Hypersensitive response induced resistance in plants by seed treatment

FIELD OF THE INVENTION

[0003] The present invention relates to imparting hypersensitive response induced resistance to plants by treatment of seeds.

BACKGROUND OF THE INVENTION

[0004] Living organisms have evolved a complex array of biochemical pathways that enable them to recognize and respond to signals from the environment. These pathways include receptor organs, hormones, second messengers, and enzymatic modifications. At present, little is known about the signal transduction pathways that are activated during a plant's response to attack by a pathogen, although this knowledge is central to an understanding of disease susceptibility and resistance. A common form of plant resistance is the restriction of pathogen proliferation to a small zone surrounding the site of infection. In many cases, this restriction is accompanied by localized death (i.e., necrosis) of host tissues. Together, pathogen restriction and local tissue necrosis characterize the hypersensitive response. In addition to local defense responses, many plants respond to infection by activating defenses in uninfected parts of the plant. As a result, the entire plant is more resistant to a secondary infection. This systemic acquired resistance can persist for several weeks or more (R. E. F. Matthews, Plant Virology (Academic Press, New York, ed. 2, 1981)) and often confers cross-resistance to unrelated pathogens (J. Kuc, in Innovative Approaches to Plant Disease Control, I. Chet, Ed. (Wiley, N.Y., 1987), pp. 255-274, which is hereby incorporated by reference). See also Kessman, et al., “Induction of Systemic Acquired Disease Resistance in Plants By Chemicals,” Ann. Rev. Phytopathol. 32:439-59 (1994), Ryals, et al., “Systemic Acquired Resistance,” The Plant Cell 8:1809-19 (October 1996), and Neuenschwander, et al., “Systemic Acquired Resistance,” Plant-Microbe Interactions vol. 1, G. Stacey, et al. ed. pp. 81-106 (1996), which are hereby incorporated by reference.

[0005] Expression of systemic acquired resistance is associated with the failure of normally virulent pathogens to ingress the immunized tissue (Kuc, J., “Induced Immunity to Plant Disease,” Bioscience, 32:854-856 (1982), which is hereby incorporated by reference). Establishment of systemic acquired resistance is correlated with systemic increases in cell wall hydroxyproline levels and peroxidase activity (Smith, J. A., et al., “Comparative Study of Acidic Peroxidases Associated with Induced Resistance in Cucumber, Muskmelon and Watermelon,” Physiol. Mol. Plant Pathol. 14:329-338 (1988), which is hereby incorporated by reference) and with the expression of a set of nine families of so-called systemic acquired resistance gene (Ward, E. R., et al., “Coordinate Gene Activity in Response to Agents that Induce Systemic Acquired Resistance,” Plant Cell 3:49-59 (1991), which is hereby incorporated by reference). Five of these defense gene families encode pathogenesis-related proteins whose physiological functions have not been established. However, some of these proteins have antifungal activity in vitro (Bol, J. F., et al., “Plant Pathogenesis-Related Proteins Induced by Virus Infection,” Ann. Rev. Phytopathol. 28:113-38 (1990), which is hereby incorporated by reference) and the constitutive expression of a bean chitinase gene in transgenic tobacco protects against infection by the fungus Rhizoctonia solani (Broglie, K., et al., “Transgenic Plants with Enhanced Resistance to the Fungal Pathogen Rhizoctonia Solani,” Science 254:1194-1197 (1991), which is hereby incorporated by reference), suggesting that these systemic acquired resistance proteins may contribute to the immunized state (Uknes, S., et al., “Acquired Resistance in Arabidopsis,” Plant Cell 4:645-656 (1992), which is hereby incorporated by reference)

[0006] Salicylic acid appears to play a signal function in the induction of systemic acquired resistance since endogenous levels increase after immunization (Malamy, J., et al., “Salicylic Acid: A Likely Endogenous Signal in the Resistance Response of Tobacco to Viral Infection,” Science 250:1002-1004 (1990), which is hereby incorporated by reference) and exogenous salicylate induces systemic acquired resistance genes (Yalpani, N., et al., “Salicylic Acid is a Systemic Signal and an Inducer of Pathogenesis-Related Proteins in Virus-Infected Tobacco,” Plant Cell 3:809-818 (1991), which is hereby incorporated by reference), and acquired resistance (Uknes, S., et al., “Acquired Resistance in Arabidopsis,” Plant Cell 4:645-656 (1992), which is hereby incorporated by reference). Moreover, transgenic tobacco plants in which salicylate is destroyed by the action of a bacterial transgene encoding salicylate hydroxylase do not exhibit systemic acquired resistance (Gaffney, T., et al., “Requirement of Salicylic Acid for the Induction of Systemic Acquired Resistance,” Science 261:754-56 (1993), which is hereby incorporated by reference). However, this effect may reflect inhibition of a local rather than a systemic signal function, and detailed kinetic analysis of signal transmission in cucumber suggests that salicylate may not be essential for long-distance signaling (Rasmussen, J. B., et al., “Systemic Induction of Salicylic Acid Accumulation in Cucumber after Inoculation with Pseudomonas Syringae pv. Syringae,” Plant Physiol. 97:1342-1347) (1991), which is hereby incorporated by reference).

[0007] Immunization using biotic agents has been extensively studied. Green beans were systemically immunized against disease caused by cultivar-pathogenic races of Colletotrichum lindemuthianum by prior infection with either cultivar-nonpathogenic races (Rahe, J. E., “Induced Resistance in Phaseolus Vulgaris to Bean Anthracnose,” Phytopatholoqy 59:1641-5 (1969); Elliston, J., et al., “Induced Resistance to Anthracnose at a Distance from the Site of the Inducing Interaction,” Phytopathology 61:1110-12 (1971); Skipp, R., et al., “Studies on Cross Protection in the Anthracnose Disease of Bean,” Physiological Plant Pathology 3:299-313 (1973), which are hereby incorporated by reference), cultivar-pathogenic races attenuated by heat in host tissue prior to symptom appearance (Rahe, J. E., et al., “Metabolic Nature of the Infection-Limiting Effect of Heat on Bean Anthracnose,” Phytopathology 60:1005-9 (1970), which is hereby incorporated by reference) or nonpathogens of bean. The anthracnose pathogen of cucumber, Colletotrichum lagenarium, was equally effective as non-pathogenic races as an inducer of systemic protection against all races of bean anthracnose. Protection was induced by C. lagenarium in cultivars resistant to one or more races of C. lindemuthianum as well as in cultivars susceptible to all reported races of the fungus and which accordingly had been referred to as ‘lacking genetic resistance’ to the pathogen (Elliston, J., et al., “Protection of Bean Against Anthracnose by Colletotrichum Species Nonpathogenic on Bean,” Phytopathologische Zeitschrift 86:117-26 (1976); Elliston, J., et al., “A Comparative Study on the Development of Compatible, Incompatible and Induced Incompatible Interactions Between Collectotrichum Species and Phaseolus Vulgaris,” Phytopathologische Zeitschrift 87:289-303 (1976), which are hereby incorporated by reference). These results suggest that the same mechanisms may be induced in cultivars reported as ‘possessing’ or ‘lacking’ resistance genes (Elliston, J., et al., “Relation of Phytoalexin Accumulation to Local and Systemic Protection of Bean Against Anthracnose,” Phytopathologische Zeitschrift 88:114-30 (1977), which is hereby incorporated by reference). It also is apparent that cultivars susceptible to all races of C. lindemuthianum do not lack genes for induction of resistance mechanisms against the pathogen.

[0008] Kuc, J., et al., “Protection of Cucumber Against Collectotrichum Lagenarium by Colletotrichum Lagenarium,” Physiological Plant Pathology 7:195-9 (1975), which is hereby incorporated by reference), showed that cucumber plants could be systemically protected against disease caused by Colletotrichum lagenarium by prior inoculation of the cotyledons or the first true leaf with the same fungus. Subsequently, cucumbers have been systemically protected against fungal, bacterial, and viral diseases by prior localized infection with either fungi, bacteria, or viruses (Hammerschmidt, R., et al., “Protection of Cucumbers Against Colletotrichum Lagenarium and Cladosporium Cucumerinum,” Phytopathology 66:790-3 (1976); Jenns, A. E., et al., “Localized Infection with Tobacco Necrosis Virus Protects Cucumber Against Colletotrichum Lagenarium,” Physiological Plant Pathology 11:207-12 (1977); Caruso, F. L., et al. “Induced Resistance of Cucumber to Anthracnose and Angular Leaf Spot by Pseudomonas Lachrymans and Colletotrichum Lagenarium,” Physiological Plant Pathology 14:191-201 (1979); Staub, T., et al., “Systemic Protection of Cucumber Plants Against Disease Caused by Cladosporium Cucumerinum and Colletotrichum Lagenarium by Prior Localized Infection with Either Fungus,” Physiological Plant Pathology, 17:389-93 (1980); Bergstrom, G. C., et al., “Effects of Local Infection of Cucumber by Colletotrichum Lagenarium, Pseudomonas Lachrymans or Tobacco Necrosis Virus on Systemic Resistance to Cucumber Mosaic Virus,” Phytopathology 72:922-6 (1982); Gessler, C., et al., “Induction of Resistance to Fusarium Wilt in Cucumber by Root and Foliar Pathogens,” Phytopathology 72:1439-41 (1982); Basham, B., et al., “Tobacco Necrosis Virus Induces Systemic Resistance in Cucumbers Against Sphaerotheca Fuliginea,” Physiological Plant Pathology 23:137-44 (1983), which are hereby incorporated by reference). Non-specific protection induced by infection with C. lagenarium or tobacco necrosis virus was effective against at least 13 pathogens, including obligatory and facultative parasitic fungi, local lesion and systemic viruses, wilt fungi, and bacteria. Similarly, protection was induced by and was also effective against root pathogens. Other curcurbits, including watermelon and muskmelon have been systemically protected against C. lagenarium (Caruso, F. L., et al., “Protection of Watermelon and Muskmelon Against Colletotrichum Lagenarium by Colletotrichum Lagenarium,” Phytopathology 67:1285-9 (1977), which is hereby incorporated by reference).

[0009] Systemic protection in tobacco has also been induced against a wide variety of diseases (Kuc, J., et al., “Immunization for Disease Resistance in Tobacco,” Recent Advances in Tobacco Science 9:179-213 (1983), which is hereby incorporated by reference). Necrotic lesions caused by tobacco mosaic virus enhanced resistance in the upper leaves to disease caused by the virus (Ross, A. F., et al., “Systemic Acquired Resistance Induced by Localized Virus Infections in Plants,” Virology 14:340-58 (1961); Ross, A. F., et al., “Systemic Effects of Local Lesion Formation,” In: Viruses of Plants pp. 127-50 (1966), which are hereby incorporated by reference). Phytophthora parasitica var. nicotianae, P. tabacina and Pseudomonas tabaci and reduced reproduction of the aphid Myzus persicae (McIntyre, J. L.,. et al., “Induction of Localized and Systemic Protection Against Phytophthora Parasitica var. nicotianae by Tobacco Mosaic Virus Infection of Tobacco Hypersensitive to the Virus,” Physiological Plant Pathology 15:321-30 (1979); McIntyre, J. L., et al., “Effects of Localized Infections of Nicotiana Tabacum by Tobacco Mosaic Virus on Systemic Resistance Against Diverse Pathogens and an Insect,” Phytopathology 71:297-301 (1981), which are hereby incorporated by reference). Infiltration of heat-killed Pseudomonas tabacin (Lovrekovich, L., et al., “Induced Reaction Against Wildfire Disease in Tobacco Leaves Treated with Heat-Killed Bacteria,” Nature 205:823-4 (1965), which is hereby incorporated by reference), and Pseudomonas solanacearum (Sequeira, L, et al., “Interaction of Bacteria and Host Cell Walls: Its Relation to Mechanisms of Induced Resistance,” Physiological Plant Pathology 10:43-50 (1977), which is hereby incorporated by reference), into tobacco leaves induced resistance against the same bacteria used for infiltration. Tobacco plants were also protected by the nematode Pratylenchus penetrans against P. parasitica var. nicotiana (McIntyre, J. L., et al. “Protection of Tobacco Against Phytophthora Parasitica Var. Nicotianae by Cultivar-Nonpathogenic Races, Cell-Free Sonicates and Pratylenchus Penetrans,” Phytopathology 68:235-9 (1978), which is hereby incorporated by reference).

