln Thr Val Leu His 245 250 255 Asp Thr Ile Thr Val Lys Ala Gly Gln Val Phe Asp Gly Lys Gly Gln 260 265 270 Thr Phe Thr Ala Gly Ser Glu Leu Gly Asp Gly Gly Gln Ser Glu Asn 275 280 285 Gln Lys Pro Leu Phe Ile Leu Glu Asp Gly Ala Ser Leu Lys Asn Val 290 295 300 Thr Met Gly Asp Asp Gly Ala Asp Gly Ile His Leu Tyr Gly Asp Ala 305 310 315 320 Lys Ile Asp Asn Leu His Val Thr Asn Val Gly Glu Asp Ala Ile Thr 325 330 335 Val Lys Pro Asn Ser Ala Gly Lys Lys Ser His Val Glu Ile Thr Asn 340 345 350 Ser Ser Phe Glu His Ala Ser Asp Lys Ile Leu Gln Leu Asn Ala Asp 355 360 365 Thr Asn Leu Ser Val Asp Asn Val Lys Ala Lys Asp Phe Gly Thr Phe 370 375 380 Val Arg Thr Asn Gly Gly Gln Gln Gly Asn Trp Asp Leu Asn Leu Ser 385 390 395 400 His Ile Ser Ala Glu Asp Gly Lys Phe Ser Phe Val Lys Ser Asp Ser 405 410 415 Glu Gly Leu Asn Val Asn Thr Ser Asp Ile Ser Leu Gly Asp Val Glu 420 425 430 Asn His Tyr Lys Val Pro Met Ser Ala Asn Leu Lys Val Ala Glu 435 440 445 7 5517 DNA Erwinia amylovora 7 atggaattaa aatcactggg aactgaacac aaggcggcag tacacacagc ggcgcacaac 60 cctgtggggc atggtgttgc cttacagcag ggcagcagca gcagcagccc gcaaaatgcc 120 gctgcatcat tggcggcaga aggcaaaaat cgtgggaaaa tgccgagaat tcaccagcca 180 tctactgcgg ctgatggtat cagcgctgct caccagcaaa agaaatcctt cagtctcagg 240 ggctgtttgg ggacgaaaaa attttccaga tcggcaccgc agggccagcc aggtaccacc 300 cacagcaaag gggcaacatt gcgcgatctg ctggcgcggg acgacggcga aacgcagcat 360 gaggcggccg cgccagatgc ggcgcgtttg acccgttcgg gcggcgtcaa acgccgcaat 420 atggacgaca tggccgggcg gccaatggtg aaaggtggca gcggcgaaga taaggtacca 480 acgcagcaaa aacggcatca gctgaacaat tttggccaga tgcgccaaac gatgttgagc 540 aaaatggctc acccggcttc agccaacgcc ggcgatcgcc tgcagcattc accgccgcac 600 atcccgggta gccaccacga aatcaaggaa gaaccggttg gctccaccag caaggcaaca 660 acggcccacg cagacagagt ggaaatcgct caggaagatg acgacagcga attccagcaa 720 ctgcatcaac agcggctggc gcgcgaacgg gaaaatccac cgcagccgcc caaactcggc 780 gttgccacac cgattagcgc caggtttcag cccaaactga ctgcggttgc ggaaagcgtc 840 cttgagggga cagataccac gcagtcaccc cttaagccgc aatcaatgct gaaaggaagt 900 ggagccgggg taacgccgct ggcggtaacg ctggataaag gcaagttgca gctggcaccg 960 gataatccac ccgcgctcaa tacgttgttg aagcagacat tgggtaaaga cacccagcac 1020 tatctggcgc accatgccag cagcgacggt agccagcatc tgctgctgga caacaaaggc 1080 cacctgtttg atatcaaaag caccgccacc agctatagcg tgctgcacaa cagccacccc 1140 ggtgagataa agggcaagct ggcgcaggcg ggtactggct ccgtcagcgt agacggtaaa 1200 agcggcaaga tctcgctggg gagcggtacg caaagtcaca acaaaacaat gctaagccaa 1260 ccgggggaag cgcaccgttc cttattaacc ggcatttggc agcatcctgc tggcgcagcg 1320 cggccgcagg gcgagtcaat ccgcctgcat gacgacaaaa ttcatatcct gcatccggag 1380 ctgggcgtat ggcaatctgc ggataaagat acccacagcc agctgtctcg ccaggcagac 1440 ggtaagctct atgcgctgaa agacaaccgt accctgcaaa acctctccga taataaatcc 1500 tcagaaaagc tggtcgataa aatcaaatcg tattccgttg atcagcgggg gcaggtggcg 1560 atcctgacgg atactcccgg ccgccataag atgagtatta tgccctcgct ggatgcttcc 1620 ccggagagcc atatttccct cagcctgcat tttgccgatg cccaccaggg gttattgcac 1680 gggaagtcgg agcttgaggc acaatctgtc gcgatcagcc atgggcgact ggttgtggcc 1740 gatagcgaag gcaagctgtt tagcgccgcc attccgaagc aaggggatgg aaacgaactg 1800 aaaatgaaag ccatgcctca gcatgcgctc gatgaacatt ttggtcatga ccaccagatt 1860 tctggatttt tccatgacga ccacggccag cttaatgcgc tggtgaaaaa taacttcagg 1920 cagcagcatg cctgcccgtt gggtaacgat catcagtttc accccggctg gaacctgact 1980 gatgcgctgg ttatcgacaa tcagctgggg ctgcatcata ccaatcctga accgcatgag 2040 attcttgata tggggcattt aggcagcctg gcgttacagg agggcaagct tcactatttt 2100 gaccagctga ccaaagggtg gactggcgcg gagtcagatt gtaagcagct gaaaaaaggc 2160 ctggatggag cagcttatct actgaaagac ggtgaagtga aacgcctgaa tattaatcag 2220 agcacctcct ctatcaagca cggaacggaa aacgtttttt cgctgccgca tgtgcgcaat 2280 aaaccggagc cgggagatgc cctgcaaggg ctgaataaag acgataaggc ccaggccatg 2340 gcggtgattg gggtaaataa atacctggcg ctgacggaaa aaggggacat tcgctccttc 2400 cagataaaac ccggcaccca gcagttggag cggccggcac