EP1127145A2

EP1127145A2 - HYPERSENSITIVE RESPONSE ELICITOR FROM $i(AGROBACTERIUM VITIS)

Info

Publication number: EP1127145A2
Application number: EP99961589A
Authority: EP
Inventors: Thomas J. Burr; Thomas C. Herlache; Hongsheng Zhang
Original assignee: Cornell Research Foundation Inc
Current assignee: Cornell Research Foundation Inc
Priority date: 1998-11-06
Filing date: 1999-11-05
Publication date: 2001-08-29
Also published as: WO2000028056A2; WO2000028056A3; WO2000028056A9; AU1813500A

Abstract

The present invention is directed to an isolated protein or polypeptide from Agrobacterium associated with production of a hypersensitive response, particularly Agrobacterium vitis. Also disclosed are isolated DNA molecules which encode such proteins or polypeptides. The protein or polypeptide in accordance with the present invention and the isolated DNA molecule that encode them have the following activities: imparting disease resistance to plants, enhancing plant growth, improving nutritional values, imparting stress tolerance, and/or controlling insects on plants. This can be achieved by applying the protein or polypeptide in a non-infectious from to plants or plant seeds under conditions effective to impart disease resistance, to enhance plant growth, to improve nutritional values, to enhance stress tolerance, and/or to control insects on plants or plants grown from the plant seeds. Alternatively, transgenic plants or plant seeds transformed with a DNA molecule encoding the protein and polypeptide can be provided and the transgenic plants or plants resulting from the transgenic plant seeds are grown under conditions effective to impart disease resistance, to enhance plant growth, to improve nutritional values, to enhance stress tolerance, and/or to control insects on plants or plants grown from the plant seeds.

Description

HYPERSENSITIVE RESPONSE ELICITOR FROM AGROBACTERIUM VITIS

The present application claims the benefit of U.S. Provisional Patent Application Serial No. 60/107,387, filed November 6, 1998, and U.S. Provisional Patent Application Serial No. 60/158,410, filed October 7, 1999, which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a protein or polypeptide from Agrobacterium associated with production of a hypersensitive response.

BACKGROUND OF THE INVENTION

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. During compatible interactions on host plants, bacterial populations increase dramatically and progressive symptoms occur. During incompatible interactions, bacterial populations do not increase, and progressive symptoms do not occur. 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. Horsfall and E.B. Cowling, ed. Academic Press New York (1980); Klement, "Hypersensitivity," pages 149-177 in: Phvtopafhogenic Prokaryotes. Vol. 2, M.S. Mount and G.H. Lacy, ed.

Academic Press, New York (1982)). The hypersensitive response elicited by bacteria is readily observed as a tissue collapse if high concentrations (> 10 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 Phytopathogenic

Prokaryotes. Vol. 2., M.S. Mount and G.H. Lacy, ed. Academic Press, New York, these pathogens also cause physiologically similar, albeit delayed, necroses in their interactions with compatible hosts. Furthermore, the ability to produce the hypersensitive response or pathogenesis has been dependent to date on a common set of genes, denoted hrp (Lindgren et al., J. Bacteriol. 168:512-22 (1986); Willis et al., Mol. Plant-Microbe Interact. 4:132-138 (1991)). Consequently, the hypersensitive response may hold clues to both the nature of plant defense and the basis for bacterial pathogenicity.

The 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

Salmonella spp. to secrete proteins essential in animal diseases (Van Gijsegem et al.. Trends Microbiol. 1 :175-180 (1993)). In E. amylovora, P. syringae, and P. solanacearum, hrp genes have been shown to control the production and secretion of glycine-rich, protein elicitors of the hypersensitive response (He et al., Cell 73:1255-1266 (1993), Wei et al., J. Bacteriol. 175:7958-7967 (1993); Arlat et al., ΕMBO J. 13:543-553 (1994)).

The first of these proteins was discovered in E. amylovora Εa321, 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 popA 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.

Other plant pathogenic hypersensitive response elicitors have been isolated, cloned, and sequenced from plant pathogenic bacteria including: Erwinia chrysanthemi (Bauer et al., MPMI 8(4): 484-91 (1995)); Erwinia carotovora (Cui et al., MPMI 9(7): 565-73 (1996)); Erwinia stewartii (Ahmad et. al., "Harpin is Not Necessary for the Pathogenicity of Erwinia stewartii on

Maize," 8th Int'l. Cong. Molec. Plant-Microb. Inter. July 14-19, 1996 and Ahmad et. al., Ann. Mtg. Am. Phytopath. Soc. July 27-31 , 1996); and Pseudomonas syringae pv. syringae (WO 94/26782 to Cornell Research Foundation, Inc.). Not all hypersensitive response (i.e. HR) elicitors are proteinaceous, however. 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.

SUMMARY OF THE INVENTION

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.

Like other elicitors, the A. vitis elicitor functions in nonhost plants by causing a rapid hypersensitive response that results in walling-off and killing of the pathogen. On grape plants, 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). However, when grape shoot explants are inoculated with another bacterium that produces an HR elicitor, such as Pseudomonas syringae, necrosis is not induced. Therefore 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows tobacco HR induced by a diverse group of A. vitis strains. Strains are designated on the leaf panels.

Figure 2 shows that a contact period between bacterium and plant of 6- 12 hours is necessary for the A. vitis hypersensitive response (HR) on tobacco (IM = Induction Medium; PDA = potato-dextrose agar).

Figure 3 shows grape necrosis pheno types 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 75 kanamycin resistance gene. Lanes 1 - 10 correspond to strains F2/5, 6, 675, 816, 832, 852, 901 , 1 123, 1 154, 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 A. vitis strains and A. 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 7 shows growth of F2/5 and mutants on grape shoot explants. Explants were inoculated as described below and five were analyzed for each bacterium at each timepoint. A one-way analysis of variance indicated that except for mutant 832, populations were not significantly different (P = 0.5) (CFU/ml = colony-forming unit/ml).

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 . vitis sequence. The aligned sequences (species, protein, Genbank accession number) are: E coli FlhA P76298, Y. enterocolitica FlhA Q56887, B. subtilis FlhA P35620, C. jejuni FlhA A49217, H. pylori FlhA O06758, C. cresentus FlhA Q03845, S. flexneri FlhA P35533, E. amylovora HrcV (Hrpl) P35654, P. syringae pvsyringae HrcV (Hrpl) P35655, Y. enterocolitica LcrD P21210, R. solanacearum HrcV (HrpO) P35656, X. campestris HrcV (HrpC2) P80150, Rhizobium sp. NGR243 Y4YR P55726.

Figure 10 shows a Southern blot of Eco 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 TrøJ-containing EcoRI loci of F2/5 associated with tobacco HR and grape necrogenesis.

DETAILED DESCRIPTION OF THE INVENTION

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.

These 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.

These 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. 11 (referred to herein as 1123-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.

These 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

753-1076 (SEQ. ID. No. 17) and 1352-1729 (SEQ. ID. No. 18) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 19 (referred to herein as 1 154-2-F). 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.

These 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.

These 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.

These proteins or polypeptides are encoded by open reading frames 176-1 198 (SEQ. ID. No. 28), 317-1198 (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.

These proteins or polypeptides are encoded by open reading frames 75-707 (SEQ. ID. No. 33), 90-707 (SEQ. ID. No. 34), and 1183-2001 (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.

