Hypersensitive response elicitor-induced stress resistance

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




FIELD OF THE INVENTION




The present invention relates to imparting stress resistance to plants with a hypersensitive response elicitor.




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.




Under riatural 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


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.




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.




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.




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.




Air pollution in the form of ozone, carbon dioxide, carbon monoxide, sulfur dioxide, NO


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.




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.




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 polllution 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). 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 licitor 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.




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:


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


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 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


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














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











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 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


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 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











Another potentially suitable hypersensitive response elicitor from


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 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











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:













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.




Another potentially suitable hypersensitive response elicitor from


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 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











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               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                1595













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











This protein or polypeptide is about 198 kDa and has a pI of 8.98.




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 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











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:













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











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 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











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. Further information about the hypersensitive response elicitor derived from


Pseudomonas syringae


is found in He, S. Y., H. C. Huang, and A. Collmer, “


Pseudomonas syringae


pv. syringae Harpin


Pss


: 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 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











Another potentially suitable hypersensitive response elicitor from


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 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











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 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                 111













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











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 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:














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











Further information regarding the hypersensitive response elicitor polypeptide or protein derived from


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


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 Ala Ile Leu Ala







1               5                   10                  15













Ala Ile Ala Leu Pro Ala Tyr Gln Asp Tyr






            20                  25











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 Gln Leu Asp Gln







1               5                   10                  15













Leu Leu Ala Met






            20











Isolation of


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 N


Ecc


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,” 8


th 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


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


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.




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. 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.




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.




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.




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).




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.




Another example of suitable fragments of a hypersensitive response elicitor which do elicit a hypersensitive response are


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


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.




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.,


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.




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. W. Studier et. al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,”


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.




Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation).




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 promoters. Furthermore, eucaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eucaryotic cells.




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-Dalgarno (“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,


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 promotors may be used. For instance, when cloning in


E. Coli


, its bacteriophages, or plasmids, promoters such as the T7 phage promotor, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the P


R


and P


L


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.




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


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.




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.




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.




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.




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:


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.




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.




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. In this embodiment, the composition contains greater than 500 nM of the elicitor.




Although not required, this composition may contain additional additives including fertilizer, insecticide, fungicide, nematacide, and mixtures thereof. Suitable fertilizers include (NH


4


)


2


NO


3


. 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.




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




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. 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.




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.,


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.,


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.




After transformation, the transformed plant cells must be regenerated.




Plant rege neration from cultured protoplasts is described in Evans et al.,


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. I, 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.




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.




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.




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.




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.).











EXAMPLES




Example 1




Hypersensitive Response Elicitor-treated Cotton is More Resistant to the Damage Caused by Insecticide Stress




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. 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-100™) 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




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).












TABLE 1











Aphid count per leaf on cotton after treatment with Asana XL ® or






HP-1000 ™













Number of aphids per leaf


1









No. sprays applied/days after treatment
















Treatment




Rate


2






1/14 DAT 1




2/28 DAT 1




3/35 DAT 1




4/42 DAT 1









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













1


Means followed by different letters are significantly different according to Duncan's MRT, P = 0.05.












2


Rate for Asana XL ® is for formulated product, rate for HP-1000 ™ is for active ingredient (a.i.).













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.




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.












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




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 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.




Result




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.




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.




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).












TABLE 3











Yield Increase of Cucumber Fruit from Hypersensitive






Response Elicitor Treated Plants


















Yield/




% of the Yield






Treatment




Replicate




kg/plant




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%.




Example 3




Hypersensitive Response Elicitor-treated Pepper is More Tolerant to Herbicide Stress




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.




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.












TABLE 4











Hypersensitive Response Elicitor-Treated Peppers






are More Tolerant to Herbicide Damage.














Damage Rating



















Treatment




1




2




3




4




5




6




Damage Index %





















Hypersensitive




1




38




17




3




1




0




41






Response Elicitor






Chemicals




0




1




6




16




19




18




87











Damage Rating: 1. No damage; 2. 0-20% leaves damaged; 3. 20-40% leaves damaged; 4. 40-50% leaves damaged; 6. More than 75% leaves damaged or entire plant dead.










Damage index = sum of each rating times the number of plants under the rating scale, divided by total number of plants times 6.










Damage index for hypersensitive response elicitor-treated plants = 1 × 1 + 2 × 38 + 3 × 17 + 4 × 3 + 5 × 1 + 6 × 0 × 100% = 41%













Example 4




Hypersensitive Response Elicitor-treated Pepper is More Tolerant to Herbicide Stress under Controlled Experimental Conditions




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:




1. Control, the peppers were neither treated by a hypersensitive response (“HR”) elicitor nor by LEXONE™ herbicide (DuPont Agricultural Products, Wilmington, Del.).