aaactctcag ccgcgaaggt 2460 atcagcggcg aactgaaaga cattcatgtc gaccacaagc agaacctgta tgccttgacc 2520 cacgagggag aggtgtttca tcagccgcgt gaagcctggc agaatggtgc cgaaagcagc 2580 agctggcaca aactggcgtt gccacagagt gaaagtaagc taaaaagtct ggacatgagc 2640 catgagcaca aaccgattgc cacctttgaa gacggtagcc agcatcagct gaaggctggc 2700 ggctggcacg cctatgcggc acctgaacgc gggccgctgg cggtgggtac cagcggttca 2760 caaaccgtct ttaaccgact aatgcagggg gtgaaaggca aggtgatccc aggcagcggg 2820 ttgacggtta agctctcggc tcagacgggg ggaatgaccg gcgccgaagg gcgcaaggtc 2880 agcagtaaat tttccgaaag gatccgcgcc tatgcgttca acccaacaat gtccacgccg 2940 cgaccgatta aaaatgctgc ttatgccaca cagcacggct ggcaggggcg tgaggggttg 3000 aagccgttgt acgagatgca gggagcgctg attaaacaac tggatgcgca taacgttcgt 3060 cataacgcgc cacagccaga tttgcagagc aaactggaaa ctctggattt aggcgaacat 3120 ggcgcagaat tgcttaacga catgaagcgc ttccgcgacg aactggagca gagtgcaacc 3180 cgttcggtga ccgttttagg tcaacatcag ggagtgctaa aaagcaacgg tgaaatcaat 3240 agcgaattta agccatcgcc cggcaaggcg ttggtccaga gctttaacgt caatcgctct 3300 ggtcaggatc taagcaagtc actgcaacag gcagtacatg ccacgccgcc atccgcagag 3360 agtaaactgc aatccatgct ggggcacttt gtcagtgccg gggtggatat gagtcatcag 3420 aagggcgaga tcccgctggg ccgccagcgc gatccgaatg ataaaaccgc actgaccaaa 3480 tcgcgtttaa ttttagatac cgtgaccatc ggtgaactgc atgaactggc cgataaggcg 3540 aaactggtat ctgaccataa acccgatgcc gatcagataa aacagctgcg ccagcagttc 3600 gatacgctgc gtgaaaagcg gtatgagagc aatccggtga agcattacac cgatatgggc 3660 ttcacccata ataaggcgct ggaagcaaac tatgatgcgg tcaaagcctt tatcaatgcc 3720 tttaagaaag agcaccacgg cgtcaatctg accacgcgta ccgtactgga atcacagggc 3780 agtgcggagc tggcgaagaa gctcaagaat acgctgttgt ccctggacag tggtgaaagt 3840 atgagcttca gccggtcata tggcgggggc gtcagcactg tctttgtgcc tacccttagc 3900 aagaaggtgc cagttccggt gatccccgga gccggcatca cgctggatcg cgcctataac 3960 ctgagcttca gtcgtaccag cggcggattg aacgtcagtt ttggccgcga cggcggggtg 4020 agtggtaaca tcatggtcgc taccggccat gatgtgatgc cctatatgac cggtaagaaa 4080 accagtgcag gtaacgccag tgactggttg agcgcaaaac ataaaatcag cccggacttg 4140 cgtatcggcg ctgctgtgag tggcaccctg caaggaacgc tacaaaacag cctgaagttt 4200 aagctgacag aggatgagct gcctggcttt atccatggct tgacgcatgg cacgttgacc 4260 ccggcagaac tgttgcaaaa ggggatcgaa catcagatga agcagggcag caaactgacg 4320 tttagcgtcg atacctcggc aaatctggat ctgcgtgccg gtatcaatct gaacgaagac 4380 ggcagtaaac caaatggtgt cactgcccgt gtttctgccg ggctaagtgc atcggcaaac 4440 ctggccgccg gctcgcgtga acgcagcacc acctctggcc agtttggcag cacgacttcg 4500 gccagcaata accgcccaac cttcctcaac ggggtcggcg cgggtgctaa cctgacggct 4560 gctttagggg ttgcccattc atctacgcat gaagggaaac cggtcgggat cttcccggca 4620 tttacctcga ccaatgtttc ggcagcgctg gcgctggata accgtacctc acagagtatc 4680 agcctggaat tgaagcgcgc ggagccggtg accagcaacg atatcagcga gttgacctcc 4740 acgctgggaa aacactttaa ggatagcgcc acaacgaaga tgcttgccgc tctcaaagag 4800 ttagatgacg ctaagcccgc tgaacaactg catattttac agcagcattt cagtgcaaaa 4860 gatgtcgtcg gtgatgaacg ctacgaggcg gtgcgcaacc tgaaaaaact ggtgatacgt 4920 caacaggctg cggacagcca cagcatggaa ttaggatctg ccagtcacag cacgacctac 4980 aataatctgt cgagaataaa taatgacggc attgtcgagc tgctacacaa acatttcgat 5040 gcggcattac cagcaagcag tgccaaacgt cttggtgaaa tgatgaataa cgatccggca 5100 ctgaaagata ttattaagca gctgcaaagt acgccgttca gcagcgccag cgtgtcgatg 5160 gagctgaaag atggtctgcg tgagcagacg gaaaaagcaa tactggacgg taaggtcggt 5220 cgtgaagaag tgggagtact tttccaggat cgtaacaact tgcgtgttaa atcggtcagc 5280 gtcagtcagt ccgtcagcaa aagcgaaggc ttcaataccc cagcgctgtt actggggacg 5340 agcaacagcg ctgctatgag catggagcgc aacatcggaa ccattaattt taaatacggc 5400 caggatcaga acaccccacg gcgatttacc ctggagggtg gaatagctca ggctaatccg 5460 caggtcgcat ctgcgcttac tgatttgaag aaggaagggc tggaaatgaa gagctaa 5517 8 1838 PRT Erwinia amylovora 8 Met Glu Leu Lys Ser Leu Gly Thr Glu His Lys Ala Ala Val His Thr 1 5 10 15 Ala Ala His Asn Pro Val Gly His Gly Val Ala Leu Gln Gln Gly Ser 20 25 30 Ser Ser Ser Ser Pro