These 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.

These 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.

These 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.

These 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-

F).

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.

These 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

412-900 (SEQ. ID. No. 69), 2576-3181 (SEQ. ID. No. 70), 3178-3591 (SEQ. ID. No. 71), and 4251-4838 (SEQ. ID. No. 72) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 73 (referred to herein as 852- 2-F). The fifty-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. 74.

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

33-851 (SEQ. ID. No. 74), 129-851 (SEQ. ID. No. 75), 939-2291 (SEQ. ID. No. 76), 2572-318 (SEQ. ID. No. 77), and 3280-4134 (SEQ. ID. No. 78) of a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 79 (referred to herein as 852-2-R).

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. Alternatively, 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. In this method, 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.

Likewise, library clones containing elicitor-production genes can be identified by Southern blotting. This method does not require that the organism containing the A. vitis library express the A. vitis genes cloned into the library vector. Probes for Southern blotting can be generated by restriction of the DNA sequences incorporated into this patent. Alternatively, the sequences incoφorated 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 SΕQ. ID. Nos. 6, 11, 14, 16, 24, 27, 32, 36, 39, 41, 43, 48, 51, 53, 55, 60, 65, 67, 68, 73, 79, 81, or 83 under stringent conditions characterized by a 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. The protein or polypeptide of the present invention is preferably in isolated form (i.e. separated from its host organism) and more preferably produced in purified form (preferably at least about 60%, more preferably 80%, pure) by conventional techniques. Typically, the protein or polypeptide of the present invention is produced but not secreted into the growth medium of recombinant host cells. Alternatively, the protein or polypeptide of the present invention is secreted into growth medium. In the case of unsecreted protein, to isolate the protein, the host cell (e.g., E. coli) carrying a recombinant plasmid is propagated, lysed by sonication, heat, or chemical treatment, and the homogenate is centrifuged to remove bacterial debris. Alternatively, 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. Likewise, 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. 5,316,931, which is hereby incoφorated by reference. Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtl 1, gt WES.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 incoφorated by reference), pQE, pIH821, pGEX, pET series (see Studier et al., Gene Expression Technology vol. 185 (1990), which is hereby incoφorated by reference), and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1989), which is hereby incoφorated by reference.

A variety of host- vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.

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

Transcription of DNA is dependent upon the presence of a promoter which is 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. Furthermore, 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.

Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno ("SD") sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AGGA that appears about 7 nucleotides 5' of the amino-terminal methionine start codon of the protein. The SD sequences are complementary to the 3 '-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby incoφorated by reference. Promoters vary in their "strength" (i.e. their ability to promote transcription). For the puφoses 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. WO 98/37223 to Pang et al., which is hereby incoφorated by reference). Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P_R and P promoters of coliphage lambda and others, including but not limited, to /αcUV5, ompF, bla, Ipp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacOW5 (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. In addition, 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 incoφorated 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. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp,pro, etc., are under different controls.

Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength" as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a 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. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan Ε, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DΝA or other techniques involving incoφoration of synthetic nucleotides may be used.

In an expression system, the isolated DΝA 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 DΝA molecule encoding the polypeptide or protein associated with production of a hypersensitive response elicitor may be in antisense orientation. In addition, the isolated DΝA 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. Once the isolated DΝA molecule encoding the polypeptide or protein associated with production of a hypersensitive response elicitor has been cloned into an expression system, it is ready to be incoφorated into a host cell. Such incoφoration can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.

The present invention 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. Alternatively, the protein or polypeptide associated with production of a hypersensitive response elicitor (or the elicitor itself) 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.

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. Alternatively, 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. In the latter embodiment, 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. In these embodiments, 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. Examples of suitable ornamental plants are: Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia. With regard to the use of the protein or polypeptide of the present invention in imparting disease resistance, 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, inter alia, to the following fungi by use of the method of the present invention: Fusarium oxysporum and Phytophthora infestans. With regard to the use of the protein or polypeptide of the present invention to enhance plant growth, 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. For example, 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. For example, early germination and early maturation permit crops to be grown in areas where short growing seasons would otherwise preclude their growth in that locale. Increased percentage of seed germination results in improved crop stands and more efficient seed use. Greater yield, increased size, and enhanced biomass production allow greater revenue generation from a given plot of land.

Another aspect of the present invention is directed to effecting any form of insect control for plants. For example, insect control according to the present invention 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 armyworm, cabbage looper, corn ear worm, fall armyworm, diamondback moth, cabbage root maggot, onion maggot, seed corn maggot, pickleworm (melonworm), pepper maggot, tomato pinworm, and maggots. Collectively, 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. This encompasses, for example, the ability to plant earlier in the spring and improved frost- and chill-tolerance which allow a crop to be efficiently produced in regions that are normally too cold for the plant. 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. When treating plant seeds or propagules (e.g., cuttings), in accordance with the application embodiment of the present invention, 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. Once treated with the protein or polypeptide of the present invention, the seeds can be planted in natural or artificial soil and cultivated using conventional procedures to produce plants. After plants have been propagated from seeds treated in accordance with the present invention, the plants may be treated with one or more applications of the 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. Although not required, this composition may contain additional additives including fertilizer, insecticide, fungicide, nematacide, and mixtures thereof. Suitable fertilizers include (NH₄) NO₃. An example of a suitable insecticide is Malathion. Useful fungicides include Captan.

Other suitable additives include buffering agents, wetting agents, coating agents, and abrading agents. These materials can be used to facilitate the process of the present invention. In addition, the 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. In the alternative embodiment of the present invention involving the use of transgenic plants and transgenic seeds, 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. Gen. Genetics, 202:179-85 (1985), which is hereby incoφorated by reference. The genetic material may also be transferred into the plant cell using polyethylene glycol. Krens et al., Nature, 296:72-74 (1982), which is hereby incoφorated by reference.

Another approach to transforming plant cells with a gene which imparts resistance to pathogens is particle bombardment (also known as biolistic transformation) of the host cell. This can be accomplished in one of several ways. The first involves propelling inert or biologically active particles at cells. This technique is disclosed in U.S. Patent Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which are hereby incoφorated by reference. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incoφorated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells.

Yet another method of introduction is fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies. Fraley et al, Proc. Natl. Acad. Sci. USA. 79:1859-63 (1982), which is hereby incoφorated by reference.

The DNA molecule may also be introduced into the plant cells by electroporation. Fromm et al., Proc. Natl. Acad. Sci. USA, 82:5824 (1985), which is hereby incoφorated by reference. In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate.

Another method of introducing the DNA molecule into plant cells is to infect a plant cell with Agrobacterium tumefaciens or A. rhizogenes previously transformed with the gene. Under appropriate conditions known in the art, the transformed plant cells are grown to form shoots or roots, and develop further into plants. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28°C. Agrobacterium is a representative genus of the Gram-negative family Rhizobiaceae. Its species are responsible for crown gall (A. tumefaciens, 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. In addition, 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 incoφorated 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. Schell, Science, 237:1176-83 (1987), which is hereby incoφorated by reference.

After transformation, the transformed plant cells must be regenerated.

Plant regeneration from cultured protoplasts is described in Evans et al, Handbook of Plant Cell Cultures, Vol. 1 : (MacMillan Publishing Co., New York, 1983); and Vasil I.R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. Ill (1986), which are hereby incoφorated by reference.

