WO2000028056A9 - ELICITEUR DE REACTION D'HYPERSENSIBILITE A PARTIR D'$i(AGROBACTERIUM VITIS) - Google Patents

ELICITEUR DE REACTION D'HYPERSENSIBILITE A PARTIR D'$i(AGROBACTERIUM VITIS)

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Publication number
WO2000028056A9
WO2000028056A9 PCT/US1999/026079 US9926079W WO0028056A9 WO 2000028056 A9 WO2000028056 A9 WO 2000028056A9 US 9926079 W US9926079 W US 9926079W WO 0028056 A9 WO0028056 A9 WO 0028056A9
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WIPO (PCT)
Prior art keywords
seq
plant
plants
protein
polypeptide
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PCT/US1999/026079
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English (en)
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WO2000028056A3 (fr
WO2000028056A2 (fr
Inventor
Thomas J Burr
Thomas C Herlache
Hongsheng Zhang
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Cornell Res Foundation Inc
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Application filed by Cornell Res Foundation Inc filed Critical Cornell Res Foundation Inc
Priority to AU18135/00A priority Critical patent/AU1813500A/en
Priority to EP99961589A priority patent/EP1127145A2/fr
Publication of WO2000028056A2 publication Critical patent/WO2000028056A2/fr
Publication of WO2000028056A3 publication Critical patent/WO2000028056A3/fr
Publication of WO2000028056A9 publication Critical patent/WO2000028056A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to a protein or polypeptide from Agrobacterium associated with production of a hypersensitive response.
  • Interactions between bacterial pathogens and plants generally fall into two categories: (1) compatible (pathogen-host), leading to intercellular bacterial growth, symptom development, and disease development in the host plant; and (2) incompatible (pathogen-nonhost), resulting in the hypersensitive response, a particular type of incompatible interaction occurring without progressive disease symptoms.
  • compatible pathogen-host
  • pathogen-nonhost pathogen-nonhost
  • the hypersensitive response is a rapid, localized necrosis that is associated with the active defense of plants against many pathogens (Kiraly, "Defenses Triggered by the Invader: Hypersensitivity," pages 201-224 in: Plant Disease: An Advanced Treatise, Vol. 5, J.G.
  • the hypersensitive response elicited by bacteria is readily observed as a tissue collapse if high concentrations (> 10 7 cells/ml) of a pathogen like Pseudomonas syringae or Erwinia amylovora are infiltrated into the leaves of nonhost plants (necrosis occurs in isolated plant cells at lower levels of inoculum) (Klement, Nature 199:299-300; Klement et al., Phytopathology 54:474-477 (1963); Turner et al., Phytopathology 64:885-890 (1974); Klement, "Hypersensitivity," supra).
  • the capacities to elicit the hypersensitive response in a nonhost and be pathogenic in a host appear linked. As noted by Klement, "Hypersensitivity,” pages 149-177 in Phvtopatho enie
  • hrp genes are widespread in Gram-negative plant pathogens, where they are clustered, conserved, and in some cases interchangeable (Willis et al., Mol. Plant-Microbe Interact. 4:132-138 (1991); Bonas, pages 79-98 in: Current Topics in Microbiology and Immunology: Bacterial Pathogenesis of Plants and Animals - Molecular and Cellular Mechanisms, J.L. Dangl, ed. Springer-Verlag, Berlin (1994)).
  • Several hrp genes encode components of a protein secretion pathway similar to one used by Yersinia, Shigella, and
  • E. amylovora ⁇ a321 a bacterium that causes fire blight of rosaceous plants, and was designated harpin (Wei et al., Science 257:85-88 (1992)). Mutations in the encoding hrpN gene revealed that harpin is required for E. amylovora to elicit a hypersensitive response in nonhost tobacco leaves and incite disease symptoms in highly susceptible pear fruit.
  • the P. solanacearum GMI1000 PopAl protein has similar physical properties and also elicits the hypersensitive response in leaves of tobacco, which is not a host of that strain (Arlat et al., EMBO J. 13:543-53 (1994)). However, P. solanacearum pop A mutants still elicit the hypersensitive response in tobacco and incite disease in tomato. Thus, the role of these glycine-rich hypersensitive response elicitors can vary widely among Gram-negative plant pathogens.
  • the oomycete fungus Phytophora infestans, elicits an HR on tobacco, because it produces an elicitor derived from the fatty acid(s) linolenic acid and/or arachinodate (Choi et al., PNAS, 91 :2329-2333 (1994)).
  • the present invention seeks the protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis.
  • the present invention is directed to isolated proteins or polypeptides from Agrobacterium associated with production of a hypersensitive response, particularly Agrobacterium vitis. Also disclosed are DNA molecules encoding such proteins or polypeptides as well as expression systems, host cells, and plants containing such molecules. Uses of the proteins or polypeptides themselves and the DNA molecules encoding them in imparting disease resistance to plants, enhancing plant growth, improving nutritional values, enhancing stress tolerance, and controlling insects are disclosed.
  • the A. vitis elicitor functions in nonhost plants by causing a rapid hypersensitive response that results in walling-off and killing of the pathogen.
  • the A. vitis elicitor induces a restricted necrosis of tissues, resulting in plant cell death and induction of pathogen resistance (e.g., resistance to Downey Mildew).
  • pathogen resistance e.g., resistance to Downey Mildew
  • plant receptor molecules for bacterial elicitors differ between species and the A. vitis elicitor may be uniquely effective for Vitis spp. and possible other plant genera. In addition to grape disease resistance, this will make the A. vitis elicitor useful for inducing localized cell death following insect and nematode feeding.
  • Figure 1 shows tobacco HR induced by a diverse group of A. vitis strains. Strains are designated on the leaf panels.
  • Figure 3 shows grape necrosis phenotypes of A. vitis F2/5 (full necrosis) and Tn5 mutant 6 (reduced necrosis) and Tn5 mutant 675 (no necrosis).
  • Figure 4 shows a Southern blot of EcoRI digested DNA from F2/5 and Tn5 mutants probed with the pUT containing mini-7>?5 kanamycin resistance gene. Lanes 1 - 10 correspond to strains F2/5, 6, 675, 816, 832, 852, 901, 1 123, 1154, and 1320.
  • Figure 5 shows Tn5 mutants infiltrated into tobacco.
  • Figure 6 shows the conservation of strain F2/5 necrosis and HR- related EcoRI loci within vitis strains and tumefaciens, A. radiobacter, and E. amylovora. The loci are listed on the left. Bacterial strains from which EcoRI digests were probed are identified at the top of each row. A. vitis strain K306 was not probed with DNA from 675.
  • Figure 8 shows the population of F2/5 and mutant 6 after infiltration of tobacco leaf panels.
  • Figures 9 A and B show cladistic analyses of the 44 amino acid region encoded by the A. vitis 134 bp F2-R2 PCR amplicon.
  • Figure 9A shows a comparison of thirteen hrcVIFlhA homologues generated with the Clustal alignment tool (DNAstar, Madison, WI). Both the flagellar and pathogenesis genes from Yersinia enterocolitica were included as internal controls on the quality of the alignment. Alignment parameters were set to gap and gap-length penalties of 10.
  • Figure 9B shows the same alignment performed with the addition of the deduced A. vitis sequence.
  • the aligned sequences (species, protein, Genbank accession number) are: E.
  • Figure 10 shows a Southern blot of Ec Rl-digested total bacterial DNA probed with the hrcVIFlhA -homologous F2-R2 134 bp PCR amplicon from A. vitis strain F2/5.
  • Lanes 1 through 6 are A. vitis strains CG49, CG78, K306, CG523, CG561 and F2/5.
  • Lanes 7, 8, 9 and 10 are A. tumefaciens strain C58, A. rhizogenes strain K84, E. amylovora strain FB01, and pCPP143.