Gln Asn Ala Ala Ala Ser Leu Ala Ala Glu Gly 35 40 45 Lys Asn Arg Gly Lys Met Pro Arg Ile His Gln Pro Ser Thr Ala Ala 50 55 60 Asp Gly Ile Ser Ala Ala His Gln Gln Lys Lys Ser Phe Ser Leu Arg 65 70 75 80 Gly Cys Leu Gly Thr Lys Lys Phe Ser Arg Ser Ala Pro Gln Gly Gln 85 90 95 Pro Gly Thr Thr His Ser Lys Gly Ala Thr Leu Arg Asp Leu Leu Ala 100 105 110 Arg Asp Asp Gly Glu Thr Gln His Glu Ala Ala Ala Pro Asp Ala Ala 115 120 125 Arg Leu Thr Arg Ser Gly Gly Val Lys Arg Arg Asn Met Asp Asp Met 130 135 140 Ala Gly Arg Pro Met Val Lys Gly Gly Ser Gly Glu Asp Lys Val Pro 145 150 155 160 Thr Gln Gln Lys Arg His Gln Leu Asn Asn Phe Gly Gln Met Arg Gln 165 170 175 Thr Met Leu Ser Lys Met Ala His Pro Ala Ser Ala Asn Ala Gly Asp 180 185 190 Arg Leu Gln His Ser Pro Pro His Ile Pro Gly Ser His His Glu Ile 195 200 205 Lys Glu Glu Pro Val Gly Ser Thr Ser Lys Ala Thr Thr Ala His Ala 210 215 220 Asp Arg Val Glu Ile Ala Gln Glu Asp Asp Asp Ser Glu Phe Gln Gln 225 230 235 240 Leu His Gln Gln Arg Leu Ala Arg Glu Arg Glu Asn Pro Pro Gln Pro 245 250 255 Pro Lys Leu Gly Val Ala Thr Pro Ile Ser Ala Arg Phe Gln Pro Lys 260 265 270 Leu Thr Ala Val Ala Glu Ser Val Leu Glu Gly Thr Asp Thr Thr Gln 275 280 285 Ser Pro Leu Lys Pro Gln Ser Met Leu Lys Gly Ser Gly Ala Gly Val 290 295 300 Thr Pro Leu Ala Val Thr Leu Asp Lys Gly Lys Leu Gln Leu Ala Pro 305 310 315 320 Asp Asn Pro Pro Ala Leu Asn Thr Leu Leu Lys Gln Thr Leu Gly Lys 325 330 335 Asp Thr Gln His Tyr Leu Ala His His Ala Ser Ser Asp Gly Ser Gln 340 345 350 His Leu Leu Leu Asp Asn Lys Gly His Leu Phe Asp Ile Lys Ser Thr 355 360 365 Ala Thr Ser Tyr Ser Val Leu His Asn Ser His Pro Gly Glu Ile Lys 370 375 380 Gly Lys Leu Ala Gln Ala Gly Thr Gly Ser Val Ser Val Asp Gly Lys 385 390 395 400 Ser Gly Lys Ile Ser Leu Gly Ser Gly Thr Gln Ser His Asn Lys Thr 405 410 415 Met Leu Ser Gln Pro Gly Glu Ala His Arg Ser Leu Leu Thr Gly Ile 420 425 430 Trp Gln His Pro Ala Gly Ala Ala Arg Pro Gln Gly Glu Ser Ile Arg 435 440 445 Leu His Asp Asp Lys Ile His Ile Leu His Pro Glu Leu Gly Val Trp 450 455 460 Gln Ser Ala Asp Lys Asp Thr His Ser Gln Leu Ser Arg Gln Ala Asp 465 470 475 480 Gly Lys Leu Tyr Ala Leu Lys Asp Asn Arg Thr Leu Gln Asn Leu Ser 485 490 495 Asp Asn Lys Ser Ser Glu Lys Leu Val Asp Lys Ile Lys Ser Tyr Ser 500 505 510 Val Asp Gln Arg Gly Gln Val Ala Ile Leu Thr Asp Thr Pro Gly Arg 515 520 525 His Lys Met Ser Ile Met Pro Ser Leu Asp Ala Ser Pro Glu Ser His 530 535 540 Ile Ser Leu Ser Leu His Phe Ala Asp Ala His Gln Gly Leu Leu His 545 550 555 560 Gly Lys Ser Glu Leu Glu Ala Gln Ser Val Ala Ile Ser His Gly Arg 565 570 575 Leu Val Val Ala Asp Ser Glu Gly Lys Leu Phe Ser Ala Ala Ile Pro 580 585 590 Lys Gln Gly Asp Gly Asn Glu Leu Lys Met Lys Ala Met Pro Gln His 595 600 605 Ala Leu Asp Glu His Phe Gly His Asp His Gln Ile Ser Gly Phe Phe 610 615 620 His Asp Asp His Gly Gln Leu Asn Ala Leu Val Lys Asn Asn Phe Arg 625 630 635 640 Gln Gln His Ala Cys Pro Leu Gly Asn Asp His Gln Phe His Pro Gly 645 650 655 Trp Asn Leu Thr Asp Ala Leu Val Ile Asp Asn Gln Leu Gly Leu His 660 665 670 His Thr Asn Pro Glu Pro His Glu Ile Leu Asp Met Gly His Leu Gly 675 680 685 Ser Leu Ala Leu Gln Glu Gly Lys Leu His Tyr Phe Asp Gln Leu Thr 690 695 700 Lys Gly Trp Thr Gly Ala Glu Ser Asp Cys Lys Gln Leu Lys Lys Gly 705 710 715 720 Leu Asp Gly Ala Ala Tyr Leu Leu Lys Asp Gly Glu Val Lys Arg Leu 725 730 735 Asn Ile Asn Gln Ser Thr Ser Ser Ile Lys His Gly Thr Glu Asn Val 740 745 750 Phe Ser Leu Pro His Val Arg Asn Lys Pro Glu Pro Gly Asp Ala Leu 755 760 765 Gln Gly Leu Asn Lys Asp Asp Lys Ala Gln Ala Met Ala Val Ile Gly 770 775 780 Val Asn Lys Tyr Leu Ala Leu Thr Glu Lys Gly Asp Ile Arg Ser Phe 785 790 795 800 Gln Ile Lys Pro Gly Thr Gln Gln Leu Glu Arg Pro Ala Gln Thr Leu 805 810 815 Ser Arg Glu Gly Ile Ser Gly Glu Leu Lys Asp Ile His Val Asp His 820 825 830 Lys Gln Asn Leu Tyr Ala Leu Thr His