It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugarcane, sugar beets, cotton, fruit trees, and legumes.

Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.

After the expression cassette is stably incoφorated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

Once transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure with the presence of the gene encoding the 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. Alternatively, transgenic seeds or propagules (e.g., cuttings) are recovered from the transgenic plants. The seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants. The transgenic plants are propagated from the planted transgenic seeds under conditions effective to impart disease resistance to plants, to enhance plant growth, to improve nutritional values, to enhance stress tolerance, and/or to control insects. While not wishing to be bound by theory, such disease resistance, growth enhancement, improved nutritional value, enhanced stress tolerance, and/or insect control may be RNA mediated, may result from expression of the polypeptide or protein, or may be caused by enzymatic production of secondary metabolites.

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.).

EXAMPLES

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.

Table 1. Bacterial strains

Species, Strain Isolated from Phenotype Reference/Source

E. coli, DH5α PG+^C amp Rodriguez- (pCPP2068) Pal enzuela et al., J. Bacteriol. 173:6547- 6552 (1991)

E. coli, HB101 Km^r Figurski et al., Proc. (pRK2013) Natl. Acad. Sci. USA 76:1648-1652 (1979)

Erwinia amylovora, Fire blight infection Burr, recovered FB01 from disease specimen

A. vitis, CG49 V. vinifera gall Tumorigenic, nop b Burr et al., Plant Disease 79:677-682 (1995)

A. vitis, CG50 Tn5 derivative of PG^", tumorigenic, Rodriguez- CG49 nop Pal enzuela et al., J. Bacteriol. 173:6547- 6552 (1991)

A. vitis, CG50 PehA- PG+, tumorigenic, Present application (pTH2068) complemented nop

CG50

A. vitis, CG78 V. vinifera gall Tumorigenic, vit Burr et al., Plant

Disease 83:102-107 (1999) A. vitis, K306 V. vinifera gall Tumorigenic, oct Kerr, A., Plant Disease 64:25-30 (1980), isolated from infected grapevine

A. vitis, 22-9 V. riparia Nontumorigenic Present application

A. vitis, CG523 V. riparia Nontumorigenic Burr et al., Plant Disease 83:102-107 (1999)

A. vitis, CG542 V. riparia Nontumorigenic Burr et al. , Plant Disease 83:102-107 (1999)

A. vitis, CG556 V. riparia Nontumorigenic, Present application

A. vitis, CG561 V. riparia Nontumorigenic, Burr et al. , Plant Disease 83:102-107 (1999)

A. vitis, F2/5 V. vinifera gall, Nontumorigenic, Staphorst et al., Curr. Microbiol. 12:45-52 (1985)

A. tumefaciens, Tumorigenic, nop Zimmerer et al., J. C58 Bacteriol. 92:746- 750 (1966)

A. rhizogenes, K84 Nontumorigenic Kerr, Plant Disease 64:25-30 (1980)

Expresses pehA from A. vitis. ^b Defines pTi plasmid type based on presence of opine synthase genes, nop = nopaline, oct = octopine/cucumopine and vit = vitopine. ^c All references are hereby incoφorated by reference.

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 incoφorated by reference), as previously described (Burr et al., Phytopath. 87:706-711 (1997), which is hereby incoφorated by reference). Mutants were grown on PDA amended with kanamycin (50 μg/ml). E coli (pCPP2068) expresses a clone of the A. vitis strain CG49 polygalacturonase pehA (Rodriguez-Palensuela et al., J. Bacteriol. 173:6547-6552 (1991), which is hereby incoφorated by reference). E. coli was grown on Luria- Bertoni agar medium (LBA) (Sambrook et al. "Molecular Cloning: A Laboratory Manual," 2^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), which is hereby incoφorated by reference) with appropriate antibiotics at 37 °C. To test the role of growth media on the induction of the tobacco HR, A. 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 incoφorated by reference).

Example 2 - Tobacco HR Assays.

Leaf panels on fully-expanded leaves of young (6 - 7 leaves) Nicotianna tabaccum cv. Havana 423, were infiltrated through a needle puncture using a needle-less syringe (Wei et al., Science, 257:85-88 (1992), which is hereby incoφorated by reference), with overnight cultures of bacteria grown on PDA. Prior to infiltration, bacteria were suspended in sterile distilled water to an OD₆₀o of 1.5 and suspensions were diluted to determine the minimum concentration necessary for HR induction. Tobacco plants were grown in the greenhouse.

Strains F2/5 and CG49 (efficient and inefficient HR inducers on N. tabaccum, respectively) 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 incoφorated by reference). Bacterial cells were made to OD o₀ 1.5 and diluted in twofold increments prior to infiltration.

The infiltration buffer has also been reported to influence the sensitivity of HR elicitation (Νissinen et al., Phytopathology, 87(7):678-684 (1997), which is hereby incoφorated by reference). To test this variable for strain F2/5, an overnight PDA culture was suspended to OD₆₀₀ 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. hypersensitive response) induction, tobacco panels were infiltrated with OD₆₀₀ 1.0 suspensions of F2/5 in sterile distilled water. At time intervals from t=0 to t=12 hours after bacterial infiltration the inoculated leaf panels were re-infiltrated with sterile 5 mg/ml tetracycline (lethal to A. vitis ). Lack of collapse in antibiotic-infiltrated panels indicated an insufficient period for bacterial initiation of the HR.

To test plant metabolic inhibitors for their ability to block A. vitis- induced collapse of tobacco leaves, leaf panels were infiltrated with inhibitor solutions, and the panels were allowed to dry before infiltration with A. vitis. The inhibitors and their concentrations were the same as those used for inhibition of host necrosis and by others for inhibition of tobacco HR by P. syringae (He et al., Cell, 73:1255-66 (1993), which is hereby incoφorated by reference). Lack of collapse in inhibitor-treated panels indicated a block in a plant metabolic pathway necessary for HR induction. All experiments were repeated at least once.

Example 3 - Tn5 Mutants.

Approximately 2000 Tn5 mutants of strain F2/5 were screened for their ability to elicit necrosis on V. vinifera cv. 'Chardonnay' green shoot explants. Explants were excised from greenhouse-grown vines and surface- disinfected with 50% v/v solution of Clorox in distilled water for 20 minutes followed by 70% ethanol for 5 minutes. They were rinsed twice in sterile distilled water and cut into approximately 1 cm-long sections. Explants were supported vertically in 10% water agar plates and their aerial ends were inoculated with 3 μl of an OD oo 1.0 suspension of the mutants (about 10⁹ CFU per ml). Necrosis development was scored over a period of 5 days. 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.

Example 4 - Southern blots.

Total bacterial DΝA was prepared as previously described (Burr et al., Plant Disease. 79:677-682 (1995), which is hereby incoφorated by reference). Ten μg of DΝA was digested to completion with EcoRI (does not cut within the transposon) and transferred to a Νytran membrane according to the TurboBlotter protocol (Schelicher and Scheull, Keene, ΝH). Bacterial DΝA was probed with a kanamycin-resistance gene probe that was generated from a pUT plasmid carrying (Burr et al., Phytopath., 87(7):706-711 (1997), which is hereby incoφorated 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 «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).

Example 5 - Conservation of Necrosis and HR Associated Loci. Total DNA from mutants was prepared as previously described.