2. Control pepper with application of 0.15 pound LEXONE™ herbicide/acre.




3. Control pepper with application of 0.3 pound LEXONE™ herbicide/acre.




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.




5. HR elicitor treatment with application of 0.15 pound LEXONE™ herbicide/acre.




6. HR elicitor treatment with application of 0.3 pound LEXONE™ herbicide/acre.




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.




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.




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 firther confirrn that HR elicitors can help crops withstand the phytotoxic effects of herbicides and are very beneficial to the agricultural industry.












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



























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 harvested







Treatment




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




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.




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.




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.












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











*Same letter indicates no statistical difference between the two treatments at the defined level; different letter indicates a statistical difference between the two treatments at the defined level.





















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. #





Avg. #








bolls in





of total







the #1 & 2




Percent




bolls per




Percent






Treatment




position




difference




5 plant




difference









MESSENGER ™ 2.2




18.4 a




+46.0%




21.4 a




+57.0%






OZ.






Check




12.6 b





13.6 b











Statistically significant




P = 0.032





P = 0.01






level











*Same letter indicates no statistical difference between the two treatments at the defined level; different letter indicates a statistical difference between the two treatments at the defined level.













Example 6




Hypersensitive Response Elicitor-treated Tomato is More Tolerant to Calcium Deficiency




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.




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.




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.












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
















*The data were collected from the fruits in 100 square foot plot













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.







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
  • 1. A method of imparting resistance to heat stress, chemical stress, salt stress, or stress caused by calcium deficiency to plants, said method comprising:applying a hypersensitive response elicitor protein or polypeptide, isolated from a Gram negative bacterium, in a non-infectious form to a plant or plant seed under conditions effective to impart resistance to heat stress, chemical stress, salt stress, or stress caused by calcium deficiency.
  • 2. A method according to claim 1, wherein the stress is chemical stress where the chemical is selected from the group consisting of insecticides, fungicides, herbicides, and heavy metals.
  • 3. A method according to claim 1, wherein the hypersensitive response elicitor protein or polypeptide is isolated from Erwinia, Pseudomnonas, or Xanthomonas.
  • 4. A method according to claim 3, wherein the hypersensitive response elicitor protein or polypeptide is isolated from Erwinia amylovora, Erwinia carotovora, Erwinia chrysanthemi, or Erwinia stewartii.
  • 5. A method according to claim 7, wherein the hypersensitive response elicitor protein or polypeptide is isolated from Pseudomonas syringae or Pseudomonas solancearum.
  • 6. A method according to claim 3, wherein the hypersensitive response elicitor protein or polypeptide is isolated from a Xanthomonas species.
  • 7. A method according to claim 1, wherein plants are treated during said applying.
  • 8. A method according to claim 1, wherein plant seeds are treated during said applying, said method further comprising:planting the seeds treated with the hypersensitive response elicitor protein or polypeptide in natural or artificial soil and propagating plants from seeds planted in soil.
  • 9. A method according to claim 1, 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.
  • 10. A method according to claim 1, wherein the plant is selected from the group consisting of Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.
  • 11. A method according to claim 1, wherein said stress resistance imparted to said plant or plant seed is resistance to heat stress.
  • 12. A method according to claim 1, wherein said stress resistance imparted to said plant or plant seed is resistance to chemical stress.
  • 13. A method according to claim 1, wherein said stress resistance imparted to said plant or plant seed is resistance to salt stress.
  • 14. A method according to claim 1, wherein said stress resistance imparted to said plant or plant seed is resistance to stress caused by calcium deficiency.
Parent Case Info

This application claims benefit of U.S. Provisional Patent Application Ser. No. 60/107,243, filed Nov. 5, 1998.

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Entry
US 5,650,387, 7/1997, Wei et al. (withdrawn)
Collmer et al., “Erwinia chysanthemi and Pseudomonas syringae: Plant Pathogens Trafficking in Extracellular Virulence Proteins,” pp. 43-78.
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Preston et al., “The HrpZ Proteins of Pseudomonas syringae pvs. syringae, glycinea, and tomato are Encoded by an Operon Containing Yersinia ysc Homologs and Elicit the Hypersensitive Response in Tomato but not Soybean,” Mol. Plant-Microbe Interact., 8(5):717-32 (1995).
Bauer et al., “Erwinia chrysanthemi hrp Genes and their Involvement in Elicitation of the Hypersensitive Response in Tobacco,” Sixth International Symposium on Molecular Plant Microbe Interactions, Abstract No. 146 (Jul. 1992).
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Provisional Applications (1)
Number Date Country
60/107243 Nov 1998 US