[0010] Cruikshank, I. A. M., et al., “The Effect of Stem Infestation of Tobacco with Peronospora Tabacina Adam on Foliage Reaction to Blue Mould,” Journal of the Australian Institute of Agricultural Science 26:369-72 (1960), which is hereby incorporated by reference, were the first to report immunization of tobacco foliage against blue mould (i.e., P. tabacina) by stem injection with the fungus, which also resulted in dwarfing and premature senescence. It was recently discovered that injection external to the xylem not only alleviated stunting but also promoted growth and development. Immunized tobacco plants, in both glasshouse and field experiments, were approximately 40% taller, had a 40% increase in dry weight, a 30% increase in fresh weight, and 4-6 more leaves than control plants (Tuzun, S., et al., “The Effect of Stem Injections with Peronospora Tabacina and Metalaxyl Treatment on Growth of Tobacco and Protection Against Blue Mould in the Field,” Phytopathology 74:804 (1984), which is hereby incorporated by reference). These plants flowered approximately 2-3 weeks earlier than control plants (Tuzun, S., et al., “Movement of a Factor in Tobacco Infected with Peronospora Tabacina Adam which Systemically Protects Against Blue Mould,” Physiological Plant Pathology 26:321-30 (1985), which is hereby incorporated by reference).

[0011] Systemic protection does not confer absolute immunity against infection, but reduces the severity of the disease and delays symptom development. Lesion number, lesion size, and extent of sporulation of fungal pathogens are all decreased. The diseased area may be reduced by more than 90%.

[0012] When cucumbers were given a ‘booster’ inoculation 3-6 weeks after the initial inoculation, immunization induced by C. lagenarium lasted through flowering and fruiting (Kuc, J., et al., “Aspects of the Protection of Cucumber Against Colletotrichum Lagenarium by Colletotrichum Lagenarium,” Phytopathology 67:533-6 (1977), which is hereby incorporated by reference). Protection could not be induced once plants had set fruit. Tobacco plants were immunized for the growing season by stem injection with sporangia of P. tabacina. However, to prevent systemic blue mould development, this technique was only effective when the plants were above 20 cm in height.

[0013] Removal of the inducer leaf from immunized cucumber plants did not reduce the level of immunization of pre-existing expanded leaves. However, leaves which subsequently emerged from the apical bud were progressively less protected than their predecessors (Dean, R. A., et al., “Induced Systemic Protection in Cucumber: Time of Production and Movement of the ‘Signal’,” Phytopathology 76:966-70 (1986), which is hereby incorporated by reference). Similar results were reported by Ross, A. F., “Systemic Effects of Local Lesion Formation,” In: Viruses of Plants pp. 127-50 (1966), which is hereby incorporated by reference, with tobacco (local lesion host) immunized against tobacco mosaic virus by prior infection with tobacco mosaic virus. In contrast, new leaves which emerged from scions excised from tobacco plants immunized by stem-injection with P. tabacina were highly protected (Tuzun, S., et al., “Transfer of Induced Resistance in Tobacco to Blue Mould (Peronospora tabacina Adam.) Via Callus,” Phytopathology 75:1304 (1985), which is hereby incorporated by reference). Plants regenerated via tissue culture from leaves of immunized plants showed a significant reduction in blue mould compared to plants regenerated from leaves of non-immunized parents. Young regenerants only showed reduced sporulation. As plants aged, both lesion development and sporulation were reduced. Other investigators, however, did not reach the same conclusion, although a significant reduction in sporulation in one experiment was reported (Lucas, J. A., et al., “Nontransmissibility to Regenerants from Protected Tobacco Explants of Induced Resistance to Peronospora Hyoscyami,” Phytopathology 75:1222-5 (1985), which is hereby incorporated by reference).

[0014] Protection of cucumber and watermelon is effective in the glasshouse and in the field (Caruso, F. L., et al., “Field Protection of Cucumber Against Colletotrichum Lagenarium by C. Lagenarium,” Phytopathology 67:1290-2 (1977), which is hereby incorporated by reference). In one trial, the total lesion area of C. lagenarium on protected cucumber was less than 2% of the lesion areas on unprotected control plants. Similarly, only 1 of 66 protected, challenged plants died, whereas 47 of 69 unprotected, challenged watermelons died. In extensive field trials in Kentucky and Puerto Rico, stem injection of tobacco with sporangia of P. tabacina was at least as effective in controlling blue mould as the best fungicide, metalaxyl. Plants were protected, leading to a yield increase of 10-25% in cured tobacco.

[0015] Induced resistance against bacteria and viruses appears to be expressed as suppression of disease symptoms or pathogen multiplication or both (Caruso, F. L., et al., “Induced Resistance of Cucumber to Anthracnose and Angular Leaf Spot by Pseudomonas Lachrymans and Colletotrichum Lagenarium,” Physiological Plant Pathology 14:191-201 (1979); Doss, M., et al., “Systemic Acquired Resistance of Cucumber to Pseudomonas Lachrymans as Expressed in Suppression of Symptoms, but not in Multiplication of Bacteria,” Acta Phytopathologia Academiae Scientiarum Hungaricae 16: (3-4), 269-72 (1981); Jenns, A. E., et al., “Non-Specific Resistance to Pathogens Induced Systemically by Local Infection of Cucumber with Tobacco Necrosis Virus, Colletotrichum Lagenarium or Pseudomonas Lachrymans,” Phytopathologia Mediterranea 18:129-34 (1979), which are hereby incorporated by reference).

[0016] As described above, research concerning systemic acquired resistance involves infecting plants with infectious pathogens. Although studies in this area are useful in understanding how systemic acquired resistance works, eliciting such resistance with infectious agents is not commercially useful, because such plant-pathogen contact can weaken or kill plants. The present invention is directed to overcoming this deficiency.

SUMMARY OF THE INVENTION

[0017] The present invention relates to a method of producing plant seeds which impart pathogen resistance to plants grown from the seeds. This method involves applying a hypersensitive response elicitor polypeptide or protein in a non-infectious form to plant seeds under conditions where the polypeptide or protein contacts cells of the plant seeds.

[0018] As an alternative to applying a hypersensitive response elicitor polypeptide or protein to plant seeds in order to impart pathogen resistance to plants grown from the seeds, transgenic seeds can be utilized. This involves providing a transgenic plant seed transformed with a DNA molecule encoding a hypersensitive response elicitor polypeptide or protein and planting that seed in soil. A plant is then propagated from the planted seed under conditions effective to impart pathogen resistance to the plant.

[0019] Another aspect of the present invention relates to a pathogen-resistance imparting plant seed to which a non-infectious hypersensitive response elicitor polypeptide or protein has been applied.

[0020] The present invention has the potential to: treat plant diseases which were previously untreatable; treat diseases systemically that one would not want to treat separately due to cost; and avoid the use of agents that have an unpredictable effect on the environment and even the plants. The present invention can impart resistance without using agents which are harmful to the environment or pathogenic to the plant seeds being treated or to plants situated near the location that treated seeds are planted. Since the present invention involves use of a natural product that is fully and rapidly biodegradable, the environment would not be contaminated.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention relates to a method of producing plant seeds which impart pathogen resistance to plants grown from the seeds. This method involves applying a hypersensitive response elicitor polypeptide or protein in a non-infectious form to a plant seed under conditions effective to impart disease resistance to a plant grown from the seed.

[0022] As an alternative to applying a hypersensitive response elicitor polypeptide or protein to plant seeds in order to impart pathogen resistance to plants grown from the seeds, transgenic seeds can be utilized. This involves providing a transgenic plant seed transformed with a DNA molecule encoding a hypersensitive response elicitor polypeptide or protein and planting that seed in soil. A plant is then propagated from the planted seed under conditions effective to impart pathogen resistance to the plant.

[0023] Another aspect of the present invention relates to a pathogen-resistance imparting plant seed to which a non-infectious hypersensitive response elicitor polypeptide or protein has been applied.

[0024] The hypersensitive response elicitor polypeptide or protein utilized in the present invention can correspond to hypersensitive response elicitor polypeptides or proteins derived from a wide variety of fungal and bacterial pathogens. Such polypeptides or proteins are able to elicit local necrosis in plant tissue contacted by the elicitor.

[0025] Examples of suitable bacterial sources of polypeptide or protein elicitors include Erwinia, Pseudomonas, and Xanthamonas species (e.g., the following bacteria: Erwinia amylovora, Erwinia chrysanthemi, Erwinia stewartii, Erwinia carotovora, Pseudomonas syringae, Pseudomonas solancearum, Xanthomonas campestris, or mixtures thereof).

[0026] An example of a fungal source of a hypersensitive response elicitor protein or polypeptide is Phytophthora. Suitable species of such fungal pathogens include Phytophthora parasitica, Phytophthora cryptogea, Phytophthora cinnamomi, Phytophthora capsici, Phytophthora megasperma, and Phytophthora citrophthora.

[0027] The embodiment of the present invention where the hypersensitive response elicitor polypeptide or protein is applied to the plant seed can be carried out in a number of ways, including: 1) application of an isolated elicitor polypeptide or protein; 2) application of bacteria which do not cause disease and are transformed with genes encoding a hypersensitive response elicitor polypeptide or protein; and 3) application of bacteria which cause disease in some plant species (but not in those to which they are applied) and naturally contain a gene encoding the hypersensitive response elicitor polypeptide or protein. In addition, seeds in accordance with the present invention can be recovered from plants which have been treated with a hypersensitive response elicitor protein or polypeptide in accordance with the present invention.

[0028] In one embodiment of the present invention, the hypersensitive response elicitor polypeptides or proteins to be applied can be isolated from their corresponding organisms and applied to plants. Such isolation procedures are well known, as described in Arlat, M., F. Van Gijsegem, J. C. Huet, J. C. Pemollet, and C. A. Boucher, “PopA1, a Protein which Induces a Hypersensitive-like Response in Specific Petunia Genotypes is Secreted via the Hrp Pathway of Pseudomonas solanacearum,” EMBO J. 13:543-553 (1994); He, S. Y., H. C. Huang, and A. Collmer, “Pseudomonas syringae pv. syringae HarpinPss: a Protein that is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants,” Cell 73:1255-1266 (1993); and Wei, Z. -M., R. J. Laby, C. H. Zumoff, D. W. Bauer, S. -Y. He, A. Collmer, and S. V. Beer, “Harpin Elicitor of the Hypersensitive Response Produced by the Plant Pathogen Erwinia amylovora, Science 257:85-88 (1992), which are hereby incorporated by reference. See also pending U.S. patent application Ser. Nos. 08/200,024 and 08/062,024, which are hereby incorporated by reference. Preferably, however, the isolated hypersensitive response elicitor polypeptides or proteins of the present invention are produced recombinantly and purified as described below.