Plasmid pBluescript, II KS⁺ (Promega, Madison WI) was prepared according to the alkaline-lysis protocol (Sambrook et al., supra, which is hereby incoφorated by reference). 10 μg 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 μg of pBlueScript DNA was digested with EcoRI and purified in a similar manner. Aliquots of EcoRI-digested mutant DNA and pBluescript DNA were mixed in a 3: 1 ratio and ligated for 4 hours with T4 DNA ligase (Promega, Madison, WI) at room temperature. Twenty μl of the ligation reaction was used to transform E coli JM109 competent cells (Strategene, Lajolla CA) by heat shock. The transformation reaction was inoculated to 15 ml Terrific broth amended with kanamycin sulfate (50 μg/ml) and ampicillin (100 μg/ml). Ampicillin and kanamycin-resistant bacteria were then dilution plated on Luria- Bertoni agar amended with the same antibiotics, X-gal (50 μg/ml) and IPTG (100 μg/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.

Subsequently, 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.

Example 6 - Detection of hrcV Homologues.

Bacterial DNA from strain F2/5 was prepared as described and 10 μg was digested with EcoRI, separated on 0.7% agarose gels, and blotted to

Nytran membranes according to manufacturers methods (Turboblotter, Schleicher and Scheull). Blots were hybridized against a 134 bp PCR product amplified from F2/5 using degenerate primers designed against hrcV which is the most highly conserved of the hrp genes (Bogdanove et al., J. Bacteriol. 178(6): 1720- 1730 (1996), which is hereby incoφorated by reference) and which is essential for function of the type III secretion pathway (Hueck, Microbiol. and Mol. Biol. Rev.," 62(2):379-433 (1998), which is hereby incoφorated by reference). The forward primer F2, designed against amino-acids 154 through 159 (MPGKQM) (SEQ. ID. No. 84) of the HrcV consensus, has the sequence 5'-

ATGCCNGGNAARCARATG-3' (SEQ. ID. No. 85). The reverse primer R2

(complementary to the coding strand), designed against amino-acids 191 through

197 of HrcV (MDGAMK (SEQ. ID. NO. 86)) and the first two bases of the F codon, has the sequence 5'-AAYTTCATNGCNCCRTCCAT-3' (SEQ. ID. No.

87). 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.

178(6): 1720- 1730 (1996), which is hereby incoφorated by reference). Random-hexamer primed, 32 P-labeled, Southern-blot probe synthesis was done using the Klenow fragment (Sambrook et al., "Molecular Cloning: A Laboratory Manual, 2^nd ed.", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), which is hereby incoφorated by reference). Hybridization was performed in a Hybaid Mini hybridization oven at 50 °C for 18 hours. The membranes were washed twice at 50 °C in 2X SSC, 0.1% sodium dodecyl sulfate (SDS) and twice in 1 X SSC, 0.1 % SDS at 50 °C, and used to expose Kodak BioMax MR film.

Example 7 - Growth on Nitrate.

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.

Example 8 - Necrosis Inhibition.

Inhibition of host necrosis by plant metabolic inhibitors was tested on stem sections of V. riparia cv St. George, and on crown and stem sections of V. labrusca cv. Concord. The inhibitors used were: cycloheximide 7.1 x 10^"5M, CoCl₂ 5xlO^"5M, Na₃VO₄5xlO^"5M (He et al., Cell, 73:1255-66 (1993), which is hereby incoφorated by reference). Plant sections were vacuum infiltrated with the appropriate chemical in the presence of the silicon-oil based spray adjuvant ' Breakthrough' (Plant Health Technologies, Boise, Idaho) to facilitate penetration of inhibitors. 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 5μl drop of an OD₆₀₀ 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).

Table 2. Frequency of HR induction by different bacterial strains on Nicotianna tabaccum cv. Havana 523^a.

Strain Tumorigenic Total % Positive

Infiltrations HR

CG49 + 31 6.5

CG78 + 33 39.4

K306 + 33 39.4

22-9 - 34 38.2

CG523 - 19 42.1

CG542 - 27 63.0

CG556 - 18 66.7

CG561 - 28 85.7

F2/5 - 37 75.7

C58 + 31 0.0 ^a Infiltrations of leaf panels were done as described in text. HR was recorded up to 48 hours.

It was observed that both the age of the bacterial culture and the inoculated plant had a strong effect on HR induction. The most consistent results were obtained with overnight PDA-grown cultures infiltrated into young (less than 8 leaves) tobacco plants. Juvenile-moφhology tobacco leaves, identified by the infrequent occurrence of secondary veins and rounded leaf tips, appear to be more sensitive than adult-moφhology leaves. Under these conditions most non-tumorigenic

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. Interestingly, 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 oo 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 ₀o 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. To determine the minimum inoculum dose necessary to elicit collapse, tobacco leaves were inoculated with a dilution series in fivefold increments from OD₆oo 1.5 of A. vitis F2/5 grown in either IM or on PDA. This strain was selected, because it produced the most consistent results and elicited the strongest response on tobacco of all strains tested. Results indicate that the minimum inoculum dose sufficient to elicit the HR is approximately 4 x 10⁸ CFU/ml for PDA- or IM-grown F2/5 on highly responsive leaves. On less responsive leaves, PDA-grown F2/5 requires between 7 x 10⁸ and 1 x 10⁹ CFU/ml to elicit a response. Growing F2/5 on an induction medium (IM) as compared to PDA did not effect the concentration of bacteria necessary to induce the HR. Similarly, diluting the bacterium in 5 mM phosphate buffer or in 5 mM MES, had no effect on the concentration of bacteria required.

To determine if 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.

Inoculum Concentration³

OD O.l IP^'1 IP^'2 IP^'3 IP^'4 IP^'5

Strain Exp. Necrosis rating (no. stem sections with galls)"

K306 1 3.7 (2) 1.8 (7) 1.9 (7) 1.4 (10) 0.6 (11) 0.3 (10)

2 2.9 (0) 3.5 (2) 1.2 (7)

3 1.8 (4) 0.8 (9)

4 2.7 (5) 1.3 (11)

CG49 4 3.0 (1) 1.8 (10)

CG78 5 2.4 (5) 0 (12) a The OD₆oo 0.1 inoculum concentrations ranged from 5 X 10 to 1 X 10^ CFU/ml. Twelve nodes were inoculated per experiment with various dilutions as indicated in table.

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).

The infiltration buffer has been reported to influence the sensitivity of HR elicitation (Nissinen et al., Phytopathology 87:678-684 (1997), which is hereby incoφorated by reference). To test if this is also true for A. vitis, F2/5 was resuspended to OD₆₀₀ 1.5, and diluted in 2-fold increments in distilled water, 5 mM pH 5.5 phosphate buffer, and 5 mM pH 5.5 MES buffer. Tobacco leaf panels were infiltrated with the water dilution series and a buffered dilution series. Results of these HR tests revealed no difference in sensitivity when the bacteria were infiltrated in buffer solutions versus distilled water. In all cases, the lowest inoculum level capable of eliciting a confluent HR was approximately 4 x 10⁸ CFU/ml.

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 Fe₂SO .7H₂0 either delayed the appearance of the HR by 24 hours or blocked it completely. Infiltration in 7.2 x 10^"3 M Fe₂SO₄.7H₂O 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.