  • Figures 11 A and B shows probes derived from 7n5-containing EcoRI loci of F2/5 associated with tobacco HR and grape necrogenesis.
  • the present invention is directed to isolated proteins or polypeptides from Agrobacterium associated with production of a hypersensitive response, particularly Agrobacterium vitis. Also disclosed are DNA molecules encoding such proteins or polypeptides as well as expression systems, host cells, and plants containing such molecules. Uses of the proteins or polypeptides themselves and the DNA molecules encoding them in imparting disease resistance to plants, enhancing plant growth, improving nutritional values, enhancing stress tolerance, and controlling insects are disclosed.
  • the first protein or polypeptide associated with production of a hypersensitive response elicitor polypeptide or protein from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 1.
  • the second protein or polypeptide associated with production of a hypersensitive response elicitor polypeptide or protein from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 2.
  • the third protein or polypeptide associated with production of a hypersensitive response elicitor polypeptide or protein from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 3.
  • the fourth protein or polypeptide associated with production of a hypersensitive response elicitor polypeptide or protein from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 4.
  • the fifth protein or polypeptide associated with production of a hypersensitive response elicitor polypeptide or protein from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 5.
  • proteins or polypeptides are encoded by open reading frames 79-753 (SEQ. ID. No. 1), 88-753 (SEQ. ID. No. 2), 856-1512 (SEQ. ID. No. 3), 970-1512 (SEQ. ID. No. 4), and 1237-1512 (SEQ. ID. No. 5) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 6 (referred to herein as l l23-F).
  • the sixth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 7.
  • the seventh protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 8.
  • the eighth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 9.
  • the ninth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 10.
  • proteins or polypeptides are encoded by open reading frames 97-798 (SEQ. ID. No. 7), 136-894 (SEQ. ID. No. 8), 895-1584 (SEQ. ID. No. 9), and 1395-1668 (SEQ. ID. No. 10) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 1 1 (referred to herein as 1 123-R).
  • the tenth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 12.
  • the eleventh protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 13.
  • proteins or polypeptides are encoded by open reading frames 196-1038 (SEQ. ID. No. 12) and 1166-1532 (SEQ. ID. No. 13) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 14 (referred to herein as 1 154-1 -F).
  • the twelfth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 15.
  • This protein or polypeptide is encoded by an open reading frame 255-1421 of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 16 (referred to herein as 1 154-1-R).
  • the thirteenth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 17.
  • the fourteenth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 18. These proteins or polypeptides are encoded by open reading frames
  • the fifteenth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 20.
  • the sixteenth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 21.
  • the seventeenth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 22.
  • the eighteenth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 23.
  • proteins or polypeptides are encoded by open reading frames 360-854 (SEQ. ID. No. 20), 1006-2070 (SEQ. ID. No. 21), 2328-2876 (SEQ. ID. No. 22), and 2713-3135 (SEQ. ID. No. 23) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 24 (referred to herein as 1 154-2-R).
  • the nineteenth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 25.
  • the twentieth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 26.
  • proteins or polypeptides are encoded by open reading frames 734-1282 (SEQ. ID. No. 25) and 1295-1669 (SEQ. ID. No. 26) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 27 (referred to herein as 1320-1-F).
  • the twenty-first protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 28.
  • the twenty-second protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 29.
  • the twenty-third protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 30.
  • the twenty-fourth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 31.
  • proteins or polypeptides are encoded by open reading frames 176-1 198 (SEQ. ID. No. 28), 317-1 198 (SEQ. ID. No. 29). 894-1418 (SEQ. ID. No. 30), and 1213-1620 (SEQ. ID. No. 31) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 32 (referred to herein as 1320-1-R).
  • the twenty-fifth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 33.
  • the twenty-sixth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 34.
  • the twenty-seventh protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 35.
  • proteins or polypeptides are encoded by open reading frames 75-707 (SEQ. ID. No. 33), 90-707 (SEQ. ID. No. 34), and 1 183-2001 (SEQ. ID. No. 35) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 36 (referred to herein as 1320-2-F).
  • the twenty-eighth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 37.
  • the twenty-ninth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 38.
  • proteins or polypeptides are encoded by open reading frames 58-1224 (SEQ. ID. No. 37) and 1857-2351 (SEQ. ID. No. 38) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 39 (referred to herein as 1320-2-R).
  • the thirtieth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 40.
  • This protein or polypeptide is encoded by an open reading frame 4- 648 of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 41 (referred to herein as 6-F).
  • the thirty-first protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 42.
  • This protein or polypeptide is encoded by an open reading frame 283-660 of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 43 (referred to herein as 6-R).
  • the thirty-second protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 44.
  • the thirty-third protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 45.
  • the thirty-fourth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 46.
  • the thirty-fifth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 47.
  • proteins or polypeptides are encoded by open reading frames 146-481 (SEQ. ID. No. 44), 337-759 (SEQ. ID. No. 45), 1006-2280 (SEQ. ID No. 46), and 1713-2027 (SEQ. ID. No. 47) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 48 (referred to herein as 675-F).
  • the thirty-sixth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 49.
  • the thirty-seventh protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 50.
  • proteins or polypeptides are encoded by open reading frames 194-1036 (SEQ. ID. No. 49) and 1143-1550 (SEQ. ID. No. 50) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 51 (referred to herein as 675-R).
  • the thirty-eighth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 52.
  • This protein or polypeptide is encoded by an open reading frame 13-633 of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 53 (referred to herein as 816-F).
  • the thirty-ninth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 54.
  • This protein or polypeptide is encoded by an open reading frame 19-633 of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 55 (referred to herein as 816-R).
  • the fortieth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 56.
  • the forty-first protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 57.
  • the forty-second protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 58.
  • the forty-third protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 59.
  • proteins or polypeptides are encoded by open reading frames 1080-1829 (SEQ. ID. No. 56), 1 191-1829 (SEQ. ID. No. 57), 1468-1782 (SEQ. ID. No. 58), and 1845-2165 (SEQ. ID. No. 59) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 60 (referred to herein as 832-
  • the forty-fourth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 61.
  • the forty-fifth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 62.
  • the forty-sixth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 63.
  • the forty-seventh protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 64.
  • proteins or polypeptides are encoded by open reading frames 50-514 (SEQ. ID. No. 61), 657-1475 (SEQ. ID. No. 62), 1092-1475 (SEQ. ID. No. 63), and 1849-2203 (SEQ. ID. No. 64) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 65 (referred to herein as 832- R).
  • the forty-eighth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 66.
  • This protein or polypeptide is encoded by an open reading frame 55-528 of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 67 (referred to herein as 852- 1-F).
  • the present invention also relates to a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 68 (referred to herein as 852- 1-R).
  • the forty-ninth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 69.
  • the fiftieth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 70.
  • the fifty-first protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 71.
  • the fifty-second protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 72. These proteins or polypeptides are encoded by open reading frames
  • the fifty-fourth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 75.
  • the fifty-fifth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 76.
  • the fifty-sixth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 77.
  • the fifty-seventh protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 78. These proteins or polypeptides are encoded by open reading frames
  • the fifty-eighth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 80.
  • This protein or polypeptide is encoded by an open reading frame 43-486 of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 81 (referred to herein as 901-F).
  • the fifty-ninth protein or polypeptide associated with production of a hypersensitive response elicitor from Agrobacterium vitis has an amino acid sequence corresponding to SEQ. ID. No. 82.
  • This protein or polypeptide is encoded by an open reading frame 7- 486 of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 83 (referred to herein as 901 -R).
  • Agrobacterium vitis genes encoding elicitor production can be identified by mutagenizing bacterial strains which produce the elicitor, resulting in mutants that have lost elicitor-production ability and thus do not result in the production of a hypersensitive response. Mutagenesis methods include, but are not limited to, transposon mutagenesis, chemical mutagenesis, and exposure to ionizing radiation. DNA from a wild-type (elicitor producing, non-mutated) strain that is carried on a suitable vector is then introduced into the mutants.