[0029] In other embodiments of the present invention, the hypersensitive response elicitor polypeptide or protein of the present invention can be applied to plant seeds by applying bacteria containing genes encoding the hypersensitive response elicitor polypeptide or protein. Such bacteria must be capable of secreting or exporting the polypeptide or protein so that the elicitor can contact plant seed cells. In these embodiments, the hypersensitive response elicitor polypeptide or protein is produced by the bacteria after application to the seeds or just prior to introduction of the bacteria to the seeds.

[0030] In one embodiment of the bacterial application mode of the present invention, the bacteria to be applied do not cause the disease and have been transformed (e.g., recombinantly) with genes encoding a hypersensitive response elicitor polypeptide or protein. For example, E. coli, which do not elicit a hypersensitive response in plants, can be transformed with genes encoding a hypersensitive response elicitor polypeptide and other related proteins required for production and secretion of the elicitor which is then applied to plant seeds. Expression of this polypeptide or protein can then be caused to occur. Bacterial species (other than E. coli) can also be used in this embodiment of the present invention.

[0031] In another embodiment of the bacterial application mode of the present invention, the bacteria do cause disease and naturally contain a gene encoding a hypersensitive response elicitor polypeptide or protein. Examples of such bacteria are noted above. However, in this embodiment these bacteria are applied to plant seeds for plants which are not susceptible to the disease carried by the bacteria. For example, Erwinia amylovora causes disease in apple or pear but not in tomato. However, such bacteria will elicit a hypersensitive response in tomato. Accordingly, in accordance with this embodiment of the present invention, Erwinia amylovora can be applied to tomato seeds to impart pathogen resistance without causing disease in plants of that species.

[0032] The hypersensitive response elicitor polypeptide or protein from Erwinia chrysanthemi has an amino acid sequence corresponding to SEQ. ID. No. 1 as follows:

1Met Gln Ile Thr Ile Lys Ala His Ile Gly Gly Asp Leu Gly Val Ser1 5 10 15 Gly Leu Gly Ala Gln Gly Leu Lys Gly Leu Asn Ser Ala Ala Ser Ser 20 25 30 Leu Gly Ser Ser Val Asp Lys Leu Ser Ser Thr Ile Asp Lys Leu Thr 35 40 45 Ser Ala Leu Thr Ser Met Met Phe Gly Gly Ala Leu Ala Gln Gly Leu 50 55 60 Gly Ala Ser Ser Lys Gly Leu Gly Met Ser Asn Gln Leu Gly Gln Ser65 70 75 80 Phe Gly Asn Gly Ala Gln Gly Ala Ser Asn Leu Leu Ser Val Pro Lys 85 90 95 Ser Gly Gly Asp Ala Leu Ser Lys Met Phe Asp Lys Ala Leu Asp Asp 100 105 110 Leu Leu Gly His Asp Thr Val Thr Lys Leu Thr Asn Gln Ser Asn Gln 115 120 125 Leu Ala Asn Ser Met Leu Asn Ala Ser Gln Met Thr Gln Gly Asn Met 130 135 140 Asn Ala Phe Gly Ser Gly Val Asn Asn Ala Leu Ser Ser Ile Leu Gly145 150 155 160Asn Gly Leu Gly Gln Ser Met Ser Gly Phe Ser Gln Pro Ser Leu Gly 165 170 175 Ala Gly Gly Leu Gln Gly Leu Ser Gly Ala Gly Ala Phe Asn Gln Leu 180 185 190 Gly Asn Ala Ile Gly Met Gly Val Gly Gln Asn Ala Ala Leu Ser Ala 195 200 205 Leu Ser Asn Val Ser Thr His Val Asp Gly Asn Asn Arg His Phe Val 210 215 220 Asp Lys Glu Asp Arg Gly Met Ala Lys Glu Ile Gly Gln Phe Met Asp225 230 235 240Gln Tyr Pro Glu Ile Phe Gly Lys Pro Glu Tyr Gln Lys Asp Gly Trp 245 250 255 Ser Ser Pro Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser Lys 260 265 270 Pro Asp Asp Asp Gly Met Thr Gly Ala Ser Met Asp Lys Phe Arg Gln 275 280 285 Ala Met Gly Met Ile Lys Ser Ala Val Ala Gly Asp Thr Gly Asn Thr 290 295 300 Asn Leu Asn Leu Arg Gly Ala Gly Gly Ala Ser Leu Gly Ile Asp Ala305 310 315 320Ala Val Val Gly Asp Lys Ile Ala Asn Met Ser Leu Gly Lys Leu Ala 325 330 335 Asn Ala

[0033] This hypersensitive response elicitor polypeptide or protein has a molecular weight of 34 kDa, is heat stable, has a glycine content of greater than 16%, and contains substantially no cysteine. The Erwinia chrysanthemi hypersensitive response elicitor polypeptide or protein is encoded by a DNA molecule having a nucleotide sequence corresponding to SEQ. ID. No. 2 as follows:

2CGATTTTACC CGGGTGAACG TGCTATGACC GACAGCATCA CGGTATTCGA CACCGTTACG60GCGTTTATGG CCGCGATGAA CCGGCATCAG GCGGCGCGCT GGTCGCCGCA ATCCGGCGTC120GATCTGGTAT TTCAGTTTGG GGACACCGGG CGTGAACTCA TGATGCAGAT TCAGCCGGGG180CAGCAATATC CCGGCATGTT GCGCACGCTG CTCGCTCGTC GTTATCAGCA GGCGGCAGAG240TGCGATGGCT GCCATCTGTG CCTGAACGGC AGCGATGTAT TGATCCTCTG GTGGCCGCTG300CCGTCGGATC CCGGCAGTTA TCCGCAGGTG ATCGAACGTT TGTTTGAACT GGCGGGAATG360ACGTTGCCGT CGCTATCCAT AGCACCGACG GCGCGTCCGC AGACAGGGAA CGGACGCGCC420CGATCATTAA GATAAAGGCG GCTTTTTTTA TTGCAAAACG GTAACGGTGA GGAACCGTTT480CACCGTCGGC GTCACTCAGT AACAAGTATC CATCATGATG CCTACATCGG GATCGGCGTG540GGCATCCGTT GCAGATACTT TTGCGAACAC CTGACATGAA TGAGGAAACG AAATTATGCA600AATTACGATC AAAGCGCACA TCGGCGGTGA TTTGGGCGTC TCCGGTCTGG GGCTGGGTGC660TCAGGGACTG AAAGGACTGA ATTCCGCGGC TTCATCGCTG GGTTCCAGCG TGGATAAACT720GAGCAGCACC ATCGATAAGT TGACCTCCGC GCTGACTTCG ATGATGTTTG GCGGCGCGCT780GGCGCAGGGG CTGGGCGCCA GCTCGAAGGG GCTGGGGATG AGCAATCAAC TGGGCCAGTC840TTTCGGCAAT GGCGCGCAGG GTGCGAGCAA CCTGCTATCC GTACCGAAAT CCGGCGGCGA900TGCGTTGTCA AAAATGTTTG ATAAAGCGCT GGACGATCTG CTGGGTCATG ACACCGTGAC960CAAGCTGACT AACCAGAGCA ACCAACTGGC TAATTCAATG CTGAACGCCA GCCAGATGAC1020CCAGGGTAAT ATGAATGCGT TCGGCAGCGG TGTGAACAAC GCACTGTCGT CCATTCTCGG1080CAACGGTCTC GGCCAGTCGA TGAGTGGCTT CTCTCAGCCT TCTCTGGGGG CAGGCGGCTT1140GCAGGGCCTG AGCGGCGCGG GTGCATTCAA CCAGTTGGGT AATGCCATCG GCATGGGCGT1200GGGGCAGAAT GCTGCGCTGA GTGCGTTGAG TAACGTCAGC ACCCACGTAG ACGGTAACAA1260CCGCCACTTT GTAGATAAAG AAGATCGCGG CATGGCGAAA GAGATCGGCC AGTTTATGGA1320TCAGTATCCG GAAATATTCG GTAAACCGGA ATACCAGAAA GATGGCTGGA GTTCGCCGAA1380GACGGACGAC AAATCCTGGG CTAAAGCGCT GAGTAAACCG GATGATGACG GTATGACCGG1440CGCCAGCATG GACAAATTCC GTCAGGCGAT GGGTATGATC AAAAGCGCGG TGGCGGGTGA1500TACCGGCAAT ACCAACCTGA ACCTGCGTGG CGCGGGCGGT GCATCGCTGG GTATCGATGC1560GGCTGTCGTC GGCGATAAAA TAGCCAACAT GTCGCTGGGT AAGCTGGCCA ACGCCTGATA1620ATCTGTGCTG GCCTGATAAA GCGGAAACGA AAAAAGAGAC GGGGAAGCCT GTCTCTTTTC1680TTATTATGCG GTTTATGCGG TTACCTGGAC CGGTTAATCA TCGTCATCGA TCTGGTACAA1740ACGCACATTT TCCCGTTCAT TCGCGTCGTT ACGCGCCACA ATCGCGATGG CATCTTCCTC1800GTCGCTCAGA TTGCGCGGCT GATGGGGAAC GCCGGGTGGA ATATAGAGAA ACTCGCCGGC1860CAGATGGAGA CACGTCTGCG ATAAATCTGT GCCGTAACGT GTTTCTATCC GCCCCTTTAG1920CAGATAGATT GCGGTTTCGT AATCAACATG GTAATGCGGT TCCGCCTGTG CGCCGGCCGG1980GATCACCACA ATATTCATAG AAAGCTGTCT TGCACCTACC GTATCGCGGG AGATACCGAC2040AAAATAGGGC AGTTTTTGCG TGGTATCCGT GGGGTGTTCC GGCCTGACAA TCTTGAGTTG2100GTTCGTCATC ATCTTTCTCC ATCTGGGCGA CCTGATCGGT T2141

[0034] The hypersensitive response elicitor polypeptide or protein derived from Erwinia amylovora has an amino acid sequence corresponding to SEQ. ID. No. 3 as follows:

3Met Ser Leu Asn Thr Ser Gly Leu Gly Ala Ser Thr Met Gln Ile Ser1 5 10 15 Ile Gly Gly Ala Gly Gly Asn Asn Gly Leu Leu Gly Thr Ser Arg Gln 20 25 30 Asn Ala Gly Leu Gly Gly Asn Ser Ala Leu Gly Leu Gly Gly Gly Asn 35 40 45 Gln Asn Asp Thr Val Asn Gln Leu Ala Gly Leu Leu Thr Gly Met Met 50 55 60 Met Met Met Ser Met Met Gly Gly Gly Gly Leu Met Gly Gly Gly Leu65 70 75 80 Gly Gly Gly Leu Gly Asn Gly Leu Gly Gly Ser Gly Gly Leu Gly Glu 85 90 95 Gly Leu Ser Asn Ala Leu Asn Asp Met Leu Gly Gly Ser Leu Asn Thr 100 105 110 Leu Gly Ser Lys Gly Gly Asn Asn Thr Thr Ser Thr Thr Asn Ser Pro 115 120 125 Leu Asp Gln Ala Leu Gly Ile Asn Ser Thr Ser Gln Asn Asp Asp Ser 130 135 140 Thr Ser Gly Thr Asp Ser Thr Ser Asp Ser Ser Asp Pro Met Gln Gln145 150 155 160Leu Leu Lys Met Phe Ser Glu Ile Met Gln Ser Leu Phe Gly Asp Gly 165 170 175 Gln Asp Gly Thr Gln Gly Ser Ser Ser Gly Gly Lys Gln Pro Thr Glu 180 185 190 Gly Glu Gln Asn Ala Tyr Lys Lys Gly Val Thr Asp Ala Leu Ser Gly 195 200 205 Leu Met Gly Asn Gly Leu Ser Gln Leu Leu Gly Asn Gly Gly Leu Gly 210 215 220 Gly Gly Gln Gly Gly Asn Ala Gly Thr Gly Leu Asp Gly Ser Ser Leu225 230 235 240Gly Gly Lys Gly Leu Gln Asn Leu Ser Gly Pro Val Asp Tyr Gln Gln 245 250 255 Leu Gly Asn Ala Val Gly Thr Gly Ile Gly Met Lys Ala Gly Ile Gln 260 265 270 Ala Leu Asn Asp Ile Gly Thr His Arg His Ser Ser Thr Arg Ser Phe 275 280 285 Val Asn Lys Gly Asp Arg Ala Met Ala Lys Glu Ile Gly Gln Phe Met 290 295 300 Asp Gln Tyr Pro Glu Val Phe Gly Lys Pro Gln Tyr Gln Lys Gly Pro305 310 315 320Gly Gln Glu Val Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser 325 330 335 Lys Pro Asp Asp Asp Gly Met Thr Pro Ala Ser Met Glu Gln Phe Asn 340 345 350 Lys Ala Lys Gly Met Ile Lys Arg Pro Met Ala Gly Asp Thr Gly Asn 355 360 365 Gly Asn Leu Gln Ala Arg Gly Ala Gly Gly Ser Ser Leu Gly Ile Asp 370 375 380 Ala Met Met Ala Gly Asp Ala Ile Asn Asn Met Ala Leu Gly Lys Leu385 390 395 400Gly Ala Ala

[0035] This hypersensitive response elicitor polypeptide or protein has a molecular weight of about 39 kDa, it has a pI of approximately 4.3, and is heat stable at 100° C. for at least 10 minutes. This hypersensitive response elicitor polypeptide or protein has substantially no cysteine. The hypersensitive response elicitor polypeptide or protein derived from Erwinia amylovora is more fully described in Wei, Z. -M., R. J. Laby, C. H. Zumoff, D. W. Bauer, S. -Y. He, A. Collmer, and S. V. Beer, “Harpin, Elicitor of the Hypersensitive Response Produced by the Plant Pathogen Erwinia amylovora,” Science 257:85-88 (1992), which is hereby incorporated by reference. The DNA molecule encoding this polypeptide or protein has a nucleotide sequence corresponding to SEQ. ID. No. 4 as follows:

4AAGCTTCGGC ATGGCACGTT TGACCGTTGG GTCGGCAGGG TACGTTTGAA TTATTCATAA60GAGGAATACG TTATGAGTCT GAATACAAGT GGGCTGGGAG CGTCAACGAT GCAAATTTCT120ATCGGCGGTG CGGGCGGAAA TAACGGGTTG CTGGGTACCA GTCGCCAGAA TGCTGGGTTG180GGTGGCAATT CTGCACTGGG GCTGGGCGGC GGTAATCAAA ATGATACCGT CAATCAGCTG240GCTGGCTTAC TCACCGGCAT GATGATGATG ATGAGCATGA TGGGCGGTGG TGGGCTGATG300GGCGGTGGCT TAGGCGGTGG CTTAGGTAAT GGCTTGGGTG GCTCAGGTGG CCTGGGCGAA360GGACTGTCGA ACGCGCTGAA CGATATGTTA GGCGGTTCGC TGAACACGCT GGGCTCGAAA420GGCGGCAACA ATACCACTTC AACAACAAAT TCCCCGCTGG ACCAGGCGCT GGGTATTAAC480TCAACGTCCC AAAACGACGA TTCCACCTCC GGCACAGATT CCACCTCAGA CTCCAGCGAC540CCGATGCAGC AGCTGCTGAA GATGTTCAGC GAGATAATGC AAAGCCTGTT TGGTGATGGG600CAAGATGGCA CCCAGGGCAG TTCCTCTGGG GGCAAGCAGC CGACCGAAGG CGAGCAGAAC660GCCTATAAAA AAGGAGTCAC TGATGCGCTG TCGGGCCTGA TGGGTAATGG TCTGAGCCAG720CTCCTTGGCA ACGGGGGACT GGGAGGTGGT CAGGGCGGTA ATGCTGGCAC GGGTCTTGAC780GGTTCGTCGC TGGGCGGCAA AGGGCTGCAA AACCTGAGCG GGCCGGTGGA CTACCAGCAG840TTAGGTAACG CCGTGGGTAC CGGTATCGGT ATGAAAGCGG GCATTCAGGC GCTGAATGAT900ATCGGTACGC ACAGGCACAG TTCAACCCGT TCTTTCGTCA ATAAAGGCGA TCGGGCGATG960GCGAAGGAAA TCGGTCAGTT CATGGACCAG TATCCTGAGG TGTTTGGCAA GCCGCAGTAC1020CAGAAAGGCC CGGGTCAGGA GGTGAAAACC GATGACAAAT CATGGGCAAA AGCACTGAGC1080AAGCCAGATG ACGACGGAAT GACACCAGCC AGTATGGAGC AGTTCAACAA AGCCAAGGGC1140ATGATCAAAA GGCCCATGGC GGGTGATACC GGCAACGGCA ACCTGCAGGC ACGCGGTGCC1200GGTGGTTCTT CGCTGGGTAT TGATGCCATG ATGGCCGGTG ATGCCATTAA CAATATGGCA1260CTTGGCAAGC TGGGCGCGGC TTAAGCTT1288

[0036] The hypersensitive response elicitor polypeptide or protein derived from Pseudomonas syringae has an amino acid sequence corresponding to SEQ. ID. No. 5 as follows:

5Met Gln Ser Leu Ser Leu Asn Ser Ser Ser Leu Gln Thr Pro Ala Met1 5 10 15 Ala Leu Val Leu Val Arg Pro Glu Ala Glu Thr Thr Gly Ser Thr Ser 20 25 30 Ser Lys Ala Leu Gln Glu Val Val Val Lys Leu Ala Glu Glu Leu Met 35 40 45 Arg Asn Gly Gln Leu Asp Asp Ser Ser Pro Leu Gly Lys Leu Leu Ala 50 55 60 Lys Ser Met Ala Ala Asp Gly Lys Ala Gly Gly Gly Ile Glu Asp Val65 70 75 80 Ile Ala Ala Leu Asp Lys Leu Ile His Glu Lys Leu Gly Asp Asn Phe 85 90 95 Gly Ala Ser Ala Asp Ser Ala Ser Gly Thr Gly Gln Gln Asp Leu Met 100 105 110 Thr Gln Val Leu Asn Gly Leu Ala Lys Ser Met Leu Asp Asp Leu Leu 115 120 125 Thr Lys Gln Asp Gly Gly Thr Ser Phe Ser Glu Asp Asp Met Pro Met 130 135 140 Leu Asn Lys Ile Ala Gln Phe Met Asp Asp Asn Pro Ala Gln Phe Pro145 150 155 160Lys Pro Asp Ser Gly Ser Trp Val Asn Glu Leu Lys Glu Asp Asn Phe 165 170 175 Leu Asp Gly Asp Glu Thr Ala Ala Phe Arg Ser Ala Leu Asp Ile Ile 180 185 190 Gly Gln Gln Leu Gly Asn Gln Gln Ser Asp Ala Gly Ser Leu Ala Gly 195 200 205 Thr Gly Gly Gly Leu Gly Thr Pro Ser Ser Phe Ser Asn Asn Ser Ser 210 215 220 Val Met Gly Asp Pro Leu Ile Asp Ala Asn Thr Gly Pro Gly Asp Ser225 230 235 240Gly Asn Thr Arg Gly Glu Ala Gly Gln Leu Ile Gly Glu Leu Ile Asp 245 250 255 Arg Gly Leu Gln Ser Val Leu Ala Gly Gly Gly Leu Gly Thr Pro Val 260 265 270 Asn Thr Pro Gln Thr Gly Thr Ser Ala Asn Gly Gly Gln Ser Ala Gln 275 280 285 Asp Leu Asp Gln Leu Leu Gly Gly Leu Leu Leu Lys Gly Leu Glu Ala 290 295 300 Thr Leu Lys Asp Ala Gly Gln Thr Gly Thr Asp Val Gln Ser Ser Ala305 310 315 320Ala Gln Ile Ala Thr Leu Leu Val Ser Thr Leu Leu Gln Gly Thr Arg 325 330 335 Asn Gln Ala Ala Ala 340

[0037] This hypersensitive response elicitor polypeptide or protein has a molecular weight of 34-35 kDa. It is rich in glycine (about 13.5%) and lacks cysteine and tyrosine. Further information about the hypersensitive response elicitor derived from Pseudomonas syringae is found in He, S. Y., H. C. Huang, and A. Collmer, “Pseudomonas syringae pv. syringae HarpinPss: a Protein that is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants,” Cell 73:1255-1266 (1993), which is hereby incorporated by reference. The DNA molecule encoding the hypersensitive response elicitor from Pseudomonas syringae has a nucleotide sequence corresponding to SEQ. ID. No. 6 as follows:

6ATGCAGAGTC TCAGTCTTAA CAGCAGCTCG CTGCAAACCC CGGCAATGGC CCTTGTCCTG60GTACGTCCTG AAGCCGAGAC GACTGGCAGT ACGTCGAGCA AGGCGCTTCA GGAAGTTGTC120GTGAAGCTGG CCGAGGAACT GATGCGCAAT GGTCAACTCG ACGACAGCTC GCCATTGGGA180AAACTGTTGG CCAAGTCGAT GGCCGCAGAT GGCAAGGCGG GCGGCGGTAT TGAGGATGTC240ATCGCTGCGC TGGACAAGCT GATCCATGAA AAGCTCGGTG ACAACTTCGG CGCGTCTGCG300GACAGCGCCT CGGGTACCGG ACAGCAGGAC CTGATGACTC AGGTGCTCAA TGGCCTGGCC360AAGTCGATGC TCGATGATCT TCTGACCAAG CAGGATGGCG GGACAAGCTT CTCCGAAGAC420GATATGCCGA TGCTGAACAA GATCGCGCAG TTCATGGATG ACAATCCCGC ACAGTTTCCC480AAGCCGGACT CGGGCTCCTG GGTGAACGAA CTCAAGGAAG ACAACTTCCT TGATGGCGAC540GAAACGGCTG CGTTCCGTTC GGCACTCGAC ATCATTGGCC AGCAACTGGG TAATCAGCAG600AGTGACGCTG GCAGTCTGGC AGGGACGGGT GGAGGTCTGG GCACTCCGAG CAGTTTTTCC660AACAACTCGT CCGTGATGGG TGATCCGCTG ATCGACGCCA ATACCGGTCC CGGTGACAGC720GGCAATACCC GTGGTGAAGC GGGGCAACTG ATCGGCGAGC TTATCGACCG TGGCCTGCAA780TCGGTATTGG CCGGTGGTGG ACTGGGCACA CCCGTAAACA CCCCGCAGAC CGGTACGTCG840GCGAATGGCG GACAGTCCGC TCAGGATCTT GATCAGTTGC TGGGCGGCTT GCTGCTCAAG900GGCCTGGAGG CAACGCTCAA GGATGCCGGG CAAACAGGCA CCGACGTGCA GTCGAGCGCT960GCGCAAATCG CCACCTTGCT GGTCAGTACG CTGCTGCAAG GCACCCGCAA TCAGGCTGCA1020GCCTGA1026