Strain CG49 frequently failed to induce HR on N. tabaccum (HR induced following 3 of 14 infiltrations) and on N. glauca (3 of 12 infiltrations) but was highly inductive on N. benthamania (11 of 11). Strain F2/5 induced HR on all infiltrated leaf panels of all three species in these experiments.

Example 10 - HR Induction Period. Leaf panels were infiltrated with OD ₀o 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. When 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. To test if this is the case with ., v/^'tw-induced collapse, 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 7.1xlO^"7M cycloheximide and allowed to dry 1 hour before infiltration with an OD₆₀₀ 1.5 suspension of A. vitis strain F2/5.

As shown in Table 4, the calcium-channel blocker cobalt chloride effectively inhibits F2/5-induced HR at a concentration of 5 xlO^"4M. 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 HφΝpss-mediated hypersensitive response. On sensitive leaves, which show a positive response to F2/5 alone, approximately 40% of leaf panels treated with cycloheximide fail to respond to F2/5 altogether, and a further 40% show a reduced response characterized by spotty collapse of the infiltrated region (Table 4).

Table 4 - Effect of Plant Metabolic Inhibitors on Tobacco HR

HR Response³

Inhibitor Positive Intermediate Negative %Reduction

Cobalt Chloride 0 2 19 100

Orthovanadate 2 6 13 90.5

Cycloheximide 9 8 4 57.1

Cumulative totals from 3 independent experiments.

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 watersoa ing was no longer apparent (approximately 30 minutes) A vitis strain T2/5 was then infiltrated into the treated area

Growth tests in potato-dextrose broth medium amended with cobalt chloride and sodium orthovanadate inhibitors indicate that 5x10^"5 M cobalt chloride is bacteriostatic and 5x10 M sodium orthovanadate has no effect on bacterial growth. After 24 hours in cobalt chloride-amended medium, viable bacteria counts remained the same as at the start of the assay. This indicates a lack of gross effects on the bacteria and supports the idea that the necrosis- blocking effects of these chemicals are due to inhibition of plant signaling pathways.

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 OD₆₀₀ 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.

Therefore, host 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 incoφorated by reference). This suggests that the necrotic response is very different from maceration by polygalacturonase. Maceration causes plant cell death and tissue dissolution by weakening the cell wall to the extent that turgor pressure causes the cell to rupture. This process does not require any active response by the plant, and is, therefore, not blocked by plant metabolic inhibitors.

Example 13 - Mutagenesis of A. vitis.

Nine Tn5 mutants were identified that exhibited altered grape necrosis and/or HR phenotypes (Figures 3, 4 and 5, Table 5).

Table 5. Necrosis and HR phenotypes of F2/5 mutants on different Vitis spp. and Nicotianna spp.

No. Explants with different necrosis ratings ^a Hypersensitive

Strain/mutant response on

V. vinifera V. labrusca V. riparia Nicotianna spp. ^b

F2/5 14-6-0 18-2-0 14-6-0 +

6 2-18-0 13-7-0 4-16-0 -

675 0-0-20 0-0-20 0-0-20 -

816 12-3-5 15-3-2 8-5-7 +

832 3-6-11 11-7-2 2-4-14 -

852 10-8-2 17-2-1 13-5-2 -

901 7-13-0 19-1-0 5-14-1 -

1123 15-4-1 7-13-0 6-11-3 - (+)^C

1154 0-0-20 0-8-12 0-0-20 -

1320 6-10-4 11-6-3 3-10-7 -

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.

Southern blots of mutants showed that at least eight of the Tn5 insertions occurred at different EcoRI-defined loci (Figure 6). Mutants differed in the degree of necrosis they caused on grape species. Seven mutants did not cause HR but still induced various levels of grape necrosis.

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).

Example 14 - Conservation of Necrosis and HR-Associated TnS Loci Within A. vitis.

Probes derived from partial sequences of necrosis and HR- associated EcoRI loci are described in Table 6 (see Figures 1 1 A and B).

Table 6. Probes derived from 7«5-containing EcoRI loci of F2/5 that are associated with tobacco HR and grape necrogenesis.

EcoRI Probe Primer sequences Homologue/% homology at locus Length nucleotide level

(bp)

6 563 5' CGAATGGTTGCTTCCAG 3' (SΕQ. ID. No homologue over 44 bp in

No. 88)

5' GCTGTACCAGCAAACAG 3' (SEQ. ID. database No.89)

675 862 5' GGAAAAATGGCCGCAGGTC 3' Rhizobium etli. pyruvate

(SEQ. ID. No.90)

5' CGAGATCCCGCTTCAGA 3' (SEQ. carboxylase PVC gene/84% ID. No. 91 )

816 332 5' GAACGCGACGAACGCCATCG 3' R. tropici uridylytransferase

(SEQ. ID. No. 92)

5' ACCTCCGACAGGCCGGGACGGGC 3' glnD gene/79% (SEQ. ID. No.93)

832 861 5' TGGCATCTGGACGAAGC 3' (SEQ. ID. R. leguminosarum insertion

No.94)

5' GCAGTCACCACACGTTTG 3' (SEQ. sequence ISRle39d/87% ID. No.95)

852 574 5' TCAGCCTTCAACTTGTCGTCG 3' No homologue over 40 bp in

(SEQ. ID. No.96)

5' GGATAGATGTGCGAGGTGCG 3' database (SEQ. ID. No. 97) .

Southern analysis revealed a diverse pattern of conservation and restriction- fragment length polymoφhism for loci 6, 675, 816, 832, and 852 within A. vitis (Figure 6, summarized in Table 7). Table 7. Summation of EcoRI Southern blot data⁹

A. vitis A A. Strain tumefaciens rhizogenes e

Tn5 CG49 CG78 K306 CG523 CG561 F25 C58 K84 mutant

+, 2 +, + + bands RFLP

675 + +^l + + + ND

816 +, + + + + + +/ + RFLP RFLP

832 - - +/ + -

RFLP RFLP

852 +/ + +, + + .

RFLP RFLP ^a A strain was scored as '+' for homology to a particular probe if hybridization was observed, 'RFLP' indicates a restriction-fragment length polymoφhism 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 Hindlll 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. For non-A. vitis strains, only probe 816 gave a faint band, in A. tumefaciens strain C58 and ,4. rhizogenese strain K84. None of the probes hybridized to E. amylovora strain ΕaFBOl.

Example 15 - A hrcV 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 incoφorated by reference). Southern blot experiments were performed to identify putative brc homologs in A. vitis. Initial experiments utilized a 1.6 kb EcoRI -Pstl fragment that contains the C-terminus of the E. amylovora HφJ and the highly-conserved N-terminal region of HrcV. Low- stringency blots using this probe revealed many hybridizing bands, making firm conclusions about the presence of hrcV homologues in vitis untenable. Therefore, it was decided to attempt PCR amplification of a portion of the highly- conserved HrcV N-terminus using degenerate primers based on the HrcV consensus sequence (Bogdanove et al., J. Bacteriol. 178(6): 1720- 1730 (1996), which is hereby incoφorated by reference). Degenerate primers were designed to three highly-conserved regions of the HrcV N-terminus. One primer pair, F2-R2, produced an amplification product of the predicted size (134 bp) from all A. vitis strains and a similar-sized product from . 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.

BlastX searches of the NCBI database revealed homology between the 134 bp A. vitis amplicon and type III secretory genes involved in flagellar biosynthesis and pathogenicity. The 200 bp amplicon shows homology to 3- oxoadipate CoA-succinyl transferase α-subunits. The 950 bp fragment is homologous to the β-lactamase regulatory gene mazG.