  • This pool of mutants containing wild-type DNA carried on a vector are then screened for restoration of elicitor production.
  • Genes encoding elicitor production are thereby identified by purifying the vector containing the introduced, complementing, DNA sequences. These complementing clones can be sequenced to determine the identity of the genes carried therein.
  • Suitable vectors for complementation experiments include plasmids and cosmids with origins of replication suitable for use in Agrobacterium vitis. Bacteriophage capable of infecting A. vitis may also be used for this purpose.
  • A. vitis DNA from an elicitor-producing strain can be screened for its ability to encode production of an active elicitor in another organism such as, but not limited to, E. coli.
  • Suitable organisms for this method do not elicit grape necrosis or the tobacco HR.
  • A. vitis DNA is restricted into fragments and cloned into a suitable vector, creating a library.
  • This A. vitis library is introduced into the alternative organism (such as E. coli) in a manner such that individual E. coli transformants each carry a different fragment of cloned A. vitis DNA.
  • the transformed strains are then tested for their ability to elicit grape necrosis or the tobacco HR. Transformed strains that are able to cause either of these responses must, therefore, contain some of the A. vitis genes responsible for elicitor production.
  • Suitable vectors for library construction include various plasmids, cosmids, and bacteriophage.
  • probes for Southern blotting can be generated by restriction of the DNA sequences incorporated into this patent. Alternatively, the sequences incorporated in this patent can be used as templates in a PCR reaction to generate suitable amplified fragments for probe generation. Southern blot probes are produced by labeling these restriction or PCR fragments with a radioactive nucleotide, or with nucleotides labeled in some other manner (for example, with digoxigenin or biotin for chemiluminescent detection).
  • DNA from library- containing strains is affixed to a support such as a nylon or nitrocellulose membrane, and clones containing genes with homology to those incorporated in this patent are identified by their ability to bind probe molecules. Binding of probe molecules is observed as radioactive spots on the support, or as light- emitting spots when using chemiluminescent detection methods. These clones can then be moved into a suitable expression vector for elicitor production.
  • Suitable DNA molecules are those that hybridize to a DNA molecule comprising a nucleotide sequence of 20 continuous bases of S ⁇ Q. ID. Nos.
  • hybridization buffer comprising 0.9M sodium citrate ("SSC") buffer at a temperature of 37°C and remain bound when subject to washing with SSC buffer at 37°C; these DNA molecules preferably hybridize in a hybridization buffer comprising 20% formamide in 0.9M saline/0.09M SSC buffer at a temperature of 42°C and remain bound when subject to washing at 42°C with 0.2 x SSC buffer at 42°C.
  • SSC sodium citrate
  • the protein or polypeptide of the present invention is preferably in isolated form (i.e.
  • the protein or polypeptide of the present invention is produced but not secreted into the growth medium of recombinant host cells.
  • the protein or polypeptide of the present invention is secreted into growth medium.
  • the host cell e.g., E. coli
  • the homogenate is centrifuged to remove bacterial debris.
  • the purification may begin with partitioning the starting material into aqueous and organic fractions.
  • the supernatant fraction containing the protein or polypeptide is subjected to gel filtration or another suitable purification method such as separation by HPLC.
  • derivatives of small-molecule HR elicitors with greater specificity or activity can be synthesized by using the elicitor molecule(s) as the starting material for combinatorial chemical synthesis. Said derivatives are identified by their improved performance (lower phytotoxicity, improved stability, enhanced disease control, etc.) using assays standard in the art.
  • U.S. Patent 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. In addition, recombinant genes can be expressed in a viral system using subgenomic promoters as described in U.S. Patent No.
  • Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtl 1, gt W ⁇ S.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, 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 Studier et al., Gene Expression Technology vol.
  • viral vectors such as lambda vector system gtl 1, gt W ⁇ S.tB, Charon 4, and plasmid vectors such as pBR322,
  • 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, New York (1989), which is hereby incorporated by reference.
  • host-vector systems may be utilized to express the protein-encoding sequence(s).
  • the vector system must be compatible with the host cell used.
  • Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria.
  • the expression elements of these vectors vary in their strength and specificities. Depending upon the host- vector system utilized, any one of a number of suitable transcription and translation elements can be used.
  • mRNA messenger RNA
  • telomere a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis.
  • the DNA sequences of eucaryotic promoters differ from those of procaryotic promoters.
  • eucaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters may not be recognized and may not function in eucaryotic cells.
  • 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 AGGA that appears about 7 nucleotides 5' of the amino-terminal methionine start codon of the protein.
  • SD Shine-Dalgarno
  • 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.
  • ribosomal RNA Ribonucleic acid
  • Promoters vary in their "strength" (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it may be desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene (see PCT International Application No.
  • promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P R and P L promoters of coliphage lambda and others, including but not limited, to / ⁇ cUV5, ompF, bla, Ipp, and the like, may be used to direct high levels of transcription of adjacent DNA segments.
  • trp-lacOY5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • pathogen inducible and wound inducible promoters may be used (see, e.g., U.S. Patent No. 5,866,776 to de Wit et al.; U.S. Patent No. 5,743,477 to Walsh et al. (proteinase inhibitor II promoter); U.S. Patent No. 5,689,056 to Cramer et al.; U.S. Patent No. 5,677,175 to Hodges et al.; Martini et al., Mol. Gen. Genet. 263:179 (1993), which are hereby incorporated by reference).
  • Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operations, the addition of specific inducers is necessary for efficient transcription of the inserted DNA.
  • the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside).
  • IPTG isopropylthio-beta-D-galactoside
  • Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength" as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively.
  • the DNA expression vector which contains a promoter, 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.
  • ATG initiation codon
  • any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan ⁇ , D, C, B or A genes.
  • any SD-ATG combination produced by recombinant D ⁇ A or other techniques involving incorporation of synthetic nucleotides may be used.
  • the isolated D ⁇ A molecule encoding the polypeptide or protein associated with production of a hypersensitive response elicitor may be in sense orientation and correct reading frame. Alternatively, the isolated D ⁇ A molecule encoding the polypeptide or protein associated with production of a hypersensitive response elicitor may be in antisense orientation. In addition, the isolated D ⁇ A molecule encoding the polypeptide or protein associated with production of a hypersensitive response elicitor may be nontranslatable by, for example, inserting translation stop codes into the template.
  • the present invention further relates to methods of imparting disease resistance to plants, enhancing plant growth, improving nutritional values, enhancing stress tolerance, and/or effecting insect control for plants. These methods involve applying or expressing the polypeptide or protein associated with production of a hypersensitive response elicitor (or the elicitor itself) in a non- infectious form to all or part of a plant or a plant seed under conditions effective for the protein or polypeptide to impart disease resistance, enhance growth, improve nutritional values, enhance stress tolerance, and/or control insects.
  • the protein or polypeptide associated with production of a hypersensitive response elicitor can be applied to or expressed in part of the plants such that seeds recovered from such plants themselves are able to impart disease resistance in plants, to enhance plant growth, to improve nutritional values, to enhance stress tolerance, and/or to effect insect control.
  • transgenic plants or plant seeds can be utilized as an alternative to applying a polypeptide or protein to plants or plant seeds in order to impart disease resistance in plants, to effect plant growth, improve nutritional values, enhance stress tolerance, and/or to control insects on the 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 polypeptide or protein associated with production of a hypersensitive response elicitor and growing the plant under conditions effective to permit that DNA molecule to impart disease resistance to plants, to enhance plant growth, to improve nutritional values, to enhance stress tolerance, and/or to control insects.
  • a transgenic plant seed transformed with a DNA molecule encoding a polypeptide or protein associated with production of a hypersensitive response elicitor 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 disease resistance to plants, to enhance plant growth, to improve nutritional values, to enhance stress tolerance, and/or to control insects.