Glu Gly Glu Val Phe His Gln 835 840 845 Pro Arg Glu Ala Trp Gln Asn Gly Ala Glu Ser Ser Ser Trp His Lys 850 855 860 Leu Ala Leu Pro Gln Ser Glu Ser Lys Leu Lys Ser Leu Asp Met Ser 865 870 875 880 His Glu His Lys Pro Ile Ala Thr Phe Glu Asp Gly Ser Gln His Gln 885 890 895 Leu Lys Ala Gly Gly Trp His Ala Tyr Ala Ala Pro Glu Arg Gly Pro 900 905 910 Leu Ala Val Gly Thr Ser Gly Ser Gln Thr Val Phe Asn Arg Leu Met 915 920 925 Gln Gly Val Lys Gly Lys Val Ile Pro Gly Ser Gly Leu Thr Val Lys 930 935 940 Leu Ser Ala Gln Thr Gly Gly Met Thr Gly Ala Glu Gly Arg Lys Val 945 950 955 960 Ser Ser Lys Phe Ser Glu Arg Ile Arg Ala Tyr Ala Phe Asn Pro Thr 965 970 975 Met Ser Thr Pro Arg Pro Ile Lys Asn Ala Ala Tyr Ala Thr Gln His 980 985 990 Gly Trp Gln Gly Arg Glu Gly Leu Lys Pro Leu Tyr Glu Met Gln Gly 995 1000 1005 Ala Leu Ile Lys Gln Leu Asp Ala His Asn Val Arg His Asn Ala Pro 1010 1015 1020 Gln Pro Asp Leu Gln Ser Lys Leu Glu Thr Leu Asp Leu Gly Glu His 1025 1030 1035 1040 Gly Ala Glu Leu Leu Asn Asp Met Lys Arg Phe Arg Asp Glu Leu Glu 1045 1050 1055 Gln Ser Ala Thr Arg Ser Val Thr Val Leu Gly Gln His Gln Gly Val 1060 1065 1070 Leu Lys Ser Asn Gly Glu Ile Asn Ser Glu Phe Lys Pro Ser Pro Gly 1075 1080 1085 Lys Ala Leu Val Gln Ser Phe Asn Val Asn Arg Ser Gly Gln Asp Leu 1090 1095 1100 Ser Lys Ser Leu Gln Gln Ala Val His Ala Thr Pro Pro Ser Ala Glu 1105 1110 1115 1120 Ser Lys Leu Gln Ser Met Leu Gly His Phe Val Ser Ala Gly Val Asp 1125 1130 1135 Met Ser His Gln Lys Gly Glu Ile Pro Leu Gly Arg Gln Arg Asp Pro 1140 1145 1150 Asn Asp Lys Thr Ala Leu Thr Lys Ser Arg Leu Ile Leu Asp Thr Val 1155 1160 1165 Thr Ile Gly Glu Leu His Glu Leu Ala Asp Lys Ala Lys Leu Val Ser 1170 1175 1180 Asp His Lys Pro Asp Ala Asp Gln Ile Lys Gln Leu Arg Gln Gln Phe 1185 1190 1195 1200 Asp Thr Leu Arg Glu Lys Arg Tyr Glu Ser Asn Pro Val Lys His Tyr 1205 1210 1215 Thr Asp Met Gly Phe Thr His Asn Lys Ala Leu Glu Ala Asn Tyr Asp 1220 1225 1230 Ala Val Lys Ala Phe Ile Asn Ala Phe Lys Lys Glu His His Gly Val 1235 1240 1245 Asn Leu Thr Thr Arg Thr Val Leu Glu Ser Gln Gly Ser Ala Glu Leu 1250 1255 1260 Ala Lys Lys Leu Lys Asn Thr Leu Leu Ser Leu Asp Ser Gly Glu Ser 1265 1270 1275 1280 Met Ser Phe Ser Arg Ser Tyr Gly Gly Gly Val Ser Thr Val Phe Val 1285 1290 1295 Pro Thr Leu Ser Lys Lys Val Pro Val Pro Val Ile Pro Gly Ala Gly 1300 1305 1310 Ile Thr Leu Asp Arg Ala Tyr Asn Leu Ser Phe Ser Arg Thr Ser Gly 1315 1320 1325 Gly Leu Asn Val Ser Phe Gly Arg Asp Gly Gly Val Ser Gly Asn Ile 1330 1335 1340 Met Val Ala Thr Gly His Asp Val Met Pro Tyr Met Thr Gly Lys Lys 1345 1350 1355 1360 Thr Ser Ala Gly Asn Ala Ser Asp Trp Leu Ser Ala Lys His Lys Ile 1365 1370 1375 Ser Pro Asp Leu Arg Ile Gly Ala Ala Val Ser Gly Thr Leu Gln Gly 1380 1385 1390 Thr Leu Gln Asn Ser Leu Lys Phe Lys Leu Thr Glu Asp Glu Leu Pro 1395 1400 1405 Gly Phe Ile His Gly Leu Thr His Gly Thr Leu Thr Pro Ala Glu Leu 1410 1415 1420 Leu Gln Lys Gly Ile Glu His Gln Met Lys Gln Gly Ser Lys Leu Thr 1425 1430 1435 1440 Phe Ser Val Asp Thr Ser Ala Asn Leu Asp Leu Arg Ala Gly Ile Asn 1445 1450 1455 Leu Asn Glu Asp Gly Ser Lys Pro Asn Gly Val Thr Ala Arg Val Ser 1460 1465 1470 Ala Gly Leu Ser Ala Ser Ala Asn Leu Ala Ala Gly Ser Arg Glu Arg 1475 1480 1485 Ser Thr Thr Ser Gly Gln Phe Gly Ser Thr Thr Ser Ala Ser Asn Asn 1490 1495 1500 Arg Pro Thr Phe Leu Asn Gly Val Gly Ala Gly Ala Asn Leu Thr Ala 1505 1510 1515 1520 Ala Leu Gly Val Ala His Ser Ser Thr His Glu Gly Lys Pro Val Gly 1525 1530 1535 Ile Phe Pro Ala Phe Thr Ser Thr Asn Val Ser Ala Ala Leu Ala Leu 1540 1545 1550 Asp Asn Arg Thr Ser Gln Ser Ile Ser Leu Glu Leu Lys Arg Ala Glu 1555 1560 1565 Pro Val Thr Ser Asn Asp Ile Ser Glu Leu Thr Ser Thr Leu Gly Lys 1570 1575 1580 His Phe Lys Asp Ser Ala Thr Thr Lys Met Leu Ala Ala Leu Lys Glu 1585 1590 1595 1600 Leu Asp Asp Ala Lys Pro Ala Glu Gln Leu His Ile Leu Gln Gln His 1605 