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). The exceptions are Shigella flexneri MxiA, which forms its own branch, and the 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 brcF-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 ΕaFBOl , and hybridizes to the expected brc -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. Both HR and grape necrosis were inhibited by pre-treatment of plants with different eukaryotic metabolic inhibitors (Herlache, "Biochemical and Molecular Genetic Investigations of ^'the Agrobacterium F/tύ-Grapevine Interaction," Ph.D. Thesis. Cornell University (1999), which is hereby incoφorated by reference).

Although 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 10⁹ CFU per ml for the HR (Νissinen et al., Phytopathology. 87(7):678-684 (1997), which is hereby incoφorated by reference) and the non-macerogenic out -mutant E. chrysanthemi requires about 5x10 CFU per ml (Bauer et al., Mol. Plant-Microbe Interact., 7(5):573-581 (1994), which is hereby incoφorated by reference). Growth media and suspension buffers are known to affect the inoculum dose for HR (Wei et al., J. Bacteriol., 174(6): 1875- 1882 (1992), which is hereby incoφorated by reference); however, the growth media and buffers that were tested thus far did not affect the inoculum dose necessary for A. vitis HR. Several lines of evidence demonstrate that the A. vitis HR collapse is not an artifact caused by high inoculum doses. Equivalent concentrations of the closely-related A. tumefaciens strain C58 fail to elicit a HR or grape necrosis, neither do E. coli strains expressing A. vitis plant-cell-wall degrading enzymes. Furthermore, several A. vitis strains tested in this report are weak elicitors of the HR, and these strains occasionally failed to elicit necrosis in leaves responsive to the more-consistent HR-eliciting strains such as F2/5 and CG561. This last observation demonstrates that there is nothing inordinately phytotoxic about high inoculum doses of A. vitis.

Infiltration in mannitol or mannitol plus ammonium nitrate did not interfere with HR induction by A. vitis strain F2/5. This suggests that a low C:N ratio in the infiltrated area is not inhibitory to HR development and further suggests that sensing of low nitrogen availability is not important in regulation of the A. vitis HR-inducing mechanism. A low C:N ratio in the rhizosphere prevents the induction of chlorosis in bean plants treated with Rhizobium tropici strain CIAT899 (O'Connell et al., Appl. and Env. Micro. 59:2184-2189 (1993), which is hereby incoφorated by reference), and readily-utilized nitrogen sources repress E. 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 incoφorated by reference). This suggests that the A. vitis HR-induction mechanism is regulated differently than the R. tropici chlorosis mechanism and the E. amylovora Hφ 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. vitis strains were generally less efficient than nontumorigenic strains at inducing HR on N. tabaccum. It was previously demonstrated that prior infiltration of tobacco with tumorigenic A. tumefaciens or Pseudomonas syringae pv. savastanoi inhibits HR caused by P. syringae pv.phaseolicola (Robinette et al., J. Bacteriol., 172:5742-5749 (1990), which is hereby incoφorated by reference). In this case, inhibition was dependent on the presence of functional tms genes. It may be that tumorigenic strains have evolved toward a reduced HR eliciting ability to avoid killing plant cells targeted for transformation. However, it has also been shown that strain CG49, which is a weak HR inducer on N. tabaccum, is highly efficient on N. benthamiana. Therefore, 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. In most cases 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 incoφorated by reference). Mutations in the secretion system render the mutant unable to elicit an HR and to cause disease on host plants. The HR induced by C michigense subsp. 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 incoφorated 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. Plant-Microbe Interact., 3(2): 1 12-121 (1990), which is hereby incoφorated by reference). 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 incoφorated by reference). An A. vitis PCR product with homology to a highly- conserved Ν-terminal region of hrcV was identified. This sequence is also related to the Ν-terminus of E. coliflhA and the Rhizobium sp. ΝGR243 Y4YR nolT gene. It is speculated that the band that hybridizes to the brcF-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 that affected the HR and necrosis phenotypes were identified in at least 8 different EcoRI-defined loci showing that these phenotypes are probably associated with several genes. In all but one case, single mutations affected both necrosis and HR indicating that these phenotypes share an underlying mechanism.

The interesting array of phenotypes of the vitis mutants on different Vitis and Nicotianna species suggests that plant species are responding differently to either multiple elicitors or to different moieties on the same elicitor. For example the phenotype of mutants 816 (necrosis altered but still able to induce tobacco HR) shows that HR- associated factor(s) alone is not sufficient for necrosis. In contrast, the other mutants that have altered necrosis phenotypes fail to cause HR. Again, these observations are different from what one would be expected if A. vitis had the classical hrp systems found in P. syringae pv. syringae or E amylovora. 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.

Because all A. vitis strains cause grape necrosis, it was expected that related loci would be highly-conserved within strains. Southern blots however, reveal a suφrising 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. This correlates well with the observed poor HR-eliciting efficiency of these. A. vitis strain CG49 is the weakest HR elicitor (Herlache, "Biochemical and Molecular Genetic Investigations of the Agrobacterium ztw-Grapevine Interaction," Ph.D. Thesis, Cornell University (1999), which is hereby incoφorated by reference), and this strain has the most changes at these loci.

A model of the observed mutant phenotypes can be built around the well-characterized production of nodulation signals by Rhizobium species (Pueppke, Crit. Rev. Biotechnology, 16:1-51 (1996), which is hereby incorporated by reference), which are close relatives of Agrobacterium. 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 nodPO (Horvath et al., Cell. (1987), which is hereby incoφorated by reference) or changes in the lipid tail caused by mutation in nodFE (Spaink et al., Nature 354:125-130 (1991), which is hereby incoφorated 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 suφrising 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). In the proposed model, 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

Interact., 7:684 et seq. (1994), which is hereby incoφorated by reference) or some peripheral moiety necessary for recognition by grape and tobacco.

The fact that A. vitis 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 inteφretation. Growth studies with cobalt chloride indicated that, when used at concentrations that are inhibitory to necrosis, it is bacteriostatic. If induction of host necrosis requires that bacteria multiply inplanta, then cobalt inhibition may be due to lack of bacterial division and not cobalt's blocking of plant calcium-signal transduction. This, however, seems unlikely because a very high inoculum dose (approximately 10⁸ CFU/ml) was used. This is near the typically observed population density reported at the peak of many bacterial infections, for example with P. syringae leaf infections (Klement et al., Phytopathology, 54:474-477 (1964), which is hereby incoφorated by reference) and would, therefore, seem to preclude the necessity for population increase to provoke symptoms. Sodium orthovanadate, although not bacteriostatic, caused tissue burning at inoculation sites. However, this burning occurred at locations different from the tissue that initially responds to A. vitis. The observed inhibition of host symptoms is real and not due to killing of plant cells by sodium orthovanadate. Cycloheximide at 7.1xl0^"9 M, which prevents translation by 80S ribosomes, blocked necrosis on a wide variety of hosts and on different tissue types, and had no obvious effect on plant tissue. 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 incoφorated by reference).