  • the embodiment of the present invention where the polypeptide or protein associated with a hypersensitive response elicitor is applied to the plant or plant seed can be carried out in a number of ways, including: 1) application of an isolated protein or polypeptide or 2) application of bacteria which do not cause disease and are transformed with a gene encoding the protein or polypeptide.
  • the protein or polypeptide can be applied to plants or plant seeds by applying bacteria containing the DNA molecule encoding the polypeptide or protein. It is preferred that such bacteria be capable of secreting or exporting the protein or polypeptide so that the protein or polypeptide can contact plant or plant seed cells.
  • the protein or polypeptide is produced by the bacteria inplanta 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 disease resistance, enhance growth, improve nutritional values, enhance stress tolerance, and/or control insects.
  • 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, turnip, cauliflower, broccoli, 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.
  • 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, turnip, cauliflower, broccoli, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear
  • Suitable ornamental plants are: Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.
  • absolute immunity against infection may not be conferred, but the severity of the disease is reduced and symptom development is delayed. Lesion number, lesion size, and extent of sporulation of fungal pathogens are all decreased.
  • This method of imparting disease resistance has the potential for treating previously untreatable diseases, treating diseases systemically which might not be treated separately due to cost, and avoiding the use of infectious agents or environmentally harmful materials.
  • the method of imparting pathogen resistance to plants in accordance with the present invention is useful in imparting resistance to a wide variety of pathogens including viruses, bacteria, and fungi.
  • Resistance, inter alia, to the following viruses can be achieved by the method of the present invention: Tobacco mosaic virus and Tomato mosaic virus.
  • Resistance, inter alia, to the following bacteria can also be imparted to plants in accordance with present invention: Pseudomonas solanacearum, Pseudomonas syringae pv. tabaci, and Xanthamonas campestris pv. pelargonii.
  • Plants can be made resistant, wter alia, to the following fungi by use of the method of the present invention: Fusarium oxysporum and Phytophthora infestans.
  • various forms of plant growth enhancement or promotion can be achieved. This can occur as early as when plant growth begins from seeds or later in the life of a plant.
  • plant growth according to the present invention encompasses greater yield, increased quantity of seeds produced, increased percentage of seeds germinated, increased plant size, greater biomass, more and bigger fruit, earlier fruit coloration, and earlier fruit and plant maturation. As a result, the present invention provides significant economic benefit to growers.
  • insect control encompasses preventing insects from contacting plants to which the hypersensitive response elicitor has been applied, preventing direct insect damage to plants by feeding injury, causing insects to depart from such plants, killing insects proximate to such plants, interfering with insect larval feeding on such plants, preventing insects from colonizing host plants, preventing colonizing insects from releasing phytotoxins, etc.
  • the present invention also prevents subsequent disease damage to plants resulting from insect infection.
  • the present invention is effective against a wide variety of insects.
  • European corn borer is a major pest of corn (dent and sweet corn) but also feeds on over 200 plant species including green, wax, and lima beans and edible soybeans, peppers, potato, and tomato plus many weed species.
  • Additional insect larval feeding pests which damage a wide variety of vegetable crops include the following: beet army worm, cabbage looper, corn ear worm, fall army worm, diamondback moth, cabbage root maggot, onion maggot, seed corn maggot, pickleworm (melonworm), pepper maggot, tomato pinworm, and maggots.
  • This group of insect pests represents the most economically important group of pests for vegetable production worldwide.
  • the present invention can be used to enhance cold tolerance and improve nutritional value of transgenic plants.
  • the present invention may also enhance processability and nutritional value by, for example, leading to an altered oil content in the transgenic crop.
  • the method of the present invention involving application of the hypersensitive response elicitor polypeptide or protein, which 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 polypeptide or protein associated with production of a hypersensitive response elicitor into the plant.
  • Suitable application methods include high or low pressure spraying, injection, and leaf abrasion proximate to when protein or polypeptide application takes place.
  • the 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 protein or polypeptide with cells of the plant or plant seed.
  • the seeds can be planted in natural or artificial soil and cultivated using conventional procedures to produce plants.
  • the plants may be treated with one or more applications of the protein or polypeptide to impart disease resistance to plants, to enhance plant growth, to improve nutritional values, to enhance stress tolerance, and/or to control insects on the plants.
  • the 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 protein or polypeptide 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 polypeptide or protein associated with production of a hypersensitive response elicitor. Suitable carriers include water, aqueous solutions, slurries, or dry powders.
  • this composition may contain additional additives including fertilizer, insecticide, fungicide, nematacide, and mixtures thereof.
  • Suitable fertilizers include (NH ) 2 NO 3 .
  • An example of a suitable insecticide is Malathion.
  • Useful fungicides include Captan.
  • Suitable additives include buffering agents, wetting agents, coating agents, and abrading agents. These materials can be used to facilitate the process of the present invention.
  • the protein or polypeptide associated with production of a hypersensitive response elicitor can be applied to plant seeds with other conventional seed formulation and treatment materials, including clays and polysaccharides.
  • a protein or polypeptide need not be applied topically to the plants or seeds. Instead, transgenic plants transformed with a DNA molecule encoding such a protein or polypeptide 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 mechanically transfer the recombinant DNA. Crossway, Mol.
  • 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.
  • particle bombardment also known as biolistic transformation
  • this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof.
  • the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA.
  • the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle.
  • Biologically active particles e.g., dried bacterial cells containing the vector and heterologous DNA
  • the DNA molecule may also be introduced into the plant cells by electroporation. Fromm et al., Proc. Natl. Acad. Sci. USA, 82:5824 (1985), which is hereby incorporated by reference. In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate.
  • Agrobacterium tumefaciens o A. rhizogenes previously transformed with the gene.
  • the transformed plant cells are grown to form shoots or roots, and develop further into plants.
  • 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, A. vitis, A. rubi) 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.
  • assaying for the presence of opines can be used to identify transformed tissue.
  • the DNA molecule of the present invention may also be introduced into a plant via whisker-mediated transformation which is described in U.S. Patent Nos. 5,302,532 and 5,464,765, which are hereby incorporated by reference.
  • 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 T - DNA of Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome.
  • the transformed plant cells After transformation, the transformed plant cells must be regenerated.
  • Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants.
  • the culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
  • the expression cassette After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure with the presence of the gene encoding the protein or polypeptide associated with production of a hypersensitive response elicitor resulting in disease resistance, enhanced plant growth, improved nutritional value, enhanced stress tolerance, and/or control of insects on the plant.
  • transgenic seeds or propagules are recovered from the transgenic plants.
  • the seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants.
  • the transgenic plants are propagated from the planted transgenic seeds under conditions effective to impart disease resistance to plants, to enhance plant growth, to improve nutritional values, to enhance stress tolerance, and/or to control insects.
  • RNA mediated may result from expression of the polypeptide or protein, or may be caused by enzymatic production of secondary metabolites.
  • transgenic plants and plant seeds 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 disease resistance, enhance growth, improve nutritional value, impart stress resistance, and/or control insects. Such plants may also be treated with conventional plant treatment agents (e.g., insecticides, fertilizers, etc.).
  • conventional plant treatment agents e.g., insecticides, fertilizers, etc.
  • Example 1 Bacterial Strains and Media.
  • the A. vitis strains used in this study represent both tumorigenic and non-tumorigenic strains isolated from cultivated Vitis vinifera and wild V. riparia grapevines and are listed in Table 1.
  • A. vitis strains were propagated on potato-dextrose agar (PDA) (Difco, Detroit, MI) at 28 °C. Tn5 mutagenesis of strain F2/5 was accomplished through conjugal mating with E. coli strain SI 7-1 pSUP2021 (Simon et al., Bio/Technology 1 :784- 791 (1983), which is hereby incorporated by reference), as previously described (Burr et al., Phytopath. 87:706-71 1 (1997), which is hereby incorporated by reference). Mutants were grown on PDA amended with kanamycin (50 ⁇ g/ml). E. coli (pCPP2068) expresses a clone of the A.