[0038] The hypersensitive response elicitor polypeptide or protein derived from Pseudomonas solanacearum has an amino acid sequence corresponding to SEQ. ID. No. 7 as follows:

7Met Ser Val Gly Asn Ile Gln Ser Pro Ser Asn Leu Pro Gly Leu Gln1 5 10 15 Asn Leu Asn Leu Asn Thr Asn Thr Asn Ser Gln Gln Ser Gly Gln Ser 20 25 30 Val Gln Asp Leu Ile Lys Gln Val Glu Lys Asp Ile Leu Asn Ile Ile 35 4 45 Ala Ala Leu Val Gln Lys Ala Ala Gln Ser Ala Gly Gly Asn Thr Gly 50 55 60 Asn Thr Gly Asn Ala Pro Ala Lys Asp Gly Asn Ala Asn Ala Gly Ala65 70 75 80 Asn Asp Pro Ser Lys Asn Asp Pro Ser Lys Ser Gln Ala Pro Gln Ser 85 90 95 Ala Asn Lys Thr Gly Asn Val Asp Asp Ala Asn Asn Gln Asp Pro Met 100 105 110 Gln Ala Leu Met Gln Leu Leu Glu Asp Leu Val Lys Leu Leu Lys Ala 115 120 125 Ala Leu His Met Gln Gln Pro Gly Gly Asn Asp Lys Gly Asn Gly Val 130 135 140 Gly Gly Ala Asn Gly Ala Lys Gly Ala Gly Gly Gln Gly Gly Leu Ala145 150 155 160Glu Ala Leu Gln Glu Ile Glu Gln Ile Leu Ala Gln Leu Gly Gly Gly 165 170 175 Gly Ala Gly Ala Gly Gly Ala Gly Gly Gly Val Gly Gly Ala Gly Gly 180 185 190 Ala Asp Gly Gly Ser Gly Ala Gly Gly Ala Gly Gly Ala Asn Gly Ala 195 200 205 Asp Gly Gly Asn Gly Val Asn Gly Asn Gln Ala Asn Gly Pro Gln Asn 210 215 220 Ala Gly Asp Val Asn Gly Ala Asn Gly Ala Asp Asp Gly Ser Glu Asp225 230 235 240Gln Gly Gly Leu Thr Gly Val Leu Gln Lys Leu Met Lys Ile Leu Asn 245 250 255 Ala Leu Val Gln Met Met Gln Gln Gly Gly Leu Gly Gly Gly Asn Gln 260 265 270 Ala Gln Gly Gly Ser Lys Gly Ala Gly Asn Ala Ser Pro Ala Ser Gly 275 280 285 Ala Asn Pro Gly Ala Asn Gln Pro Gly Ser Ala Asp Asp Gln Ser Ser 290 295 300 Gly Gln Asn Asn Leu Gln Ser Gln Ile Met Asp Val Val Lys Glu Val305 310 315 320Val Gln Ile Leu Gln Gln Met Leu Ala Ala Gln Asn Gly Gly Ser Gln 325 330 335 Gln Ser Thr Ser Thr Gln Pro Met 340

[0039] It is encoded by a DNA molecule having a nucleotide sequence corresponding SEQ. ID. No. 8 as follows:

8ATGTCAGTCG GAAACATCCA GAGCCCGTCG AACCTCCCGG GTCTGCAGAA CCTGAACCTC60AACACCAACA CCAACAGCCA GCAATCGGGC CAGTCCGTGC AAGACCTGAT CAAGCAGGTC120GAGAAGGACA TCCTCAACAT CATCGCAGCC CTCGTGCAGA AGGCCGCACA GTCGGCGGGC180GGCAACACCG GTAACACCGG CAACGCGCCG GCGAAGGACG GCAATGCCAA CGCGGGCGCC240AACGACCCGA GCAAGAACGA CCCGAGCAAG AGCCAGGCTC CGCAGTCGGC CAACAAGACC300GGCAACGTCG ACGACGCCAA CAACCAGGAT CCGATGCAAG CGCTGATGCA GCTGCTGGAA360GACCTGGTGA AGCTGCTGAA GGCGGCCCTG CACATGCAGC AGCCCGGCGG CAATGACAAG420GGCAACGGCG TGGGCGGTGC CAACGGCGCC AAGGGTGCCG GCGGCCAGGG CGGCCTGGCC480GAAGCGCTGC AGGAGATCGA GCAGATCCTC GCCCAGCTCG GCGGCGGCGG TGCTGGCGCC540GGCGGCGCGG GTGGCGGTGT CGGCGGTGCT GGTGGCGCGG ATGGCGGCTC CGGTGCGGGT600GGCGCAGGCG GTGCGAACGG CGCCGACGGC GGCAATGGCG TGAACGGCAA CCAGGCGAAC660GGCCCGCAGA ACGCAGGCGA TGTCAACGGT GCCAACGGCG CGGATGACGG CAGCGAAGAC720CAGGGCGGCC TCACCGGCGT GCTGCAAAAG CTGATGAAGA TCCTGAACGC GCTGGTGCAG780ATGATGCAGC AAGGCGGCCT CGGCGGCGGC AACCAGGCGC AGGGCGGCTC GAAGGGTGCC840GGCAACGCCT CGCCGGCTTC CGGCGCGAAC CCGGGCGCGA ACCAGCCCGG TTCGGCGGAT900GATCAATCGT CCGGCCAGAA CAATCTGCAA TCCCAGATCA TGGATGTGGT GAAGGAGGTC960GTCCAGATCC TGCAGCAGAT GCTGGCGGCG CAGAACGGCG GCAGCCAGCA GTCCACCTCG1020ACGCAGCCGA TGTAA1035

[0040] Further information regarding the hypersensitive response elicitor polypeptide or protein derived from Pseudomonas solanacearum is set forth in Arlat, M., F. Van Gijsegem, J. C. Huet, J. C. Pemollet, and C. A. Boucher, “PopA1, a Protein which Induces a Hypersensitive-like Response in Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum,” EMBO J. 13:543-533 (1994), which is hereby incorporated by reference.

[0041] The hypersensitive response elicitor polypeptide or protein from Xanthomonas campestris pv. glycines has an amino acid sequence corresponding to SEQ. ID. No. 9 as follows:

9Thr Leu Ile Glu Leu Met Ile Val Val Ala Ile Ile Ala Ile Leu Ala1 5 10 15Ala Ile Ala Leu Pro Ala Tyr Gln Asp Tyr 20 25

[0042] This sequence is an amino terminal sequence having 26 residues only from the hypersensitive response elicitor polypeptide or protein of Xanthomonas campestris pv. glycines. It matches with fimbrial subunit proteins determined in other Xanthomonas campestris pathovars.

[0043] The hypersensitive response elicitor polypeptide or protein from Xanthomonas campestris pelargonii is heat stable, protease sensitive, and has a molecular weight of 20 kDa. It includes an amino acid sequence corresponding to SEQ. ID. No. 10 as follows:

10Ser Ser Gln Gln Ser Pro Ser Ala Gly Ser Glu Gln Gln Leu Asp Gln 1 5 10 15Leu Leu Ala Met 20

[0044] Isolation of Erwinia carotovora hypersensitive response elicitor protein or polypeptide is described in Cai, et al., “The RsmA− Mutants of Erwinia carotovora subsp. carotova Strain Ecc7l Overexpress hrpNEcc and Elicit a Hypersensitive Reaction-Like Response in Tobacco Leaves,” MPMI, 9(7):565-73 (1996), which is hereby incorporated by reference. The hypersensitive response elicitor protein or polypeptide for Erwinia stewartii is disclosed in Ahmad, et al., “Harpin is Not Necessary for the Pathogenicity of Erwinia stewartii on Maize,” 8th Int'l. Cong. Molec. Plant-Microbe Interact, Jul. 14-19, 1996 and Ahmad, et al., “Harpin is Not Necessary for the Pathogenicity of Erwinia stewartii on Maize,” Ann. Mtg. Am. Phytopath. Soc., Jul. 27-31, 1996, which are hereby incorporated by reference.

[0045] Hypersensitive response elicitor proteins or polypeptides from Phytophora parasitica, Phytophora cryptogea, Phytophora cinnamoni, Phytophora capsici, Phytophora megasperma, and Phytophora citrophthora are described in Kamoun, et al., “Extracellular Protein Elicitors from Phytophora: Host-Specificity and Induction of Resistance to Bacterial and Fungal Phytopathogens,” Molec. Plant-Microbe Interact., 6(1):15-25 (1993), Ricci, et al., “Structure and Activity of Proteins from Pathogenic Fungi Phytophora Eliciting Necrosis and Acquired Resistance in Tobacco,” Eur. J. Biochem., 183:555-63 (1989), Ricci, et al., “Differential Production of Parasiticein, an Elicitor of Necrosis and Resistance in Tobacco by Isolates of Phytophora paraticica,” Plant Path., 41:298-307 (1992), Baillieul, et al., “A New Elicitor of the Hypersensitive Response in Tobacco: A Fungal Glycoprotein Elicits Cell Death, Expression of Defense Genes, Production of Salicylic Acid, and Induction of Systemic Acquired Resistance,” Plant J., 8(4):551-60 (1995), and Bonnet, et al., “Acquired Resistance Triggered by Elicitins in Tobacco and Other Plants,” Eur. J. Plant Path., 102:181-92 (1996), which are hereby incorporated by reference.

[0046] The above elicitors are exemplary. Other elicitors can be identified by growing fungi or bacteria that elicit a hypersensitive response under which genes encoding an elicitor are expressed. Cell-free preparations from culture supernatants can be tested for elicitor activity (i.e. local necrosis) by using them to infiltrate appropriate plant tissues.

[0047] It is also possible to use fragments of the above hypersensitive response elicitor polypeptides or proteins as well as fragments of full length elicitors from other pathogens, in the method of the present invention.

[0048] Suitable fragments can be produced by several means. In the first, subclones of the gene encoding a known elicitor protein are produced by conventional molecular genetic manipulation by subcloning gene fragments. The subclones then are expressed in vitro or in viva in bacterial cells to yield a smaller protein or a peptide that can be tested for elicitor activity according to the procedure described below.

[0049] As an alternative, fragments of an elicitor protein can be produced by digestion of a full-length elicitor protein with proteolytic enzymes like chymotrypsin or Staphylococcus proteinase A, or trypsin. Different proteolytic enzymes are likely to cleave elicitor proteins at different sites based on the amino acid sequence of the elicitor protein. Some of the fragments that result from proteolysis may be active elicitors of resistance.

[0050] In another approach, based on knowledge of the primary structure of the protein, fragments of the elicitor protein gene may be synthesized by using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein. These then would be cloned into an appropriate vector for increased expression of a truncated peptide or protein.