1610 1615 Phe Ser Ala Lys Asp Val Val Gly Asp Glu Arg Tyr Glu Ala Val Arg 1620 1625 1630 Asn Leu Lys Lys Leu Val Ile Arg Gln Gln Ala Ala Asp Ser His Ser 1635 1640 1645 Met Glu Leu Gly Ser Ala Ser His Ser Thr Thr Tyr Asn Asn Leu Ser 1650 1655 1660 Arg Ile Asn Asn Asp Gly Ile Val Glu Leu Leu His Lys His Phe Asp 1665 1670 1675 1680 Ala Ala Leu Pro Ala Ser Ser Ala Lys Arg Leu Gly Glu Met Met Asn 1685 1690 1695 Asn Asp Pro Ala Leu Lys Asp Ile Ile Lys Gln Leu Gln Ser Thr Pro 1700 1705 1710 Phe Ser Ser Ala Ser Val Ser Met Glu Leu Lys Asp Gly Leu Arg Glu 1715 1720 1725 Gln Thr Glu Lys Ala Ile Leu Asp Gly Lys Val Gly Arg Glu Glu Val 1730 1735 1740 Gly Val Leu Phe Gln Asp Arg Asn Asn Leu Arg Val Lys Ser Val Ser 1745 1750 1755 1760 Val Ser Gln Ser Val Ser Lys Ser Glu Gly Phe Asn Thr Pro Ala Leu 1765 1770 1775 Leu Leu Gly Thr Ser Asn Ser Ala Ala Met Ser Met Glu Arg Asn Ile 1780 1785 1790 Gly Thr Ile Asn Phe Lys Tyr Gly Gln Asp Gln Asn Thr Pro Arg Arg 1795 1800 1805 Phe Thr Leu Glu Gly Gly Ile Ala Gln Ala Asn Pro Gln Val Ala Ser 1810 1815 1820 Ala Leu Thr Asp Leu Lys Lys Glu Gly Leu Glu Met Lys Ser 1825 1830 1835 9 420 DNA Erwinia amylovora 9 atgacatcgt cacagcagcg ggttgaaagg tttttacagt atttctccgc cgggtgtaaa 60 acgcccatac atctgaaaga cggggtgtgc gccctgtata acgaacaaga tgaggaggcg 120 gcggtgctgg aagtaccgca acacagcgac agcctgttac tacactgccg aatcattgag 180 gctgacccac aaacttcaat aaccctgtat tcgatgctat tacagctgaa ttttgaaatg 240 gcggccatgc gcggctgttg gctggcgctg gatgaactgc acaacgtgcg tttatgtttt 300 cagcagtcgc tggagcatct ggatgaagca agttttagcg atatcgttag cggcttcatc 360 gaacatgcgg cagaagtgcg tgagtatata gcgcaattag acgagagtag cgcggcataa 420 10 139 PRT Erwinia amylovora 10 Met Thr Ser Ser Gln Gln Arg Val Glu Arg Phe Leu Gln Tyr Phe Ser 1 5 10 15 Ala Gly Cys Lys Thr Pro Ile His Leu Lys Asp Gly Val Cys Ala Leu 20 25 30 Tyr Asn Glu Gln Asp Glu Glu Ala Ala Val Leu Glu Val Pro Gln His 35 40 45 Ser Asp Ser Leu Leu Leu His Cys Arg Ile Ile Glu Ala Asp Pro Gln 50 55 60 Thr Ser Ile Thr Leu Tyr Ser Met Leu Leu Gln Leu Asn Phe Glu Met 65 70 75 80 Ala Ala Met Arg Gly Cys Trp Leu Ala Leu Asp Glu Leu His Asn Val 85 90 95 Arg Leu Cys Phe Gln Gln Ser Leu Glu His Leu Asp Glu Ala Ser Phe 100 105 110 Ser Asp Ile Val Ser Gly Phe Ile Glu His Ala Ala Glu Val Arg Glu 115 120 125 Tyr Ile Ala Gln Leu Asp Glu Ser Ser Ala Ala 130 135 11 341 PRT Pseudomonas syringae 11 Met Gln Ser Leu Ser Leu Asn Ser Ser Ser Leu Gln Thr Pro Ala Met 1 5 10 15 Ala Leu Val Leu Val Arg Pro Glu Ala Glu Thr Thr Gly Ser Thr Ser 20 25 30 Ser Lys Ala Leu Gln Glu Val Val Val Lys Leu Ala Glu Glu Leu Met 35 40 45 Arg Asn Gly Gln Leu Asp Asp Ser Ser Pro Leu Gly Lys Leu Leu Ala 50 55 60 Lys Ser Met Ala Ala Asp Gly Lys Ala Gly Gly Gly Ile Glu Asp Val 65 70 75 80 Ile Ala Ala Leu Asp Lys Leu Ile His Glu Lys Leu Gly Asp Asn Phe 85 90 95 Gly Ala Ser Ala Asp Ser Ala Ser Gly Thr Gly Gln Gln Asp Leu Met 100 105 110 Thr Gln Val Leu Asn Gly Leu Ala Lys Ser Met Leu Asp Asp Leu Leu 115 120 125 Thr Lys Gln Asp Gly Gly Thr Ser Phe Ser Glu Asp Asp Met Pro Met 130 135 140 Leu Asn Lys Ile Ala Gln Phe Met Asp Asp Asn Pro Ala Gln Phe Pro 145 150 155 160 Lys Pro Asp Ser Gly Ser Trp Val Asn Glu Leu Lys Glu Asp Asn Phe 165 170 175 Leu Asp Gly Asp Glu Thr Ala Ala Phe Arg Ser Ala Leu Asp Ile Ile 180 185 190 Gly Gln Gln Leu Gly Asn Gln Gln Ser Asp Ala Gly Ser Leu Ala Gly 195 200 205 Thr Gly Gly Gly Leu Gly Thr Pro Ser Ser Phe Ser Asn Asn Ser Ser 210 215 220 Val Met Gly Asp Pro Leu Ile Asp Ala Asn Thr Gly Pro Gly Asp Ser 225 230 235 240 Gly Asn Thr Arg Gly Glu Ala Gly Gln Leu Ile Gly Glu Leu Ile Asp 245 250 255 Arg Gly Leu Gln Ser Val Leu Ala Gly Gly Gly Leu Gly Thr Pro Val 260 265 270 Asn Thr Pro Gln Thr Gly Thr Ser Ala Asn Gly Gly Gln Ser Ala Gln 275 280 285 Asp Leu Asp Gln Leu Leu Gly Gly Leu Leu Leu Lys Gly Leu Glu Ala 290 295 300 Thr Leu Lys Asp Ala Gly Gln Thr Gly Thr Asp Val Gln Ser Ser