  • PDA potato-dextrose agar
  • vitis strains were also cultured in Induction Medium (IM) adjusted to pH 5.5 (Wei et al., J. Bacteriol. 174:1875-1882 (1992); He et al., Cell 73: 1255-1266 (1993), which are hereby incorporated by reference).
  • IM Induction Medium
  • Strains F2/5 and CG49 were also infiltrated into leaves of N glauca and N benthamiana as described above. At least 2 leaf panels per leaf on five leaves of different plants were infiltrated. All experiments were repeated. To determine the effects of growth media on the inoculum dose necessary to elicit an HR, tobacco leaves were infiltrated with A. vitis F2/5 that was grown overnight on PDA or in medium IM broth (Wei et al., J. Bacteriol., 174(6): 1875-1882 (1992), which is hereby incorporated by reference). Bacterial cells were made to OD 6 oo 1-5 and diluted in twofold increments prior to infiltration.
  • the infiltration buffer has also been reported to influence the sensitivity of HR elicitation ( ⁇ issinen et al., Phytopathology, 87(7):678-684 (1997), which is hereby incorporated by reference).
  • HR elicitation ⁇ issinen et al., Phytopathology, 87(7):678-684 (1997), which is hereby incorporated by reference.
  • an overnight PDA culture was suspended to OD 6 oo 1 -5 and diluted in twofold increments in distilled water, 5mM phosphate buffer (pH 5.5) and 5mM MES buffer (pH 5.5).
  • Tobacco leaf panels were infiltrated with a dilution series of bacteria in water or buffers. To determine the duration of active pathogen translation necessary for HR (i.e.
  • F2/5 was inoculated as a positive and distilled water as a negative control. Mutants that caused necrosis which differed from the wildtype were retested. They were also tested for their ability to induce an HR on tobacco as described above. All mutants with altered necrosis and HR phenotypes were inoculated on shoot explants of V. vinifera, V labrusca, and V. riparia as described above. Ten explants were inoculated with each mutant and F2/5; the experiment was repeated. The same strains were also infiltrated into leaf panels of N tabaccum, N. glauca, and N benthamiana, as described above.
  • Total bacterial D ⁇ A was prepared as previously described (Burr et al., Plant Disease, 79:677-682 (1995), which is hereby incorporated by reference). Ten ⁇ g of D ⁇ A was digested to completion with EcoRI (does not cut within the transposon) and transferred to a ⁇ ytran membrane according to the TurboBlotter protocol (Schelich and Scheull, Keene, ⁇ H). Bacterial D ⁇ A was probed with a kanamycin-resistance gene probe that was generated from a pUT plasmid carrying ⁇ Tn5 (Burr et al., Phytopath.. 87(7):706-71 1 (1997), which is hereby incorporated by reference).
  • Southern blots were performed in a Hybaid (Franklin, MA) Mini-4 hybridization oven. Probe annealing and wash temperatures were 65 °C. Blots were washed twice for 15 minutes with 2X SSC, and twice for 15 minutes with 0.5X SSC. Probes were labeled, and blots developed, according to the Genius non-radioactive system (Boehringer-Mannheim, Indianapolis, IN). Probes for analysis of conservation of the Tn5 loci within A. vitis were generated by PCR. Primers were designed based on partial sequence data of cloned 7 «5-containing EcoRI fragments from Tn5 mutant strains. Southern blots were performed in a Hybaid Mini-4 hybridization oven.
  • Probe annealing and wash temperatures were 65 °C. Blots were washed twice for 15 minutes with 2X SSC, and twice for 15 minutes with 0.5X SSC. Probes were generated, and blots developed, according to the Genius non-radioactive system instructions (Boehringer-Mannheim, Indianapolis, IN).
  • Plasmid pBluescript, II KS + (Promega, Madison WI) was prepared according to the alkaline-lysis protocol (Sambrook et al., supra, which is hereby incorporated by reference). 10 ⁇ g of DNA from mutants was cut to completion with EcoRI and phenol-chloroform extracted to remove the restriction enzyme. The digested DNA was then precipitated with 2 volumes of 95% ethanol and 0.2 volumes of 7.5 M ammonium acetate. 10 ⁇ g of pBlueScript DNA was digested with EcoRI and purified in a similar manner.
  • Ampicillin and kanamycin-resistant bacteria were then dilution plated on Luria- Bertoni agar amended with the same antibiotics, X-gal (50 ⁇ g/ml) and IPTG (100 ⁇ g/ml) and grown at 27 °C for 48 hours. White colonies, indicating the presence of cloned DNA carrying the Tn5, were passaged to new LB plus kanamycin plates. Plasmid DNA from these strains was purified and about 500 ng of each was digested with EcoRI and separated on a 0.7% agarose gel to verify the presence of insert DNA.
  • probes were derived from partial sequences of PCR amplicons of the cloned 7>?5-containing EcoRI fragments. PCR was done using 35 cycles of 94 °C for 1 minute, 94 °C for 1 minute, 52 °C for 1 minute, 72 °C for 7 minutes. Probes were used to analyze the presence of the loci in a diverse group of A. vitis strains. Southern blots were performed as described above.
  • Bacterial DNA from strain F2/5 was prepared as described and 10 ⁇ g was digested with EcoRI, separated on 0.7% agarose gels, and blotted to
  • the expected product size of a HrcV amplification is 134 bp based on a consensus of 45 aa in this region of the gene (Bogdanove et al., J. Bacteriol.
  • the membranes were washed twice at 50 °C in 2X SSC, 0.1% sodium dodecyl sulfate (SDS) and twice in IX SSC, 0.1 % SDS at 50 °C, and used to expose Kodak BioMax MR film.
  • F2/5 necrosis (nee) mutants were tested for their ability to assimilate nitrate as a sole nitrogen source by substituting sodium nitrate for ammonium sulfate in AB minimal media.
  • F2/5 and its nee mutant derivatives were plated on media with both nitrogen sources, and were grown for 5 days at 25 °C. The strains were passaged three times on each medium, and growth on nitrate was assessed after the third passage.
  • Plant sections were also infiltrated with a water blank containing 'Breakthrough' as a negative control.
  • the tissue pieces were allowed to dry for approximately 1 hour prior to inoculation.
  • a 5 ⁇ l drop of an OD 60 Q 1.0 or 0.1 water suspension of A. vitis F2/5 was placed on the cut tissue end, and necrosis was assessed for 4 days post-inoculation (dpi). All experiments were repeated at least once.
  • Example 9 Tobacco HR.
  • Leaf panels of Nicotianna tabaccum cv. Havana-423 plants were infiltrated with a diverse group of tumorigenic and non-tumorigenic A. vitis strains which induced a hypersensitive response at various frequencies ( Figure 1 , Table 2).
  • A. vitis strains consistently yielded a reaction where at least part of the infiltrated area collapsed.
  • the tested tumorigenic strains induce collapse less frequently than the non-tumorigenic strains.
  • the HR initiates quickly (less than 24 hours), and the collapsed area is dry and leathery.
  • the PG minus strain A. vitis 22-9 is capable of eliciting rapid collapse indicating that PG is not associated with this response.
  • A. vitis strains CG49, CG78, K306, CG523, and CG542 were found to be inconsistent HR elicitors when inoculated from overnight PDA cultures at OD 600 1.5. To test the role of growth media in this variability, these strains were grown on PDA and in IM broth, with constant shaking overnight. Bacteria were grown in IM broth, because some strains grow poorly on solid IM. IM and PDA cultures were suspended to OD 60 o 1.5 in sterile distilled water and infiltrated into tobacco leaves. Growth in IM appears to improve the elicitation of collapse, as strains that failed to elicit collapse from PDA caused collapse when grown in IM. IM itself does not cause a response.