[0051] An example of a suitable fragment is the popA1 fragment of the hypersensitive response elicitor polypeptide or protein from Pseudomonas solanacearum. See Arlat, M., F. Van Gijsegem, J. C. Huet, J. C. Pemollet, and C. A. Boucher, “PopA1, a Protein Which Induces a Hypersensitive-like Response in Specific Petunia Genotypes is Secreted via the Hrp Pathway of Pseudomonas solanacearum,” EMBO J. 13:543-53 (1994), which is hereby incorporated by reference. As to Erwinia amylovora, a suitable fragment can be, for example, either or both the polypeptide extending between and including amino acids 1 and 98 of SEQ. ID. NO. 3 and the polypeptide extending between and including amino acids 137 and 204 of SEQ. ID. No. 3.

[0052] Variants may be made by, for example, the deletion or addition of amino acids that have minimal influence on the properties, secondary structure and hydropathic nature of the polypeptide. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide.

[0053] The protein or polypeptide of the present invention is 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 E. coli. Alternatively, the protein or polypeptide of the present invention is secreted into the growth medium. In the case of unsecreted protein, to isolate the protein, the E. coli host cell carrying a recombinant plasmid is propagated, homogenized, and the homogenate is centrifuged to remove bacterial debris. The supernatant is then subjected to heat treatment and the hypersensitive response elicitor protein is separated by centrifugation. The supernatant fraction containing the polypeptide or protein of the present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction may be further purified by ion exchange or HPLC.

[0054] Alternatively, the hypersensitive response elicitor protein can be prepared by chemical synthesis using conventional techniques.

[0055] The DNA molecule encoding the hypersensitive response elicitor polypeptide or protein can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present) The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.

[0056] U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture.

[0057] Recombinant genes may also be introduced into viruses, such as vaccina virus. Recombinant viruses can be generated by transection of plasmids into cells infected with virus.

[0058] Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/− or KS +/− (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see F. W. Studier et. al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technology vol. 185 (1990), which is hereby incorporated by reference), and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference.

[0059] 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.

[0060] Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation).

[0061] Transcription of DNA is dependent upon the presence of a promotor which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promoters differ from those of procaryotic promotors. Furthermore, eucaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eucaryotic cells.

[0062] Similarly, translation of MRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby incorporated by reference.

[0063] Promotors vary in their “strength” (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promotor or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.

[0064] Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unless specifically induced. In certain operations, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.

[0065] Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promotor, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (SD) sequence about 7-9 bases 5′ to the initiation codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.

[0066] Once the isolated DNA molecule encoding the hypersensitive response elicitor polypeptide or protein has been cloned into an expression system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.

[0067] The method of the present invention can be utilized to treat seeds for a wide variety of plants to impart pathogen resistance to the plants. Suitable seeds are for plants which are dicots and monocots. More particularly, useful crop plants can include: rice, wheat, barley, rye, oats, cotton, sunflower, canola, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane. Examples of suitable ornamental plants are: rose, Saintpaulia, petunia, Pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.

[0068] 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.

[0069] Resistance, inter alia, to the following viruses can be achieved by the method of the present invention: Tobacco mosaic virus, cucumber mosaic virus, potato x virus, potato y virus, and tomato mosaic virus.

[0070] Resistance, inter alia, to the following bacteria can also be imparted to plants in accordance with the present invention: Pseudomonas solancearum, Pseudomonas syringae pv. tabaci, and Xanthamonas campestris pv. pelargonii.

[0071] 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.

[0072] The embodiment of the present invention involving applying the hypersensitive response elicitor polypeptide or protein to all or part of the plant seeds being treated can be carried out through a variety of procedures. Suitable application methods include high or low pressure spraying, injection, coating, dusting, and immersion. Other suitable application procedures can be envisioned by those skilled in the art. Once treated with the hypersensitive response elicitor of the present invention, the seeds can be planted and cultivated using conventional procedures to produce plants. After plants have been propagated from seeds treated in accordance with the present invention, the plants may be treated with one or more applications of the hypersensitive response elicitor protein or polypeptide to enhance hypersensitive response induced resistance in the plants. See U.S. patent application Ser. No. 08/475,775, which is hereby incorporated by reference. Such propagated plants, which are resistant to disease, may, in turn, be useful in producing seeds or propagules (e.g. cuttings) that produce resistant plants.

[0073] The hypersensitive response elicitor polypeptide or protein can be applied to plant seeds in accordance with the present invention alone or in a mixture with other materials.

[0074] A composition suitable for treating plant seeds in accordance with the present invention contains a hypersensitive response elicitor polypeptide or protein in a carrier. Suitable carriers include water, aqueous solutions, slurries, or dry powders. In this embodiment, the composition contains greater than 0.5 nM hypersensitive response elicitor polypeptide or protein.

[0075] Although not required, this composition may contain additional additives including fertilizer, insecticide, fungicide, nematicide, herbicide, and mixtures thereof. Suitable fertilizers include (NH4)2NO3. An example of a suitable insecticide is Malathion. Useful fungicides include Captan.

[0076] Other suitable additives include buffering agents, wetting agents, coating agents, and abrading agents. These materials can be used to facilitate the process of the present invention. In addition, the hypersensitive response elicitor polypeptide or protein can be applied to plant seeds with other conventional seed formulation and treatment materials, including clays and polysaccharides.

[0077] In the alternative embodiment of the present invention involving the use of transgenic seeds, a hypersensitive response elicitor polypeptide or protein need not be applied topically to the seeds. Instead, transgenic plants transformed with a DNA molecule encoding a hypersensitive response elicitor polypeptide or protein are produced according to procedures well known in the art, such as biolistics or Agrobacterium mediated transformation. Examples of suitable hypersensitive response elicitor polypeptides or proteins and the nucleic acid sequences for their encoding DNA are disclosed supra. As is conventional in the art, such transgenic plants would contain suitable vectors with various promoters including pathogen-induced promoters, and other components needed for transformation, transcription, and, possibly, translation. Such transgenic plants themselves could be grown under conditions effective to be imparted with pathogen resistance. In any event, once transgenic plants of this type are produced, transgenic seeds are recovered. These seeds can then be planted in the soil and cultivated using conventional procedures to produce plants. The plants are propagated from the planted transgenic seeds under conditions effective to impart pathogen resistance to the plants.

[0078] When transgenic plant seeds are used in accordance with the present invention, they additionally can be treated with the same materials (noted above) as are used to treat the seeds to which a hypersensitive response elicitor polypeptide or protein is applied. These other materials, including hypersensitive response elicitors, can be applied to the transgenic plant seeds by high or low pressure spraying, injection, coating, dusting, and immersion. Similarly, transgenic plants additionally may be treated with one or more applications of the hypersensitive response elicitor to enhance hypersensitive response induced resistance in the plants. Such plants may also be treated with conventional plant treatment agents (e.g., insecticides, fertilizers, etc.). The transgenic plants of the present invention are useful in producing seeds or propagules (e.g. cuttings) from which disease resistant plants grow.

EXAMPLES

Example 1

Effect of Treating Seeds with Hypersensitive Response Elicitor Protein

[0079] Marglobe tomato seeds were submerged in hypersensitive response elicitor protein (ca. 26 μgm/ml) from Erwinia amylovora solution or buffer in beakers on day 0 for 24 hours at 28° C. in a growth chamber. After soaking seeds in hypersensitive response elicitor protein from Erwinia amylovora or buffer, they were sown in germination pots with artificial soil on day 1. Seedlings were transplanted to individual pots at the two-true-leaf stage on day 12. After transplanting, some plants that arose from treated seed also were sprayed with hypersensitive response elicitor protein (ca. 13 μgm/ml) from Erwinia amylovora (Treatments 3 and 4).

[0080] Tomato treated as noted in the preceding paragraph were inoculated with Burkholderia (Pseudomonas) solanacearum K60 strain (See Kelman, “The Relationship of Pathogenicity in Pseudomonas solanacearum to Colony Appearance on a Tetrazolium Medium,” Phytopathology 44:693-95 (1954)) on day 23 by making vertical cuts through the roots and potting medium of tomato plants (on a tangent 2 cm from the stem and 2 times/pot) and putting 10 ml (5×108 cfu/ml) suspension into the soil.

[0081] The above procedure involved use of 10 seeds treated with hypersensitive response elicitor protein from Erwinia amylovora per treatment.

[0082] Treatments:

[0083] 1. Seeds soaked in hypersensitive response elicitor protein from Erwinia amylovora (ca. 26 μmg/ml).

[0084] 2. Seeds soaked in buffer (SmM KPO4, pH 6.8)

[0085] 3. Seeds soaked in hypersensitive response elicitor protein from Erwinia amylovora (ca. 26 μmg/ml) and seedlings sprayed with hypersensitive response elicitor protein from Erwinia amylovora (ca. 13 μgm/ml) at transplanting.

[0086] 4. Seeds soaked in buffer and seedlings sprayed with hypersensitive response elicitor protein from Erwinia amylovora (ca. 13 μgm/ml) at transplanting.

[0087] The results of these treatments are set forth in Tables 1-4.

11TABLE 1Infection Data - 28 Days After SeedTreatment and 5 Days After InoculationNumber of Plants of Given Disease Rating*Treatm.Plants0123451101000000210 910000310 9100004101000000*Disease Scale: Grade 0: No symptoms Grade 1: One leaf partially wilted. Grade 2: 2-3 leaves wilted. Grade 3: All except the top 2-3 leaves wilted. Grade 4: All leaves wilted. Grade 5: Plant Dead

[0088]

12

TABLE 2

Infection Data - 31 Days After Seed

Treatment and 8 Days After Inoculation

Number of Plants of Given Disease

Rating*

Treatm.
Plants
0
1
2
3
4
5

1
10
6
4
0
0
0
0

2
10
4
3
2
1
0
0

3
10
8
2
0
0
0
0

4
10
7
2
1
0
0
0

[0089]

13

TABLE 3

Infection Data - 35 Days After Seed

Treatment and 12 Days After Inoculation

Number of Plants of Given Disease

Rating*

Treatm.
Plants
0
1
2
3
4
5

1
10
5
3
0
1
1
0

2
10
1
3
3
2
1
0

3
10
4
3
3
0
0
0

4
10
3
3
3
1
0
0

[0090]

14

TABLE 4

Disease Indices of Seed Treatment

With Hypersensitive Response Elicitor Protein

Disease Index (%)*

Treatment
Inoculation
Day
Day
Day

Day 0
Day 14
Day 23
28
31
35

1.
Hypersensitive

Inoculate
0
8
20

response

elicitor

protein seed

soak

2.
Buffer seed

Inoculate
2
20
38

soak

3.
Hypersensitive
Spray
Inoculate
2
4
18

response
Hypersensitive

elicitor
response

protein seed
elicitor

soak
protein

4.
Buffer seed
Spray
Inoculate
0
8
24

soak
Hypersensitive

response

elicitor

protein

*The Disease Index was determined using the procedure set forth in N. N. Winstead, et al., “Inoculation Techniques for Evaluating Resistance to Pseudomonas Solanacearurn,” Phytopathology 42:628-34 (1952), particularly at page 629.

[0091] The above data shows that the hypersensitive response elicitor protein was more effective than buffer as a seed treatment in reducing disease index and was as effective as spraying leaves of young plants with hypersensitive response elicitor protein.