Ala 305 310 315 320 Ala Gln Ile Ala Thr Leu Leu Val Ser Thr Leu Leu Gln Gly Thr Arg 325 330 335 Asn Gln Ala Ala Ala 340 12 1026 DNA Pseudomonas syringae 12 atgcagagtc tcagtcttaa cagcagctcg ctgcaaaccc cggcaatggc ccttgtcctg 60 gtacgtcctg aagccgagac gactggcagt acgtcgagca aggcgcttca ggaagttgtc 120 gtgaagctgg ccgaggaact gatgcgcaat ggtcaactcg acgacagctc gccattggga 180 aaactgttgg ccaagtcgat ggccgcagat ggcaaggcgg gcggcggtat tgaggatgtc 240 atcgctgcgc tggacaagct gatccatgaa aagctcggtg acaacttcgg cgcgtctgcg 300 gacagcgcct cgggtaccgg acagcaggac ctgatgactc aggtgctcaa tggcctggcc 360 aagtcgatgc tcgatgatct tctgaccaag caggatggcg ggacaagctt ctccgaagac 420 gatatgccga tgctgaacaa gatcgcgcag ttcatggatg acaatcccgc acagtttccc 480 aagccggact cgggctcctg ggtgaacgaa ctcaaggaag acaacttcct tgatggcgac 540 gaaacggctg cgttccgttc ggcactcgac atcattggcc agcaactggg taatcagcag 600 agtgacgctg gcagtctggc agggacgggt ggaggtctgg gcactccgag cagtttttcc 660 aacaactcgt ccgtgatggg tgatccgctg atcgacgcca ataccggtcc cggtgacagc 720 ggcaataccc gtggtgaagc ggggcaactg atcggcgagc ttatcgaccg tggcctgcaa 780 tcggtattgg ccggtggtgg actgggcaca cccgtaaaca ccccgcagac cggtacgtcg 840 gcgaatggcg gacagtccgc tcaggatctt gatcagttgc tgggcggctt gctgctcaag 900 ggcctggagg caacgctcaa ggatgccggg caaacaggca ccgacgtgca gtcgagcgct 960 gcgcaaatcg ccaccttgct ggtcagtacg ctgctgcaag gcacccgcaa tcaggctgca 1020 gcctga 1026 13 1729 DNA Pseudomonas syringae 13 tccacttcgc tgattttgaa attggcagat tcatagaaac gttcaggtgt ggaaatcagg 60 ctgagtgcgc agatttcgtt gataagggtg tggtactggt cattgttggt catttcaagg 120 cctctgagtg cggtgcggag caataccagt cttcctgctg gcgtgtgcac actgagtcgc 180 aggcataggc atttcagttc cttgcgttgg ttgggcatat aaaaaaagga acttttaaaa 240 acagtgcaat gagatgccgg caaaacggga accggtcgct gcgctttgcc actcacttcg 300 agcaagctca accccaaaca tccacatccc tatcgaacgg acagcgatac ggccacttgc 360 tctggtaaac cctggagctg gcgtcggtcc aattgcccac ttagcgaggt aacgcagcat 420 gagcatcggc atcacacccc ggccgcaaca gaccaccacg ccactcgatt tttcggcgct 480 aagcggcaag agtcctcaac caaacacgtt cggcgagcag aacactcagc aagcgatcga 540 cccgagtgca ctgttgttcg gcagcgacac acagaaagac gtcaacttcg gcacgcccga 600 cagcaccgtc cagaatccgc aggacgccag caagcccaac gacagccagt ccaacatcgc 660 taaattgatc agtgcattga tcatgtcgtt gctgcagatg ctcaccaact ccaataaaaa 720 gcaggacacc aatcaggaac agcctgatag ccaggctcct ttccagaaca acggcgggct 780 cggtacaccg tcggccgata gcgggggcgg cggtacaccg gatgcgacag gtggcggcgg 840 cggtgatacg ccaagcgcaa caggcggtgg cggcggtgat actccgaccg caacaggcgg 900 tggcggcagc ggtggcggcg gcacacccac tgcaacaggt ggcggcagcg gtggcacacc 960 cactgcaaca ggcggtggcg agggtggcgt aacaccgcaa atcactccgc agttggccaa 1020 ccctaaccgt acctcaggta ctggctcggt gtcggacacc gcaggttcta ccgagcaagc 1080 cggcaagatc aatgtggtga aagacaccat caaggtcggc gctggcgaag tctttgacgg 1140 ccacggcgca accttcactg ccgacaaatc tatgggtaac ggagaccagg gcgaaaatca 1200 gaagcccatg ttcgagctgg ctgaaggcgc tacgttgaag aatgtgaacc tgggtgagaa 1260 cgaggtcgat ggcatccacg tgaaagccaa aaacgctcag gaagtcacca ttgacaacgt 1320 gcatgcccag aacgtcggtg aagacctgat tacggtcaaa ggcgagggag gcgcagcggt 1380 cactaatctg aacatcaaga acagcagtgc caaaggtgca gacgacaagg ttgtccagct 1440 caacgccaac actcacttga aaatcgacaa cttcaaggcc gacgatttcg gcacgatggt 1500 tcgcaccaac ggtggcaagc agtttgatga catgagcatc gagctgaacg gcatcgaagc 1560 taaccacggc aagttcgccc tggtgaaaag cgacagtgac gatctgaagc tggcaacggg 1620 caacatcgcc atgaccgacg tcaaacacgc ctacgataaa acccaggcat cgacccaaca 1680 caccgagctt tgaatccaga caagtagctt gaaaaaaggg ggtggactc 1729 14 424 PRT Pseudomonas syringae 14 Met Ser Ile Gly Ile Thr Pro Arg Pro Gln Gln Thr Thr Thr Pro Leu 1 5 10 15 Asp Phe Ser Ala Leu Ser Gly Lys Ser Pro Gln Pro Asn Thr Phe Gly 20 25 30 Glu Gln Asn Thr Gln Gln Ala Ile Asp Pro Ser Ala Leu Leu Phe Gly 35 40 45 Ser Asp Thr Gln Lys Asp Val Asn Phe Gly Thr Pro Asp Ser Thr Val 50 55 60 Gln Asn Pro Gln Asp Ala Ser Lys Pro Asn Asp Ser Gln Ser Asn Ile 65 70 75 80 Ala Lys Leu Ile