  • inoculum concentration affects induction of necrosis or tumorigenicity by A. vitis
  • grape stem sections were inoculated with various concentrations of tumorigenic A. vitis strains, and necrosis and tumors were scored 12-14 dpi.
  • Inoculum concentration affected the degree of necrosis and tumor formation on cut nodes. Generally, high inoculum concentrations resulted in the greatest necrosis and little tumorigenesis whereas lower inoculum concentrations resulted in less necrosis and increased tumorigenesis (Table 3).
  • Table 3 Relationship of inoculum concentration to grape necrosis and tumorigenesis on V. vinifera cv. 'Chardonnay' shoot pieces.
  • Necrosis was rated from 0 to 5 based on the degree of necrotic ingress after 12 - 13 days. The rating was determined by dividing the sum of the ratings by the total number of stem sections (12).
  • Sensing nitrogen and available iron levels are often important cues in induction of pathogenicity genes.
  • Infiltration of F2/5 in 54 mM mannitol (high carbon to nitrogen ratio ) or 54 mM mannitol, 86 mM ammonium nitrate (low carbon to nitrogen ratio) did not inhibit HR elicitation.
  • Infiltration in 7.2 x 10 "4 M Fe 2 SO 4 .7H 2 0 either delayed the appearance of the HR by 24 hours or blocked it completely.
  • Infiltration in 7.2 x 10 "3 M Fe 2 SO 4 .7H 2 O always blocked the HR completely. None of these infiltration solutions (without bacteria) caused any visible effect on the plant for at least 4 days post inoculation.
  • Example 10 HR Induction Period.
  • Leaf panels were infiltrated with OD 00 1.25 suspensions of F2/5 followed by re-infiltration with tetracycline from 2 to 12 hours after bacterial infiltration to determine the induction period of tobacco Havanna-423 responding to A. vitis.
  • the antibiotic was infiltrated from 0 to 5 hours after the bacterium, no HR developed. From 6 to 1 1 hours, an increasing degree of necrotic flecking was observed within the leaf panels and at 12 hours a confluent collapse was observed (Figure 2).
  • Example 11 Effect of Eukaryotic Metabolic Inhibitors.
  • An HR is an active response by the plant, and can be blocked with inhibitors of plant metabolism and signal transduction.
  • tobacco leaf panels were infiltrated with various eukaryotic metabolic inhibitors 20-30 minutes prior to infiltration with F2/5. They were infiltrated with either 5x10 "4 M cobalt chloride, 5x10 "5 M sodium orthovanadate, or 7.1x10 " M cycloheximide and allowed to dry 1 hour before infiltration with an OD 60 o 1.5 suspension of A. vitis strain F2/5.
  • the calcium-channel blocker cobalt chloride effectively inhibits F2/5-induced HR at a concentration of 5 xlO "4 M.
  • Sodium orthovanadate a general ATPase/phosphatase inhibitor, is also effective at blocking the HR.
  • Cycloheximide an inhibitor of 80S ribosomes, is less effective at inhibiting tobacco leaf-panel collapse, even at threefold higher concentration than is reported to be sufficient to inhibit a H ⁇ pss-mediated hypersensitive response.
  • the HR response was rated in comparison to an untreated F2/5 positive control Positive responses were equivalent to the control, intermediate responses had spotty collapse within the infiltrated panel, and negative responses had no collapse.
  • Inhibitor solutions were infiltrated into the leaf and allowed to dry until watersoaking was no longer apparent (approximately 30 minutes) A vilis strain F2/5 was then infiltrated into the treated area
  • Example 12 Inhibition of Grape Necrosis.
  • Vitis vinifera shoot sections were cut longitudinally and divided into small (0.5 cm) sections. Stem sections were soaked in inhibitor solutions or a distilled water control for 1 hour prior to treatment with A. vitis strain F2/5 suspended to OD 60 o 0.1 in either water or inhibitor solutions. Stem sections were allowed to dry briefly prior to inoculation, and were maintained on moist filter paper in Petri plates. Necrosis was assessed over the next 48 hours. In negative controls without bacteria, cobalt chloride, cycloheximide, and sodium orthovanadate did not cause any visible response by the plant tissue. Cycloheximide and cobalt chloride treatment inhibited F2/5-induced necrosis for up to 48 hours post-inoculation. Orthovanadate treatment gave a moderate reduction in necrosis, with 14 of 19 stem sections showing reduced or no necrosis.
  • necrosis induced by A. vitis can be suppressed by treating the inoculated tissue with inhibitors of plant signal transduction and translation (He et al., Mol. Plant-Microbe Interact.. 7(2):289-292 (1994), which is hereby inco ⁇ orated by reference).
  • Table shows combined results from four independent experiments in which five shoot explants were inoculated with each bacterium as described in text. First number are shoots that showed black necrosis, second number are those with reduced, brown necrosis, and third is number that showed no necrosis. b Ability to induce a hypersensitive response was evaluated on N. tabaccum, N. rustica, N. benthamiana, and N glauca. c Mutant 1123 caused an HR on N. glauca only.
  • mutant 832 All the mutants, with the exception of mutant 832, appeared to grow normally on AB agar plates with either ammonium of nitrate as the sole nitrogen source. The 832 mutant grew slowly in these assays. All of the mutants grew at rates similar to F2/5 in half-strength PD broth and on grape shoot explants except for mutant 832, which grew slower ( Figure 7). In contrast, when tobacco leaf panels were infiltrated with F2/5 and HR-minus mutant 6, F2/5 was nondetectable in the collapsed leaf tissue at 72 hours whereas mutant 6 grew over time (Figure 8).
  • a strain was scored as '+' for homology to a particular probe if hybridization was observed, 'RFLP' indicates a restriction-fragment length polymo ⁇ hism in the hybridizing band, and '-' indicates lack of hybridization. ND — not done.
  • b Strain K306 was not tested on the EcoRI blot shown in the figure, but has a fragment of identical size to F2/5 on a Hindl ⁇ l blot.
  • the tumorigenic strains CG49, CG78. and K306 have more RFLPs and deletions (at least 2 each) than the non-tumorigenic strains CG523 and CG561 and only one locus, 675, was detected in all of the A. vitis strains.
  • Example 15 - A hrc V Homologue in A. vitis The hrcV gene is highly conserved in and necessary for function of type III secretion systems (Hueck, Microbiol. and Mol. Biol. Rev., 62(2):379-433 (1998), which is hereby inco ⁇ orated by reference). Southern blot experiments were performed to identify putative ⁇ rcFhomologs mA. vitis. Initial experiments utilized a 1.6 kb EcoRI -Pstl fragment that contains the C-terminus of the E. amylovora H ⁇ J and the highly-conserved N-terminal region of HrcV.
  • vitis strains and a similar-sized product from A. tumefaciens strain C58, A. rhizogenes strain K84, and from the E. amylovora hrcV clone on pCPP143.
  • the F2-R2 primers also produced strong amplicons of approximately 200 bp and 950 bp from A. vitis strains.
  • the three A. vitis amplicons were gel purified from a 2% low-melt agarose gel and sequenced directly.
  • the 134-bp region of hrcV between the F2-R2 primer annealing sites has a useful degree of variability for similarity studies. Seven non-flagellar HrcV-homologues and six flagellar homologues from different bacteria and the 44 amino acid A. vitis F2-R2 amplicon were used in Clustal similarity analysis. The LcrD and FlhA proteins from Yersinia enterocolitica were used as internal controls on the quality of the alignment and they fall into the appropriate flagella and pathogenicity classes. When the 44 amino acid region corresponding to the A. vitis sequence is aligned without utilizing the A. vitis sequence most of the pathogenicity genes form a closely related group ( Figure 9A).
  • Shigella flexneri MxiA which forms its own branch
  • Rhizobium sp. NGR243 which clusters with the flagellar homologues.