Example 2

Effect of Treating Tomato Seeds with Hypersensitive Response Elicitor Protein From pCPP2139 Versus pCPP50 Vector on Southern Bacteria Wilt of Tomato

[0092] Marglobe tomato seeds were submerged in hypersensitive response elicitor protein from pCPP2139 or in pCPP50 vector solution (1:50, 1:100 and 1:200) in beakers on day 0 for 24 hours at 28° C. in a growth chamber. After soaking seeds in hypersensitive response elicitor protein or vector, they were sown in germination pots with artificial soil on day 0. Ten uniform appearing plants were chosen randomly from each of the following treatments:

15TreatmentContentStrainDilutionHarpin1.DH5α (pCPP2139)1:50 8μg/ml2.DH5α (pCCP50)1:50 03.DH5α (pCPP2139)1:1004μg/ml4.DH5α (pCPP50)1:10005.DH5α (pCPP2139)1:2002μg/ml6.DH5α (pCPP50)1:2000

[0093] The resulting seedlings were inoculated with Pseudomonas solanacearum K60 by dipping the roots of tomato seedling plants for about 30 seconds in a 40 ml (1×108 cfu/ml) suspension. The seedlings were then transplanted into the pots with artificial soil on day 12.

[0094] The results of these treatments are set forth in Tables 5-8.

16TABLE 516 Days After Seed Treatment and3 Days After InoculationNumber of Plants of Given DiseaseRating*Treatm.Plant012345110730000210550000310640000410640000510740000610460000

[0095]

17

TABLE 6

19 Days After Seed Treatment and

6 Days After Inoculation

Number of Plants of Given Disease

Rating*

Treatm.
Plant
0
1
2
3
4
5

1
10
6
0
0
0
0
0

2
10
2
0
2
2
1
3

3
10
2
0
2
0
2
4

4
10
3
1
2
0
2
2

5
10
2
1
0
2
2
3

6
10
1
0
1
1
3
4

[0096]

18

TABLE 7

21 Days After Seed Treatment and

8 Days After Inoculation

Number of Plants of Given Disease

Rating*

Treatm.
Plant
0
1
2
3
4
5

1
10
6
0
0
0
1
3

2
10
2
0
0
1
3
4

3
10
2
0
0
2
2
3

4
10
3
0
0
2
2
3

5
10
2
0
0
0
4
4

6
10
1
0
1
2
1
5

[0097]

19

TABLE 8

Disease Indices of Seed Treatment

With Hypersensitive Response Elicitor and Vector

Treatment
Disease Index (%)

Day 0
Day 12
Day 15
Day 18
Day 20

Hypersensitive
inoculate
6.0
32.0
38.0

response elicitor

protein seed dip

(1:50)

Vector seed dip
inoculate
10.0
58.0
70.0

(1:50)

Hypersensitive
inoculate
8.0
64.0
68.0

response elicitor

protein seed dip

(1:100)

Vector seed dip
inoculate
8.0
46.0
58.0

(1:100)

Hypersensitive
inoculate
6.0
60.00
72.0

response elicitor

protein seed dip

(1:200)

Vector seed dip
inoculate
12.0
74.0
74.0

(1:200)

[0098] The above data shows that the hypersensitive response elicitor protein is much more effective than the vector solution in preventing Tomato Southern Bacteria Wilt.

Example 3

Effect of Treating Tomato Seeds with Hypersensitive Response Elicitor Protein From pCPP2139 Versus pCPP50 Vector on Tomato Southern Bacteria Wilt

[0099] Marglobe tomato seeds were submerged in hypersensitive response elicitor protein from pCPP2139 or in pCPP50 vector solution (1:50, 1:100 and 1:200) in beakers on day 0 for 24 hours at 28° C. in a growth chamber. After soaking seeds in the hypersensitive response elicitor protein or vector, the seeds were sown in germination pots with artificial soil on day 1. Ten uniform appearing plants were chosen randomly from each of the following treatments:

20HypersensitiveResponse ElicitorTreatmentStrainDilutionContent1.DH5α (pCPP2139)1:50 8μg/ml2.DH5α (pCCP50)1:50 03.DH5α (pCPP2139)1:1004μg/ml4.DH5α (pCPP50)1:10005.DH5α (pCPP2139)1:2002μg/ml6.DH5α (pCPP50)1:2000

[0100] The resulting seedlings were inoculated with Pseudomonas solanacearum K60 by dipping the roots of tomato seedling plants for about 30 seconds in a 40 ml (1×105 cfu/ml) suspension. The seedlings were then transplanted into the pots with artificial soil on day 12.

[0101] The results of these treatments are set forth in Tables 9-12.

21TABLE 916 Days After Seed Treatment and3 Days After InoculationNumber of Plants of Given DiseaseRating*Treatm.Plant012345110820000210730000310730000410730000510820000610730000

[0102]

22

TABLE 10

19 Days After Seed Treatment and

6 Days After Inoculation

Number of Plants of Given Disease

Rating*

Treatm.
Plant
0
1
2
3
4
5

1
10
5
0
0
1
2
2

2
10
1
0
1
2
3
3

3
10
4
1
0
0
2
3

4
10
2
0
2
1
2
3

5
10
1
0
1
1
4
3

6
10
1
0
0
2
4
3

[0103]

23

TABLE 11

21 Days After Hypersensitive Response

Elicitor Protein Seed Treatment and

8 Days After Inoculation

Number of Plants of Given Disease

Rating*

Treatm.
Plant
0
1
2
3
4
5

1
10
5
0
0
0
2
3

2
10
2
0
2
0
2
4

3
10
5
0
0
0
2
3

4
10
2
0
2
0
2
4

5
10
1
0
1
0
2
6

6
10
1
0
0
0
2
7

[0104]

24

TABLE 12

Disease Indices of Seed Treatment

With Hypersensitive Response Elicitor Protein and Vector

Day 1
Day 13
Day 16
Day 19
Day 21

Hypersensitive
inoculate
4.0
42.0
46.0

response elicitor

protein seed dip

(1:50)

Vector seed dip
inoculate
6.0
70.0
64.0

(1:50)

Hypersensitive
inoculate
6.0
48.0
46.0

response elicitor

protein seed dip

(1:100)

Vector seed dip
inoculate
6.0
60.0
64.0

(1:100)

Hypersensitive
inoculate
4.0
72.0
80.0

response elicitor

protein seed dip

(1:200)

Vector seed dip
inoculate
6.0
74.0
86.0

(1:200)

[0105] The above data shows that the hypersensitive response elicitor protein is much more effective in preventing Tomato Southern Bacteria Wilt.

Example 4

Effect of Treating Tomato Seeds with Hypersensitive Response Elicitor Protein From pCPP2139 Versus pCPP50 Vector on Southern Bacteria Wilt of Tomato

[0106] Marglobe tomato seeds were submerged in hypersensitive response elicitor protein from pCPP2139 or in pCPP50 vector solution (1:25, 1:50 and 1:100) in beakers on day 0 for 24 hours at 28° C. in a growth chamber. After soaking seeds in hypersensitive response elicitor protein or vector, they were sown in germination pots with artificial soil on day 1. Ten uniform appearing plants were chosen randomly from each of the following treatments:

25TreatmentContentStrainDilutionHarpin1.DH5α (pCPP2139)1:2516μg/ml2.DH5α (pCCP50)1:2503.DH5α (pCPP2139)1:508μg/ml4.DH5α (pCPP50)1:5005.DH5α (pCPP2139) 1:1002μg/ml6.DH5α (pCPP50) 1:1000

[0107] The resulting seedlings were inoculated with Pseudomonas solanacearum K60 by dipping the roots of tomato seedling plants for about 30 seconds in a 40 ml (1×108 cfu/ml) suspension. The seedlings were then transplanted into the pots with artificial soil on day 14.

[0108] The results of these treatments are set forth in Tables 13-16.

26TABLE 1319 Days After Seed Treatment and4 Days After InoculationNumber of Plants of Given DiseaseRating*Treatm.Plants012345110820000210522100310910000410521200510531100610612100

[0109]

27

TABLE 14

21 Days After Seed Treatments and

6 Days After Inoculation

Number of Plants of Given Disease

Rating*

Treatm.
Plants
0
1
2
3
4
5

1
10
6
3
0
0
1
0

2
10
3
2
1
0
0
0

3
10
6
3
1
0
0
0

4
10
3
2
1
2
2
0

5
10
5
1
2
2
0
0

6
10
3
1
3
2
1
0

[0110]

28

TABLE 15

23 Days After Seed Treatment and

8 Days After Inoculation

Number of Plants of Given Disease

Rating*

Treatm.
Plants
0
1
2
3
4
5

1
10
7
2
0
0
0
1

2
10
2
2
2
3
0
1

3
10
7
2
0
1
0
0

4
10
2
1
2
3
0
2

5
10
3
1
2
3
0
1

6
10
2
2
2
3
0
1

[0111]

29

TABLE 16

Disease Indices of Seed Treatment

With Hypersensitive Elicitor Protein and Vector

Treatment
Disease Index (%)

Day 1
Day 15
Day 19
Day 21
Day 23

Hypersensitive
inoculate
4.0
14.0
14.0

response elicitor

protein seed dip

(1:25)

Vector seed dip
inoculate
18.0
28.0
40.0

(1:25)

Hypersensitive
inoculate
2.0
10.0
10.0

response elicitor

protein seed dip

(1:50)

Vector seed dip
inoculate
20.0
36.0
48.0

(1:50)

Hypersensitive
inoculate
16.0
22.0
38.0

response elicitor

protein seed dip

(1:100)

Vector seed dip
inoculate
16.0
34.0
40.0

(1:100)

[0112] The above data shows that the hypersensitive response elicitor protein is much more effective than the vector solution in preventing Tomato Southern Bacteria Wilt. A hypersensitive response protein concentration of 1:50 is particularly effective in disease control.

Example 5

Effect of Treating Tomato Seeds with Hypersensitive Response Elicitor Protein From pCPP2139 Versus pCPP50 Vector on Southern Bacteria Wilt of Tomato

[0113] Marglobe tomato seeds were submerged in hypersensitive response elicitor protein from pCPP2139 or pCPP50 vector solution (1:25, 1:50 and 1:100) in beakers on day 0 for 24 hours at 28° C. in a growth chamber. After soaking seeds in hypersensitive response elicitor protein or vector, they were sown in germination pots with artificial soil on day 1. Ten uniform appearing plants were chosen randomly from each of the following treatments:

30TreatmentContentStrainDilutionHarpin1.DH5α (pCPP2139)1:2516μg/ml2.DH5α (pCCP50)1:2503.DH5α (pCPP2139)1:508μg/ml4.DH5α (pCPP50)1:5005.DH5α (pCPP2139) 1:1004μg/ml6.DH5α (pCPP50) 1:1000

[0114] The resulting seedlings were inoculated with Pseudomonas solanacearum K60 by dipping the roots of tomato seedling plants for about 30 seconds in a 40 ml (1×106 cfu/ml) suspension. The seedlings were then transplanted into the pots with artificial soil on day 14.

[0115] The results of these treatments are set forth in Tables 17-20.