Like an HR elicited by E amylovora, the collapse elicited by A. vitis can be blocked by plant metabolic inhibitors (He et al., Mol. Plant- Microbe Interact., 7(2):289-292 (1994), which is hereby incoφorated by reference). Cobalt chloride provided excellent collapse inhibition at the same concentration~5xl0^"4 M~reported to inhibit the Erwinia HR (He et al., Cell, 73:1255-66 (1993); Wei et al., Science. 257:85-88 (1992), which are hereby incoφorated by reference). Sodium orthovanadate also provided very good inhibition. Cycloheximide at 7.1xl0^"9 M gave some inhibition of collapse. 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, inplanta multiplication is likely not necessary for macroscopic HR-elicitation because of the high inoculum doses used. This indicates that A. v/^'t/^'s-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. v/t/s-induced collapse. Tetracycline applied to the infiltrated area before 4 hours post-inoculation totally blocks the A. v/tz^'s-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 A. vitis.

It is interesting that 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. Also, 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 incoφorated by reference). Inhibition was dependent on the presence of functional tms genes. Thus, 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 incoφorated 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/tw-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. K /s-Induced Resistance in Vitis vinifera Against Plasmopara viticola.

The phenomenon of systemic acquired resistance (SAR) can reduce the plant diseases caused by a broad range of pathogens. SAR in grape (Vitis vinifera) was induced by inoculating leaves with vitis strain F2/5. Leaves were sprayed with F2/5 (10⁹ cfu/ml) in adjuvant Break-thru. Localized necrotic lesions were visible within 4 days. Disease suppression was evaluated on excised leaf discs by subsequent challenge with Plasmopara viticola. A reproducile, induced resistance response occurred 7 days after treatment with Agrobacterium vitis. Maximum disease severity reduction was 36% after treatment with A. vitis. The results of this experiment are shown below in Table 8:

Table 8 - Agrobacterium Induced Resistance

Trt incidence S dia s sa S (mm) (mm²)

Check 9.1 0.7 a 2.5 0.5 a 65 20 a upper (S)

Check 9.8 0.3 ac 3.3 0.4 be 94 22 be lower (D)

A. vitis 8.1 0.6 bde 1.8 0.4 bde 42 15 bde upper (S)

A. vitis 9.4 0.5 acf 2.7 .05 acf 71 19 adf lower (D)

A. vitis significantly reduces disease on both directly treated and "systemically protected" leaves.

24% severity reduction on directly treated leaves as compared to checks. 36% severity reduction on "systemically protected" leaves.

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.

Example 18 - Agrobacterium vitis Elicits a Hypersensitive Response on Tobacco.

Agrobacterium vitis is the primary causal agent of crown gall on grape. One unique characteristic of A. vitis 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. When A. vitis strains were infiltrated into tobacco leaves, an HR was elicited. The A. vitis- induced HR requires an inoculum dose of about 4x10 CFU/ml, which is greater than required for HR induction by some other Gram-negative bacteria such as Pseudomonas syringae pv. syringae. Following tobacco leaf infiltration, 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 inplanta 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.

Although the invention has been described in detail for the puφose of illustration, it is understood that such detail is solely for that puφose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims

WHAT IS CLAIMED:

1. An isolated protein or polypeptide from Agrobacterium associated with production of a hypersensitive response.

2. An isolated protein or polypeptide according to claim 1, wherein the protein or polypeptide is derived from Agrobacterium vitis.

3. An isolated protein or polypeptide according to claim 1 , wherein the protein or polypeptide has an amino acid sequence of SEQ. ID. No. 1 or SEQ. ID. No. 2 or SEQ. ID. No. 3 or SEQ. ID. No. 4 or SEQ. ID. No. 5 or

SEQ. ID. No. 7 or SEQ. ID. No. 8 or SEQ. ID. No. 9 or SEQ. ID. No. 10 or SEQ.

ID. No. 12 or SEQ. ID. No. 13 or SEQ. ID. No. 15 or SEQ. ID. No. 17 or SEQ.

ID. No. 18 or SEQ. ID. No. 20 or SEQ. ID. No. 21 or SEQ. ID. No. 22 or SEQ. ID. No. 23 or SEQ. ID. No. 25 or SEQ. ID. No. 26 or SEQ. ID. No. 28 or SEQ.

ID. No. 29 or SEQ. ID. No. 30 or SEQ. ID. No. 31 or SEQ. ID. No. 33 or SEQ.

ID. No. 34 or SEQ. ID. No. 35 or SEQ. ID. No. 37 or SEQ. ID. No. 38 or SEQ.

ID. No. 40 or SEQ. ID. No. 42 or SEQ. ID. No. 44 or SEQ. ID. No. 45 or SEQ.

ID. No. 46 or SEQ. ID. No. 47 or SEQ. ID. No. 49 or SEQ. ID. No. 50 or SEQ. ID. No. 52 or SEQ. ID. No. 54 or SEQ. ID. No. 56 or SEQ. ID. No. 57 or SEQ.

ID. No. 58 or SEQ. ID. No. 59 or SEQ. ID. No. 61 or SEQ. ID. No. 62 or SEQ.

ID. No. 63 or SEQ. ID. No. 64 or SEQ. ID. No. 66 or SEQ. ID. No. 69 or SEQ.

ID. No. 70 or SEQ. ID. No. 71 or SEQ. ID. No. 72 or SEQ. ID. No. 74 or SEQ.

ID. No. 75 or SEQ. ID. No. 76 or SEQ. ID. No. 77 or SEQ. ID. No. 78 or SEQ. ID. No. 80 or SEQ. ID. No. 82.

4. An isolated DNA molecule encoding a protein or polypeptide according to claim 1.

5. An isolated DNA molecule according to claim 4, wherein the protein or polypeptide is from an Agrobacterium vitis.

6. An isolated DNA molecule according to claim 4, wherein the protein or polypeptide has an amino acid sequence of SEQ. ID. No. 1 or SEQ. ID. No. 2 or SEQ. ID. No. 3 or SEQ. ID. No. 4 or SEQ. ID. No. 5 or SEQ. ID. No. 7 or SEQ. ID. No. 8 or SEQ. ID. No. 9 or SEQ. ID. No. 10 or SEQ. ID. No. 12 or SEQ. ID. No. 13 or SEQ. ID. No. 15 or SEQ. ID. No. 17 or SEQ. ID. No. 18 or SEQ. ID. No. 20 or SEQ. ID. No. 21 or SEQ. ID. No. 22 or SEQ. ID. No. 23 or SEQ. ID. No. 25 or SEQ. ID. No. 26 or SEQ. ID. No. 28 or SEQ. ID. No. 29 or SEQ. ID. No. 30 or SEQ. ID. No. 31 or SEQ. ID. No. 33 or SEQ. ID. No. 34 or SEQ. ID. No. 35 or SEQ. ID. No. 37 or SEQ. ID. No. 38 or SEQ. ID. No. 40 or SEQ. ID. No. 42 or SEQ. ID. No. 44 or SEQ. ID. No. 45 or SEQ. ID. No. 46 or SEQ. ID. No. 47 or SEQ. ID. No. 49 or SEQ. ID. No. 50 or SEQ. ID. No. 52 or SEQ. ID. No. 54 or SEQ. ID. No. 56 or SEQ. ID. No. 57 or SEQ. ID. No. 58 or SEQ. ID. No. 59 or SEQ. ID. No. 61 or SEQ. ID. No. 62 or SEQ. ID. No. 63 or SEQ. ID. No. 64 or SEQ. ID. No. 66 or SEQ. ID. No. 69 or SEQ. ID. No. 70 or SEQ. ID. No. 71 or SEQ. ID. No. 72 or SEQ. ID. No. 74 or SEQ. ID. No. 75 or SEQ. ID. No. 76 or SEQ. ID. No. 77 or SEQ. ID. No. 78 or SEQ. ID. No. 80 or SEQ. ID. No. 82.