  • the Y. enterocolitica proteins fall into the appropriate classes, and are therefore distinguishable from one another. If the analysis is repeated with the same alignment parameters but including 44 aa -A. vitis F2-R2 amplicon, then the A. vitis sequence is found to be most similar to the flagellar alleles ( Figure 9B). In this alignment, however, the S. flexneri and Rhizobium sequences have traded places, with Shigella MxiA clustering with the pathogenicity alleles and the Rhizobium sequence forming its own branch. This alignment still differentiates the Y. enterocolitica FlhA and LcrD proteins.
  • a probe made from the /zrc -homologous amplicon hybridized to an approximately 3.1 kb band from all the tested A. vitis strains ( Figure 10).
  • A. vitis strain K306 has a second band of approximately 4.6 kb that hybridizes to the 134 bp probe.
  • A. rhizogenes strain K84 has hybridizing sequences of 4.1, 4.5, 5.3, and 7.0 kb, suggesting that there is a family of related genes in this strain.
  • the 134 bp probe also detects a family of homologous sequences of approximately 2, 2.6, 3.6, and 9 kb in E amylovora strain ⁇ aFBOl , and hybridizes to the expected Arc -containing EcoRI band from pCPP143. A signal was not detected from C58 DNA.
  • Example 16 An HR-Like Collapse is Induced in Tobacco by A. vitis. All A. vitis strains were able to elicit a rapid HR on N. tabaccum at various frequencies. The reaction bears similarities to HRs elicited by other plant pathogenic bacteria in that collapse is noticeable within 18 hours of infiltration, and becomes dry and brown within 24 to 48 hours. Because of the appearance and integrity of the infiltrated tissue, collapse is probably not due to maceration by polygalacturonase. This is consistent with the ability of the PG(-) strain 22-9 to elicit collapse.
  • A. vitis requires a higher inoculum dose to induce HR than E. amylovora or P. syringae, it is similar to that required by other recently described bacterial HR reactions on plants.
  • the Gram-positive bacterium C. michiganensis subsp. sepedonicus requires 1.3 x 10 9 CFU per ml for the HR ( ⁇ issinen et al., Phytopathology. 87(7):678-684 (1997), which is hereby inco ⁇ orated by reference) and the non-macerogenic out -mutant E. chrysanthemi requires about 5x10 CFU per ml (Bauer et al., Mol.
  • amylovora Hrp gene transcription (Wei et al., Science, 257:85-88 (1992); Wei et al., J. Bacteriol., 174(6): 1875-1882 (1992), which are hereby inco ⁇ orated by reference).
  • A. vitis HR-induction mechanism is regulated differently than the R. tropici chlorosis mechanism and the E. amylovora H ⁇ system.
  • Infiltration of F2/5 in dilute iron solutions delays or blocks the ability of A. vitis to induce an HR. This indicates that sensing of low iron availability may be an important environmental cue to A. vitis that leads to expression of its HR- inducing mechanism. It is interesting that tumorigenic A.
  • strain CG49 which is a weak HR inducer on N tabaccum
  • different A. vitis strains may produce different elicitors or elicitors that are structurally related, but differ in ways that affect HR frequency and host range.
  • bacterial genes that are associated with HR elicitation have been found to encode proteins that comprise the type III secretion system or the elicitor itself (Bonas et al., Plant Journal. 12(1): 1-7 (1997), which is hereby inco ⁇ orated by reference). Mutations in the secretion system render the mutant unable to elicit an HR and to cause disease on host plants.
  • sepidonicus may be an exception, as the type III system is involved in protein transit through the outer membrane of Gram negative bacteria (Charkowski et al.. J. Bacteriol., 179:3866-3874 (1997), which is hereby inco ⁇ orated by reference). Clavibacter, lacking this membrane, may therefore have different secretion machinery. Also, P. syringae pv. syringae AvrD elicitor apparently does not require export through a type III secretion apparatus. This protein is known to enzymatically catalyze production of small-molecule elicitors called syringolides, that can induce an HR even when produced in type III secretion-deficient E. coli (Keen et al., Mol.
  • HrcV is a member of a well- conserved superfamily of genes involved in the type III secretion system (Bogdanove et al., J. Bacteriol. 178(6): 1720- 1730 (1996), which is hereby inco ⁇ orated by reference).
  • An A. vitis PCR product with homology to a highly- conserved ⁇ -terminal region of hrcV was identified. This sequence is also related to the ⁇ -terminus of E. coliflhA and the Rhizobium sp. ⁇ GR243 Y4YR nolT gene.
  • the band that hybridizes to the ⁇ rcF-homologous sequence is an A. vitis flagellar gene, since there is only one homologue present in most strains. This is consistent with the cladistic analysis of the cognate regions from 13 HrcV/FlhA homologues, in which the A. vitis amplicon clusters with the flhA genes.
  • Mutations in the 675 locus is epistatic to the other loci, in that a single mutation knocks out all necrosis and tobacco HR. This phenotype may result if this locus is involved in elicitor secretion or in an early step in the synthesis of a family of elicitor molecules, or in an essential upstream regulatory function.
  • A. vitis strains cause grape necrosis, it was expected that related loci would be highly-conserved within strains. Southern blots however, reveal a su ⁇ rising degree of variability at the mutated loci within A. vitis. There is a greater degree of conservation within the non-tumorigenic than within tumorigenic strains. Of the five loci, only 675 is conserved within all the strains, suggesting that it is essential for necrosis and HR. Alternatively, with the exception of locus 832, all of the other loci may be required and the mutations that result in RFLPs are silent. Each tumorigenic strain has at least two RFLPs or deletions within the set of five tested loci.
  • A. vitis strain CG49 is the weakest HR elicitor (Herlache, "Biochemical and Molecular Genetic Investigations of the Agrobacterium Vitis-G pevi Interaction," Ph.D. Thesis. Cornell University (1999), which is hereby inco ⁇ orated by reference), and this strain has the most changes at these loci.
  • Rhizobium species Paneppke, Crit. Rev. Biotechnology, 16:1-51 (1996), which is hereby incorporated by reference
  • This model could explain the variable reactions of A. vitis mutants on different Vitis and Nicotianna species.
  • Lipo-chitin oligomers (LCOs) produced by Rhizobium fulfill a number of signaling functions between bacteria and plants, causing responses such as root hair deformation and nodule meristem initiation.
  • Subtle LCO structural changes such as sulfurylation of the oligochitin moiety by nodH and nodPQ (Horvath et al., Cell. (1987), which is hereby inco ⁇ orated by reference) or changes in the lipid tail caused by mutation in nodFE (Spaink et al., Nature 354:125-130 (1991), which is hereby inco ⁇ orated by reference), affect host specificity of the signal.
  • Such alterations in nod-factor structure can affect host range at the species or cultivar level in su ⁇ rising ways. For example, mutation of R. leguminosarum bv.
  • trifolii nodE results in severe inhibition of clover nodulation but enhances nodulation of vetch and other species that are not normal hosts (Spaink et al., EMBO. 8:281 1-2818 (1989), which is hereby incorporated by reference).
  • the A. vitis mutants showing differential necrosis and HR phenotypes are affected in genes that add peripheral elements that are important for signal perception by different plant species or tissues to a core structure. Mutants that result in total loss of necrosis and HR could be involved in production of the signal molecule(s) core structure, analogous to production of the nod-factor oligochitin core by nodABC (Carlson et al., Mol. Plant-Microbe
  • necrosis induces necrosis on grape roots and crown galls on woody aerial parts of vines remains intriguing. Is necrosis associated with A. vitis host specificity and does it provide a benefit to the plant (e.g. a defense mechanism) or to the bacterium? It may be that necrosis facilitates systemic colonization of grape or that necrotic tissues provide a niche that excludes other competitive soil microbes. Host necrosis induced by A. vitis can be inhibited by chemicals that block eukaryotic metabolism and intracellular signaling. The results of host inhibition studies with cobalt chloride and sodium orthovanadate present difficulties in inte ⁇ retation.