31TABLE 1719 Days After Seed Treatment and4 Days After InoculationNumber of Plants of Given DiseaseRating*Treatm.Plants012345110820000210631000310910000410640000510621100610640000

[0116]

32

TABLE 18

21 Days After Seed Treatment and

6 Days After Inoculation

Number of Plants of Given Disease

Rating*

Treatm.
Plants
0
1
2
3
4
5

1
10
7
1
1
1
0
0

2
10
3
3
2
2
0
0

3
10
8
2
0
0
0
0

4
10
3
3
2
2
0
0

5
10
6
1
1
2
0
0

6
10
3
2
3
1
1
0

[0117]

33

TABLE 19

23 Days After Seed Treatment and

8 Days After Inoculation

Number of Plants of Given Disease

Rating*

Treatm.
Plants
0
1
2
3
4
5

1
10
7
0
2
1
0
0

2
10
3
1
2
3
0
1

3
10
8
1
0
1
0
0

4
10
3
3
1
2
0
1

5
10
3
3
0
2
1
1

6
10
3
2
0
3
0
2

[0118]

34

TABLE 20

Disease Indices of Seed Treatment

With Hypersensitive Response Elicitor Protein and Vector

Treatment
Disease Index (%)

Day 0
Day 15
Day 19
Day 21
Day 23

Hypersensitive
inoculate
4.0
12.0
14.0

response elicitor

protein seed dip

(1:25)

Vector seed dip
inoculate
10.0
26.0
38.0

(1:25)

Hypersensitive
inoculate
2.0
4.0
8.0

response elicitor

protein seed dip

(1:50)

Vector seed dip
inoculate
8.0
26.0
32.0

(1:50)

Hypersensitive
inoculate
14.0
18.0
36.0

response elicitor

protein seed dip

(1:100)

Vector seed dip
inoculate
8.0
30.0
42.0

(1:100)

[0119] The above data shows that the hypersensitive response elicitor protein is much more effective than the vector solution in preventing Tomato Southern Bacteria Wilt. A hypersensitive response elicitor protein concentration of 1:50 is more effective in disease control.

Example 6

Treating Rice Seeds with Hypersensitive Response Elicitor Protein to Reduce Rice Stem Rot

[0120] Rice seeds (variety, M-202) were submerged in two gallons of hypersensitive response elicitor protein solution at a concentration of 20 μg for 24 hours at room temperature. Rice seeds submerged in the same solution without hypersensitive response elicitor protein were used as a control. After soaking, the seeds were sown in a rice field by air plane spray. There were four replicates for both hypersensitive response elicitor protein and control treatment. The lot size of each replicate is 150 Ft2. The design of each plot was completely randomized, and each plot had substantial level contamination of Sclerotium oryzae. Three months after sowing, stem rot was evaluated according to the following rating scale: Scale 1=no disease, 2=disease present on the exterior of the leaf sheath, 3=disease penetrates leaf sheath completely but is not present on culm, 4=disease is present on culm exterior but does not penetrate to interior of culm, and 5=disease penetrates to interior of culm. 40 plants from each replicate were sampled and assessed for the disease incidence and severity. From Table 21, it is apparent that treating seeds with hypersensitive response elicitor reduced both disease incidence and severity. More particularly, regarding incidence, 67% of the plants were infected by stem rot for the control treatment, however, only 40% plants were infected for the hypersensitive response elicitor protein treatment. As to severity, the disease index* for the hypersensitive response elicitor protein treatment was 34% and 60% for the control. Accordingly, treating rice seed with hypersensitive response elicitor protein resulted in a significant reduction of stem rot disease. The hypersensitive response elicitor protein-induced resistance in rice can last a season long. In addition to disease resistance, it was also observed that hypersensitive response elicitor protein-treated rice had little or no damage by army worm (Spodoptera praefica). In addition, the treated plants were larger and had deeper green color than the control plants.

35TABLE 21Incidence and Severity of Stem Rot(Schlerotium oryzae) on Rice, M-202Disease% plants given diseases ratingindex (%)Treatment12345(severity)Harpin 20 μg/ml605 8181034Control33518281860

\begin{matrix} * Disease Index (%) \\ for the harpin treatment \end{matrix} = \frac{1 x60 + 2 x5 + 3 x8 + 4 x18 + 5 x10}{5 x100} \times 100 / 100

\begin{matrix} * Disease Index (%) \\ for the control treatment \end{matrix} = \frac{1 x33 + 2 x5 + 3 x18 + 4 x28 + 5 x18}{5 x100x100 / 100} \times 100 / 100

Example 7

Effect of Treating Onion Seed with Hypersensitive Response Elicitor Protein on the Development of Onion Smut Disease (Urocystis cepulae) and on Seedling Emergence

[0121] Onion seed, variety Pennant, (Seed Lot# 64387), obtained from the Crookham Co., Caldwell, ID 83606, treated with hypersensitive response elicitor protein or a control was planted in a natural organic or “muck” soil. Some of the seedlings that grew from the sown seed were healthy, some had lesions characteristic of the Onion Smut disease, and some of the sown seed did not produce seedlings that emerged from the soil. Thus, the effect of treating onion seed with various concentrations of hypersensitive response elicitor protein was determined.

[0122] Naturally infested muck soil was obtained from a field in Oswego County, NY, where onions had been grown for several years and where the Onion Smut disease commonly had been problematic. Buckets of muck (5-gallon plastic) were stored at 4° C. until used. The soil was mixed, sieved, and put in plastic flats 10 inches wide, 20 inches long, and 2 inches deep for use in the tests described. Based on preliminary experiments, the soil contained many propagules of the Onion Smut fungus, Urocystis cepulae, such that when onion seed was sown in the soil, smut lesions developed on many of the seedlings that emerged from the soil. In addition, the soil harbored other microorganisms, including those that cause the “damping-off” disease. Among the several fungi that cause damping off are Pythium, Fusarium, and Rhizoctonia species.

[0123] The hypersensitive response elicitor protein encoded by the hrpN gene of Erwinia amylovora was used to treat seeds. It was produced by fermentation of the cloned gene in a high-expression vector in E. coli. Analysis of the cell-free elicitor preparation by high-pressure liquid chromatography indicated its hypersensitive response elicitor protein content and on that basis appropriate dilutions were prepared in water. Seeds were soaked in a beaker containing hypersensitive response elicitor protein concentrations of 0, 5, 25, and 50 μgm/ml of hypersensitive response elicitor protein for 24 hours. They were removed, dried briefly on paper towels, and sown in the muck soil. Treated seed was arranged by row, 15 seeds in each row for each treatment; each flat contained two replicates, and there were six replicates. Thus, a total of 90 seeds were treated with each concentration of hypersensitive response elicitor protein. The flats containing the seeds were held in a controlled environment chamber operating at 60° F. (15.6° C.), with a 14-hour day/10-hour night. Observations were made on seedling emergence symptoms (smut lesions). The data were recorded 23 days after sowing.

[0124] The effect of soaking onion seed in different concentrations of hypersensitive response elicitor protein on emergence of onion seedlings and on the incidence of onion smut is shown in Table 22. Only slight differences in emergence were noted, suggesting that there is no significant effect of treating with hypersensitive response elicitor protein at the concentrations used. Among the seedlings that emerged, substantially more of the seeds that received no hypersensitive response elicitor protein exhibited symptoms of Onion Smut than seedlings that grew from seed that had been treated with hypersensitive response elicitor protein. Treating seed with 25 μgm/ml of hypersensitive response elicitor protein was the most effective concentration tested in reducing Onion Smut. Thus, this example demonstrates that treating onion seed with hypersensitive response elicitor protein reduces the Onion Smut disease.

36TABLE 22Effect of Treating Onion Seed WithHypersensitive Response Elicitor Protein (i.e. Harpin)on the Development of Onion Smut Disease(Urocystis cepulae).MeanTreatmentSeedlingsMeanEmergedharpinEmergedPercentPercentPercent(μg/ml)(of 15)EmergedHealthywith Smut05.0033.320.080.053.6724.440.959.1254.33128.850.046.2504.1727.744.056.01One seedling emerged then died.

Example 8

Effect of Treating Tomato Seed with Hypersensitive Response Elicitor Protein on the Development of Bacterial Speck of Tomato (Pseudomonas syringae pv. tomato)

[0125] Tomato seed, variety New Yorker (Seed lot# 2273-2B), obtained from Harris Seeds, Rochester, N.Y., were treated with four concentrations of hypersensitive response elicitor protein (including a no-elicitor protein, water-treated control) and planted in peatlite soil mix. After 12 days and when the seedlings were in the second true-leaf stage, they were inoculated with the Bacterial Speck pathogen. Ten days later, the treated and inoculated plants were evaluated for extent of infection. Thus, the effect of treating tomato seed with various concentrations of hypersensitive response elicitor protein on resistance to Pseudomonas syringae pv. tomato was determined.

[0126] The hypersensitive response elicitor protein encoded by the hrpN gene of Erwinia amylovora was used to treat seeds. It was produced by fermentation of the cloned gene in a high-expression vector in E. coli. Analysis of the cell-free elicitor preparation by high-pressure liquid chromatography indicated its hypersensitive response elicitor protein content and, on that basis, appropriate dilutions were prepared in water. Seeds were soaked in a beaker containing hypersensitive response elicitor protein concentrations of 0, 5, 10, and 20 μgm/ml of hypersensitive response elicitor protein for 24 hours. They were removed, dried briefly on paper towels, and sown. The soil was a mixture of peat and Pearlite™ in plastic flats 10 inches wide, 20 inches long, and 2 inches deep. Treated seed was arranged by row, 6 seeds in each row for each treatment; each flat contained two replicates, and there were four replicates and thus a total of 24 seeds that were treated with each concentration of hypersensitive response elicitor protein. The flats containing the seeds were held in a controlled environment chamber operating at 75° F. (25° C.), with a 14-hour day/10-hour night.

[0127] When twelve-days old, the tomato seedlings were inoculated with 108 colony forming units/ml of the pathogen, applied as a foliar spray. The flats containing the seedlings were covered with a plastic dome for 48 hours after inoculation to maintain high humidity. Observations were made on symptom severity using a rating scale of 0-5. The rating was based on the number of lesions that developed on the leaflets and the cotyledons and on the relative damage caused to the plant parts by necrosis that accompanied the lesions. The cotyledons and (true) leaflets were separately rated for disease severity 11 days after inoculation.

[0128] The effect of soaking tomato seed in different concentrations of hypersensitive response elicitor protein (i.e. harpin) on the development of Bacterial Speck on leaflets and cotyledons of tomato is shown in Table 23. The seedlings that grew from seed treated with the highest amount of hypersensitive response elicitor protein tested (20 μgm/ml) had fewer diseased leaflets and cotyledons than the treatments. The water-treated control seedlings did not differ substantially from the plants treated with the two lower concentrations of hypersensitive response elicitor protein. Considering the disease ratings, the results were similar. Only plants treated with the highest concentration of hypersensitive response elicitor protein had disease ratings that were less than those of the other treatments. This example demonsrates that treatment of tomato seed with hypersensitive response elicitor protein reduces the incidence and severity of Bacterial Speck of tomato.

37TABLE 23Effect of Treating Tomato Seed WithHypersensitive Response Elicitor Protein (i.e. Harpin)on the Subsequent Development of Bacterial Speck Disease(Pseudomonas syringae pv. tomato) on Tomato Cotyledonsand Tomato LeafletsCotyledonsLeafletsTreatmentMeanMeanPercentHarpinDi-PercentDiseaseDi-Di-Disease(μg/ml)seasedDiseasedRatingseasedseasedRating 06.0/9.066.60.825.8/68.837.50.5 55.3/7.372.40.822.5/68.037.50.5105.8/8.072.30.825.5/66.038.60.5205.3/8.561.80.623.8/73.532.30.4

[0129] Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

	Number	Date	Country
Parent	08984207	Dec 1997	US
Child	09766348	Jan 2001	US

Hypersensitive response induced resistance in plants by seed treatment

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