7. An isolated DNA molecule according to claim 6, wherein the DNA molecule has a nucleotide sequence of SEQ. ID. No. 6 or SEQ. ID.

No. 11 or SEQ. ID. No. 14 or SEQ. ID. No. 16 or SEQ. ID. No. 19 or SEQ. ID. No. 24 or SEQ. ID. No. 27 or SEQ. ID. No. 32 or SEQ. ID. No. 36 or SEQ. ID. No. 39 or SEQ. ID. No. 41 or SEQ. ID. No. 43 or SEQ. ID. No. 48 or SEQ. ID. No. 51 or SEQ. ID. No. 53 or SEQ. ID. No. 55 or SEQ. ID. No. 60 or SEQ. ID. No. 65 or SEQ. ID. No. 67 or SEQ. ID. No. 68 or SEQ. ID. No. 73 or SEQ. ID. No. 79 or SEQ. ID. No. 81 or SEQ. ID. No. 83, or hybridizes under stringent conditions to a nucleotide sequence of SEQ. ID. No. 6 or SEQ. ID. No. 11 or SEQ. ID. No. 14 or SEQ. ID. No. 16 or SEQ. ID. No. 19 or SEQ. ID. No. 24 or SEQ. ID. No. 27 or SEQ. ID. No. 32 or SEQ. ID. No. 36 or SEQ. ID. No. 39 or SEQ. ID. No. 41 or SEQ. ID. No. 43 or SEQ. ID. No. 48 or SEQ. ID. No. 51 or SEQ. ID. No. 53 or SEQ. ID. No. 55 or SEQ. ID. No. 60 or SEQ. ID. No. 65 or SEQ. ID. No. 67 or SEQ. ID. No. 68 or SEQ. ID. No. 73 or SEQ. ID. No. 79 or SEQ. ID. No. 81 or SEQ. ID. No. 83.

8. An expression system comprising a DNA molecule according to claim 4.

9. An expression system according to claim 8, wherein said

DNA molecule is in sense orientation and correct reading frame.

10. An expression system according to claim 8, wherein said DNA molecule is in antisense orientation.

1 1. An expression system according to claim 8, wherein said DNA molecule is nontranslatable.

12. A host cell comprising a DNA molecule according to claim 4.

13. A host cell according to claim 12, wherein the host cell is selected from the group consisting of a plant cell and a bacterial cell.

14. A host cell according to claim 12, wherein the DNA molecule is transformed in an expression system.

15. A transgenic plant transformed with the DNA molecule of claim 4.

16. A transgenic plant according to claim 15, wherein the plant is selected from the group consisting of alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, 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.

17. A transgenic plant according to claim 15, wherein the plant is selected from the group consisting of Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.

18. A transgenic plant seed comprising the DNA molecule of claim 4.

19. A transgenic plant seed according to claim 18, wherein the plant seed is selected from the group consisting of alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, 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.

20. A transgenic plant seed according to claim 18, wherein the plant seed is selected from the group consisting of Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.

21. A method of imparting disease resistance to plants comprising: applying a protein or polypeptide according to claim 1 in a non-infectious form to a plant or plant seed under conditions effective to impart disease resistance.

22. A method according to claim 21 , wherein plants are treated during said applying.

23. A method according to claim 21 , wherein plant seeds are treated during said applying, said method further comprising: planting the seeds treated with the protein or polypeptide in natural or artificial soil and propagating plants from the seeds planted in the soil.

24. A method of enhancing plant growth comprising: applying a protein or polypeptide according to claim 1 in a non-infectious form to a plant or plant seed under conditions effective to enhance plant growth.

25. A method according to claim 24, wherein plants are treated during said applying.

26. A method according to claim 24, wherein plant seeds are treated during said applying, said method further comprising: planting the seeds treated with the protein or polypeptide in natural or artificial soil and propagating plants from the seeds planted in the soil.

27. A method of insect control for plants comprising: applying a protein or polypeptide according to claim 1 in a non-infectious form to a plant or plant seed under conditions effective to control insects.

28. A method according to claim 27, wherein plants are treated during said applying.

29. A method according to claim 27, wherein plant seeds are treated during said applying, said method further comprising: planting the seeds treated with the protein or polypeptide in natural or artificial soil and propagating plants from the seeds planted in the soil.

30. A method of imparting stress resistance to plants comprising: applying a protein or polypeptide according to claim 1 in a non-infectious form to a plant or plant seed under conditions effective to impart stress resistance.

31. A method according to claim 30, wherein plants are treated during said applying.

32. A method according to claim 30, wherein plant seeds are treated during said applying, said method further comprising: planting the seeds treated with the protein or polypeptide in natural or artificial soil and propagating plants from the seeds planted in the soil.

33. A method of enhancing nutritional value of plants comprising: applying a protein or polypeptide according to claim 1 in a non-infectious form to a plant or plant seed under conditions effective to enhance nutritional value of the plant.

34. A method according to claim 33, wherein plants are treated during said applying.

35. A method according to claim 33, wherein plant seeds are treated during said applying, said method further comprising: planting the seeds treated with the protein or polypeptide in natural or artificial soil and propagating plants from the seeds planted in the soil.

36. A method of imparting disease resistance to plants comprising: providing a transgenic plant or plant seed transformed with a DNA molecule according to claim 4 and growing the transgenic plant or transgenic plants produced from the transgenic plant seeds under conditions effective to impart disease resistance.

37. A method according to claim 36, wherein a transgenic plant is provided.

38. A method according to claim 36, wherein a transgenic plant seed is provided.

39. A method of enhancing plant growth comprising: providing a transgenic plant or a plant seed transformed with a DNA molecule according to claim 4 and growing the transgenic plant or transgenic plants produced from the transgenic plant seeds under conditions effective to enhance plant growth.

40. A method according to claim 39, wherein a transgenic plant is provided.

41. A method according to claim 39, wherein a transgenic plant seed is provided.

42. A method of insect control for plants comprising: providing a transgenic plant or plant seed transformed with a DNA molecule according to claim 4 and growing the transgenic plant or transgenic plants produced from the transgenic plant seeds under conditions effective to control insects.

43. A method according to claim 42, wherein a transgenic plant is provided.

44. A method according to claim 43, wherein a transgenic plant seed is provided.

45. A method of imparting stress resistance to plants comprising: providing a transgenic plant or plant seed transformed with a DNA molecule according to claim 4 and growing the transgenic plant or transgenic plants produced from the transgenic plant seeds under conditions effective to impart stress resistance.

46. A method according to claim 45, wherein a transgenic plant is provided.

47. A method according to claim 45, wherein a transgenic plant seed is provided.

48. A method of enhancing nutritional value of a plant comprising: providing a transgenic plant or a plant seed transformed with a DNA molecule according to claim 4 and growing the transgenic plant or transgenic plants produced from the transgenic plant seeds under conditions effective to enhance nutritional value of the plant.

49. A method according to claim 48, wherein a transgenic plant is provided.

50. A method according to claim 48, wherein a transgenic plant seed is provided.