  • Cycloheximide has no effect on A. vitis, as demonstrated by its use in semi-selective media for the isolation of A. vitis from the field (Burr et al., Plant Disease. 71 (7):617-620 (1983), which is hereby inco ⁇ orated by reference).
  • Cycloheximide inhibition was less consistent than the inorganic salt inhibitors, being totally effective about 20% of the time and giving partial inhibition in the infiltrated region in an additional 40% of inoculations. None of the inhibitors had any noticeable affect on the infiltrated tobacco leaves for at least 72 hours post- inoculation. Again, the effect of these inhibitors is thought to be on the plant cell. Neither sodium orthovanadate nor cycloheximide have any observed effect on growth of A. vitis in culture. Cobalt chloride was found to be bacteriostatic at the concentration used. However, in planta multiplication is likely not necessary for macroscopic HR-elicitation because of the high inoculum doses used. This indicates that A.
  • v/ ' tw-induced tobacco collapse requires active plant metabolism like a typical HR induced by other bacteria. Besides requiring an active response by the plant, the HR also requires an adequate contact time between the plant cell and a viable bacterial cell to initiate. After this contact time has been reached the bacteria can be killed and the plant cell will still proceed on to the HR collapse. With P. syringae pv syringae, this contact time is on the order of 4 to 6 hours, after which treatment of the infiltrated area with tetracycline or rifampicin will not prevent the HR from occurring. Similar studies using A. vitis strain F2/5 indicate that the induction period is approximately 12 hours for the A. vz ' t/s-induced collapse.
  • Tetracycline applied to the infiltrated area before 4 hours post-inoculation totally blocks the A. vztw-induced HR. Tetracycline applications between 6 and 12 hours post- inoculation gave increasingly weak inhibition of the HR as indicated by the observed increase in necrotic flecking over this time period. This indicates that either subpopulations of the inoculum or of the host tissue are responding more quickly or with greater sensitivity to infiltration than others. This may be reflected in the spotty collapse that is often produced by poorly-HR inductive strains like CG78. Alternatively, barriers to bacterial infiltration within the tobacco leaf may cause bacteria to accumulate to higher, more inductive levels at locations within the leaf. At 12 hours post-inoculation, antibiotic application failed to prevent HR induction.
  • This induction period is noticeably longer than the induction period reported for Pseudomonads and Erwinia amylovora. This may be due to the former being foliar pathogens that are better adapted to attacking leaf mesophyll cells. Alternatively, these pathogens may have HR- eliciting systems that are more efficient at secreting elicitors, HR-eliciting systems that are more quickly induced, or elicitors that provoke a plant response at lower concentrations than . vitis.
  • the tested tumorigenic strains are all weak and inconsistent inducers of the tobacco HR. This could be due to weaker induction of the HR-eliciting system or lack of a highly inductive elicitor in these strains.
  • prior infiltration of tobacco with tumorigenic A. tumefaciens or Pseudomonas syringae pv. savastanoi inhibits the Pseudomonas syringae pv. phaseolicola tobacco HR (Robinette et al., J. Bacteriol.. 172(10):5742-5749 (1990), which is hereby inco ⁇ orated by reference). Inhibition was dependent on the presence of functional tms genes.
  • the presence of these genes in the tumorigenic A. vitis strains may also reduce their ability to elicit an HR.
  • These hypotheses are tantalizing, because they suggest an opposing relationship between HR elicitation and tumorigenicity. It may be that tumorigenic strains have reduced the HR eliciting function to avoid killing plant cells targeted for transformation. Alternatively, there may be a 'push-pull' relationship between necrosis and tumorigenesis such that at low population densities tumorigenesis prevails. This suggests that necrogenesis may be cell-density, or developmentally, regulated and may explain why high inoculum doses lead to increased necrosis at the expense of tumor formation.
  • Agrobacteria have been tested for their ability to elicit an HR in the past and have been reported to be negative (Klement et al.. Phytopathology, 54:474-477 (1964), which is hereby inco ⁇ orated by reference). Why would this be the case? This is probably thought to be true for several reasons, the primary reason being that it is unlikely that A. vitis strains were included in these tests. A. vitis is rarely encountered in nature, as it is found only in vineyard soils and in infected grapevines. Secondly, the A. v/t/s-induced HR requires high inoculum doses and is most consistent on young tobacco leaves, conditions which were likely different from those used by previous workers who were more familiar with Pseudomonads, etc. Example 17 - Evaluation of A. FZ/w-Induced Resistance in Vitis vinifera Against Plasmopara viticola.
  • SAR systemic acquired resistance
  • a vitis was sprayed on leaves in mixture with silicon penetrant Break-Thru. Leaves on four shoots per plant were wounded with carborundum prior to spraying, then assayed for susceptibility to P viticola.
  • Agrobacterium vitis is the primary causal agent of crown gall on grape.
  • One unique characteristic of A vitts is that it causes a grape-specific necrosis on roots, leaves, and green tissues. Necrosis was found to be inhibited by eukaryotic metabolic inhibitors; a characteristic previously demonstrated for hypersensitive-response and pathogenicity (HR) mechanisms.
  • HR hypersensitive-response and pathogenicity
  • A. vitis strains were infiltrated into tobacco leaves, an HR was elicited.
  • the A. vitis- induced HR requires an inoculum dose of about 4x10 8 CFU/ml, which is greater than required for HR induction by some other Gram-negative bacteria such as Pseudomonas syringae pv. syringae.
  • A. vitis requires 12 hours to initiate an irreversible HR. This was determined by infiltrating tobacco leaf panels with a tetracycline solution (lethal to A. vitis) at various intervals after A. vitis infiltration. This indicates the probable lack of preformed HR inducers and the need for bacterial gene expression in planta for HR elicitation. Like necrosis, the tobacco HR is blocked by the eukaryotic metabolic inhibitors cobalt chloride and sodium orthovanadate, and is reduced by cycloheximide. A. vitis is able to elicit an HR response on tobacco and that the HR mechanism may be related to the mechanism of grape necrosis.

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Abstract

L'invention concerne une protéine isolée ou un polypeptide provenant d'Agrobacterium (notamment d'Agrobacterium vitis) et associés à une réaction d'hypersensibilité ainsi que des molécules d'ADN isolées qui codent pour ces protéines ou polypeptides. La protéine ou le polypeptide de l'invention et la molécule d'ADN isolée qui les code possède les activités suivantes: conférer aux plantes une résistance aux maladies, améliorer la croissance des plantes, améliorer les valeurs nutritionnelles, conférer aux plantes une tolérance au stress et/ou combattre les insectes parasitant ces plantes. Ce résultat est possible grâce à l'application de la protéine ou du polypeptide sous une forme non infectieuse aux plantes ou à leurs graines dans des conditions efficaces pour leur conférer une résistance aux maladies, améliorer leur croissance, améliorer les valeurs nutritionnelles, leur conférer une tolérance au stress et/ou combatte les insectes parasitant ces plantes ou celles développées à partir des graines. En variante, l'invention concerne des plantes transgéniques ou des graines de plantes transformées avec une molécule d'ADN codant la protéine et le polypeptide ainsi que les plantes transgéniques ou celles développées à partir des graines transgéniques, que l'on cultive dans des conditions efficaces pour leur conférer une résistance aux maladies, améliorer la croissance des plantes, leur conférer une tolérance au stress et/ou combatte les insectes parasitant ces plantes ou celles développées à partir des graines.
PCT/US1999/026079 1998-11-06 1999-11-05 ELICITEUR DE REACTION D'HYPERSENSIBILITE A PARTIR D'$i(AGROBACTERIUM VITIS) WO2000028056A2 (fr)

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