Ser Ala Leu Ile Met Ser Leu Leu Gln Met Leu Thr 85 90 95 Asn Ser Asn Lys Lys Gln Asp Thr Asn Gln Glu Gln Pro Asp Ser Gln 100 105 110 Ala Pro Phe Gln Asn Asn Gly Gly Leu Gly Thr Pro Ser Ala Asp Ser 115 120 125 Gly Gly Gly Gly Thr Pro Asp Ala Thr Gly Gly Gly Gly Gly Asp Thr 130 135 140 Pro Ser Ala Thr Gly Gly Gly Gly Gly Asp Thr Pro Thr Ala Thr Gly 145 150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Thr Pro Thr Ala Thr Gly Gly Gly 165 170 175 Ser Gly Gly Thr Pro Thr Ala Thr Gly Gly Gly Glu Gly Gly Val Thr 180 185 190 Pro Gln Ile Thr Pro Gln Leu Ala Asn Pro Asn Arg Thr Ser Gly Thr 195 200 205 Gly Ser Val Ser Asp Thr Ala Gly Ser Thr Glu Gln Ala Gly Lys Ile 210 215 220 Asn Val Val Lys Asp Thr Ile Lys Val Gly Ala Gly Glu Val Phe Asp 225 230 235 240 Gly His Gly Ala Thr Phe Thr Ala Asp Lys Ser Met Gly Asn Gly Asp 245 250 255 Gln Gly Glu Asn Gln Lys Pro Met Phe Glu Leu Ala Glu Gly Ala Thr 260 265 270 Leu Lys Asn Val Asn Leu Gly Glu Asn Glu Val Asp Gly Ile His Val 275 280 285 Lys Ala Lys Asn Ala Gln Glu Val Thr Ile Asp Asn Val His Ala Gln 290 295 300 Asn Val Gly Glu Asp Leu Ile Thr Val Lys Gly Glu Gly Gly Ala Ala 305 310 315 320 Val Thr Asn Leu Asn Ile Lys Asn Ser Ser Ala Lys Gly Ala Asp Asp 325 330 335 Lys Val Val Gln Leu Asn Ala Asn Thr His Leu Lys Ile Asp Asn Phe 340 345 350 Lys Ala Asp Asp Phe Gly Thr Met Val Arg Thr Asn Gly Gly Lys Gln 355 360 365 Phe Asp Asp Met Ser Ile Glu Leu Asn Gly Ile Glu Ala Asn His Gly 370 375 380 Lys Phe Ala Leu Val Lys Ser Asp Ser Asp Asp Leu Lys Leu Ala Thr 385 390 395 400 Gly Asn Ile Ala Met Thr Asp Val Lys His Ala Tyr Asp Lys Thr Gln 405 410 415 Ala Ser Thr Gln His Thr Glu Leu 420 15 344 PRT Pseudomonas solanacearum 15 Met Ser Val Gly Asn Ile Gln Ser Pro Ser Asn Leu Pro Gly Leu Gln 1 5 10 15 Asn Leu Asn Leu Asn Thr Asn Thr Asn Ser Gln Gln Ser Gly Gln Ser 20 25 30 Val Gln Asp Leu Ile Lys Gln Val Glu Lys Asp Ile Leu Asn Ile Ile 35 40 45 Ala Ala Leu Val Gln Lys Ala Ala Gln Ser Ala Gly Gly Asn Thr Gly 50 55 60 Asn Thr Gly Asn Ala Pro Ala Lys Asp Gly Asn Ala Asn Ala Gly Ala 65 70 75 80 Asn Asp Pro Ser Lys Asn Asp Pro Ser Lys Ser Gln Ala Pro Gln Ser 85 90 95 Ala Asn Lys Thr Gly Asn Val Asp Asp Ala Asn Asn Gln Asp Pro Met 100 105 110 Gln Ala Leu Met Gln Leu Leu Glu Asp Leu Val Lys Leu Leu Lys Ala 115 120 125 Ala Leu His Met Gln Gln Pro Gly Gly Asn Asp Lys Gly Asn Gly Val 130 135 140 Gly Gly Ala Asn Gly Ala Lys Gly Ala Gly Gly Gln Gly Gly Leu Ala 145 150 155 160 Glu Ala Leu Gln Glu Ile Glu Gln Ile Leu Ala Gln Leu Gly Gly Gly 165 170 175 Gly Ala Gly Ala Gly Gly Ala Gly Gly Gly Val Gly Gly Ala Gly Gly 180 185 190 Ala Asp Gly Gly Ser Gly Ala Gly Gly Ala Gly Gly Ala Asn Gly Ala 195 200 205 Asp Gly Gly Asn Gly Val Asn Gly Asn Gln Ala Asn Gly Pro Gln Asn 210 215 220 Ala Gly Asp Val Asn Gly Ala Asn Gly Ala Asp Asp Gly Ser Glu Asp 225 230 235 240 Gln Gly Gly Leu Thr Gly Val Leu Gln Lys Leu Met Lys Ile Leu Asn 245 250 255 Ala Leu Val Gln Met Met Gln Gln Gly Gly Leu Gly Gly Gly Asn Gln 260 265 270 Ala Gln Gly Gly Ser Lys Gly Ala Gly Asn Ala Ser Pro Ala Ser Gly 275 280 285 Ala Asn Pro Gly Ala Asn Gln Pro Gly Ser Ala Asp Asp Gln Ser Ser 290 295 300 Gly Gln Asn Asn Leu Gln Ser Gln Ile Met Asp Val Val Lys Glu Val 305 310 315 320 Val Gln Ile Leu Gln Gln Met Leu Ala Ala Gln Asn Gly Gly Ser Gln 325 330 335 Gln Ser Thr Ser Thr Gln Pro Met 340 16 1035 DNA Pseudomonas solanacearum 16 atgtcagtcg gaaacatcca gagcccgtcg aacctcccgg gtctgcagaa cctgaacctc 60 aacaccaaca ccaacagcca gcaatcgggc cagtccgtgc aagacctgat caagcaggtc 120 gagaaggaca tcctcaacat catcgcagcc ctcgtgcaga aggccgcaca gtcggcgggc 180 ggcaacaccg gtaacaccgg caacgcgccg gcgaaggacg gcaatgccaa cgcgggcgcc 240 aacgacccga gcaagaacga cccgagcaag agccaggctc cgcagtcggc caacaagacc 300 ggcaacgtcg acgacgccaa caaccaggat ccgatgcaag cgctgatgca gctgctggaa 360 gacctggtga agctgctgaa ggcggccctg cacatgcagc agcccggcgg caatgacaag 420 ggcaacggcg tgggcggtgc caacggcgcc aagggtgccg gcggccaggg cggcctggcc 480 gaagcgctgc aggagatcga gcagatcctc gcccagctcg gcggcggcgg tgctggcgcc 540 ggcggcgcgg gtggcggtgt cggcggtgct ggtggcgcgg atggcggctc cggtgcgggt 600 ggcgcaggcg gtgcgaacgg cgccgacggc ggcaatggcg tgaacggcaa ccaggcgaac 660 ggcccgcaga acgcaggcga tgtcaacggt gccaacggcg cggatgacgg cagcgaagac 720 cagggcggcc tcaccggcgt gctgcaaaag ctgatgaaga tcctgaacgc gctggtgcag 780 atgatgcagc aaggcggcct cggcggcggc aaccaggcgc agggcggctc gaagggtgcc 840 ggcaacgcct cgccggcttc cggcgcgaac ccgggcgcga accagcccgg ttcggcggat 900 gatcaatcgt ccggccagaa caatctgcaa tcccagatca tggatgtggt gaaggaggtc 960 gtccagatcc tgcagcagat gctggcggcg cagaacggcg gcagccagca gtccacctcg 1020 acgcagccga tgtaa 1035 17 26 PRT Xanthomonas campestris pv. glycines 17 Thr Leu Ile Glu Leu Met Ile Val Val Ala Ile Ile Ala Ile Leu Ala 1 5 10 15 Ala Ile Ala Leu Pro Ala Tyr Gln Asp Tyr 20 25 18 20 PRT Xanthomonas campestris pv. pelargonii 18 Ser Ser Gln Gln Ser Pro Ser Ala Gly Ser Glu Gln Gln Leu Asp Gln 1 5 10 15 Leu Leu Ala Met 20

Claims (14)

What is claimed:
1. A method of imparting stress resistance to plants comprising:
providing a transgenic plant or plant seed transformed with a DNA molecule which encodes for a hypersensitive response elicitor protein or polypeptide and
growing the transgenic plant or plants produced from the transgenic plant seeds under conditions effective to impart stress resistance.
2. A method according to claim 1, wherein a transgenic plant is provided.
3. A method according to claim 1, wherein a transgenic plant seed is provided, said method further comprising:
planting the transgenic seeds in natural or artificial soil and propagating plants from seeds planted in soil.
4. A method according to claim 1, wherein the stress resistance is resistance to a stress selected from the group consisting of climated related stress, air pollution stress, chemical stress, and nutritional stress.
5. A method according to claim 4, wherein the stress is chemical stress where the chemical is selected from the group consisting of insecticides, fungicides, herbicides, and heavy metals.
6. A method according to claim 4, wherein the stress is climate-related stress selected from the group consisting of drought, water, frost, cold temperature, high temperature, excessive light, and insufficient light.
7. A method according to claim 4, wherein the stress is air pollution stress selected from the group consisting of carbon dioxide, carbon monoxide, sulfur dioxide, NOx, hydrocarbons, ozone, ultraviolet radiation, and acidic rain.
8. A method according to claim 4, wherein the stress is nutritional stress where the nutritional stress is caused by fertilizer, micronutrients, or macronutrients.
9. A method according to claim 4, wherein the hypersensitive response elicitor protein or polypeptide is derived from Erwinia, Pseudomonas, Xanthamonas, Phythophthera, or Clavibacter.
10. A method according to claim 9, wherein the hypersensitive response elicitor protein or polypeptide is derived from Erwinia amylovora, Erwinia carotovora, Erwinia chrysanthemi, and Erwinia stewartii.
11. A method according to claim 9, wherein the hypersensitive response elicitor protein or polypeptide is derived from Pseudomonas syringae or Pseudomonas solancearum.
12. A method according to claim 9, wherein the hypersensitive response elicitor protein or polypeptide is derived from a Xanthamonas species.
13. A method according to claim 4, wherein the plant is selected from the group consisting of alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean pea, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane.
14. A method according to claim 4, wherein the plant is selected from the group consisting of Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.
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