MXPA01003464A - Hypersensitive response elicitor from xanthomonas campestris. - Google Patents

Abstract

The present invention is directed to an isolated Xanthomonas campestris hypersensitive response elicitor protein or polypeptide. The hypersensitive response elicitor proteins or polypeptides in accordance with the present invention and the isolated DNA molecules that encode them have the following activities: imparting disease resistance to plants, enhancing plant growth, and/or to controlling insects on plants. This can be achieved by applying the hypersensitive response elicitor in a non-infectious form to plants or plant seed under conditions effective to impart disease resistance, to enhance plant growth, 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 elicitor 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, and/or to control insects on plants or plants grown from the plant seeds.

Description

PROMOTER OF THE HYPERSENSITIVE RESPONSE OF XANTHCMONAS CAMPES RIS The present invention relates to the active fragments of a hypersensitive response promoter.

BACKGROUND OF THE INVENTION The interactions between bacterial pathogens and their host or plant hosts are generally in two categories: (1) compatible (pathogen-host), which leads to intercellular bacterial development, development of symptoms, and development of the disease in the host plant; and (2) incompatible (pathogen-non-host), resulting in the hypersensitive response, a particular type of incompatible interaction that occurs, with no symptoms of progressive disease. During compatible interactions on host plants, bacterial populations increase rapidly 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, Z., REF: 128697"Defenses Triggered by the Invader: Hypersensitivity", pages 201-224 in: Plant disease: An Advanced Treatise, Vol. 5, J. G. Horsfall & E. B. Cowling, ed. Academic Press New Ycrk (1980); Klement, Z. "Hipersensibity", pages 149-177 in: Phytopathogenic Prokaryotes)), Vol. 2, M.S. Mount & G.H. Lacy, ed Academic Press, New York (1982)). The hypersensitive response promoted by bacteria is easily observed as a tissue collapse if high concentrations (> 107 cells / ml) of a pathogen of limited host range, such as Pseudomonas syringae or Erwinia amylovora are infiltrated in the leaves of non-host plants (occurs necrosis only in isolated plant cells at lower inoculum levels) (Klement Z., "Rapid Detection of Pathogenicity of Phytopathogenic Pseudomonas", Nature 199: 299-300, Klement et al., "Hypersensibity Reaction Induced by Phytopathogenic Bacteria in the Tobacco Leaf ", Phytopathology 54: 474-477 (1963); Turner et al., "The Quantitative Relation Between Plant and Bacterial Cells Involved in the Hypersensitive Reaction ", Phytopathology 64-885-890 (1974): Klein Z." Hypersensibity "pages 149-177 in Phytopathogenic Prokaryotes, Vol. 2, MS Mount and GH Lacy, ed. Academic Press, New York (1982).) The abilities to promote the hypersensitive response in a non-host and to be pathogenic in a host, seem to be linked as is noted by Klement Z. "Hypersensivity" pages 149-177 in Phytopathogenic Prokaryotes, Vol 2, MS Mount and GH Lacy, ed, Academic Press, New York, these pathogens also cause physiologically similar necrosis, although delayed, in their interactions with compatible hosts.In addition, the ability to produce the hypersensitive response or pathogenesis is dependent of a common group of genes, denoted hrp (Lindgren, PB and colcitators "Gene Cluster of Pseudomonas syringae pv. phaseolicola 'Controls Pathogenicity of Bean Plants and Hypersensivity on Nonhost Plants "J. Bacteriol. 168-512-22 (1986); illis, D. K. and collaborators, "hrp Genes of Phytopathogenic Bacteria "Mol. Plant-Microbe Interact 4: 132-138 (1991)). Consequently, the hypersensitive response can hold the keys to the nature of the defense of the plant and the basis for bacterial pathogenicity. The hrp genes are widespread in Gram-negative vegetative pathogens, where they accumulate, and are conserved and in some cases interchangeable (Willis, DK et al., "Hrp Genes of Phytopathogenic Bacteria", Mol. Plant-Microbe Interact. 132-138 (1991); Bones, U, "hrp Genes of Phytopathogenic Bacteria," pages 79-98 in: Current Topics in Microbiology and Immunology; Bacteria to Pathogenesis of Plants and Animáis - Molecular and Cellular Mechanisms, JL Dangl, ed. Springer - Verlag, Berlin (1994)). Several hrp genes code for the components of a protein secretion pathway, similar to one used by Yersinia, Shigella, and Salmonella spp., to secrete essential proteins in animal diseases (Van Gijsegem et al., "Evolution Conservation of Pathogenicity Determinants Among Plant and Animal Pathogenic Bacteria", Trends Microbiol 1: 175-180 ( 1993).) In E. amylovora, P. syringae, and P. solanacearum, hrp genes have been shown to control the production and suppression of glycine-rich protein promoters of the hypersensitive response (He, SY et al., * 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), Wei, Z, H., and collaborators, wHrpl of Erwinia amylovora Functions in Secretion of Harpin and is a Member of a New Protein Family ", J. Bacteriol 175-7958-7967 (1993); Arlat M. and collaborators X? PopAl, a protein Which Induces a Hypersensitive-like Response on Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum "EMBO J. 13: 543-553 (1994).) The first of these Protein was discovered in E. amylovora Ea321, a bacterium that causes fire blight of pink plants, and was designated harpin (Wei "*" Z-M. And collaborators "Harpin Elicitor of the Hypesensitive Response Produced by the Plant Pathogen Erwinia amylovora" Science 257: 85-88 (1992)). Mutations in the hrpN coding gene revealed that harpin is required for E. amylcvora, to promote a hypersensitive response in non-host tobacco leaves and incite disease symptoms in highly susceptible pears. The PopAl protein of P. Solanacearum GMI1000 has similar physical properties and also promotes the hypersensitive response in leaves of tobacco, which is not a host of that strain (Arlat et al. "PopAl, a Protein Which Induces a Hypersensitive-like Response on Specific Petunia Genotypes is Secreted via the Hrp Pathawy of Pseudomonas solanacearum "EMBO J. 13: 543-53 (1994)). However, the mutants of P. Solanacearum PoPAl GMI1000 also promote the hypersensitive response in tobacco and promote the disease in tomato. Thus, the role of these glycine-rich hypersensitive response promoters can vary widely in Gram negative plant pathogens. Other hypersensitive response promoters, plant pathogens, have been isolated, cloned and sequenced. These include: Erwinia chrysanthemi (Bauer et al. "Erwinia chrysanthemi HarpinE h 'Soft-Rot Pathogenesis", MPMI 8 (4): 484-91 (1995)); Erwinia carotovora (Cui, and collaborators "The Rs A" Mutants of Erwinia carot ovora subsp.carotovora Strain Ecc71 Overexpress hrpNcc and Elicit a Hypersensitive Reaction-like Response in Tobacco Leaves ", MPMI9 (7): 565-73 (1996)); Erwinia stewa rtii (Ahmad et al, "Harpin is not necessary for the Pathogenicity of Erwinia stewartii on Maize" 8th Int., Cong. Molec. Plant-Microb, inter, July 14-19, 1996 and Ahmad, et al., "Harpin is not Neccesary for the Pathogenicity of Erwinia stewartii on Maize "Ann. Mt. Am. Phytopath. Soc. July 27-31, 1996) and Pseudomonas syringae pv. Syringae (WO 94/26782 to Cornell Research Foundation, Inc.) present invention seeks to identify another protein or polypeptide promoter of the hypersensitive response.

BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to a protein or polypeptide promoter of the hypersensitive response, of Xanthomonas campestris, isolated. The promoters of the hypersensitive response according to the present invention have the following activity when used together in plants: the imparting of resistance against diseases to the plants, the increase of the growth of the plant and / or the control of insects. This involves the application of the hypersensitive response promoter in a non-infectious manner to the plants or the seeds of the plants, under conditions effective to impart resistance against the disease, to increase the growth of the plant and / or to control insects on the plants. Plants or plants developed from the seeds of plants. As an alternative to the application of the hypersensitive response promoter to plants or plant seeds in order to impart resistance against diseases, to increase the growth of plants and / or to control insects on plants, plants can be used transgenic seeds or seeds of transgenic plants. When transgenic plants are used, this involves the provision of a transgenic plant transformed with a DNA molecule that codes for a protein or polypeptide promoter of the hypersensitive response, of Xanthomonas campestris, according to the present invention, and the development of the plant under effective conditions to impart resistance against the disease, to increase the development of the plant and / or to control insects in the plants or plants developed from the seeds of plants. Alternatively, a transgenic plant seed transformed with the DNA molecule encoding such a hypersensitive response promoter can be provided and seeded in the soil. A plant is then propagated under effective conditions to impart resistance against the disease, to increase the development of the plants and / or to control insects on the plants or plants developed from the seeds of plants.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a protein or polypeptide promoter of the hypersensitive, isolated response of Xanthomonas campestris. The hypersensitive response promoters according to the present invention have the following activity when used in conjunction with plants: imparting disease resistance to plants, increasing plant development and / or controlling insects. This involves the application of the hypersensitive response promoter in a non-infectious way to plants or plant seeds, under conditions effective to impart resistance against the disease, to increase the growth of the plants and / or to control the insects on the plants. plants or plants developed from plant seeds. As an alternative to the application of the hypersensitive response promoter to plants or plant seeds, in order to impart resistance against the disease, to increase the development of the plants, and / or to control insects on the plants, they can be used transgenic plants or seeds of transgenic plants. When transgenic plants are used, this involves the provision of a transgenic plant transformed with a DNA molecule that codes for a hypersensitive response-promoting protein or polypeptide, of Xanthomonas campestris, according to the present invention, and developing the plant under effective conditions to impart resistance against the disease, to increase the development of the plant and / or to control insects in the plants or plants developed from the seeds of plants. Alternatively, a transgenic plant seed transformed with the DNA molecule encoding such a hypersensitive response promoter can be provided and planted in the soil. A plant is then propagated under effective conditions to impart resistance against the disease, to increase the development of the plant, and / or to control insects on plants or plants developed from plant seeds. The polypeptide or hypersensitive response promoter protein derived from Xanthomonas campestris has an amino acid sequence corresponding to SEQ. ID. No. 1 as follows: Met Asp Gly Lie Gly Asn His Phe Ser Asn 1 5 10 This polypeptide or hypersensitive response promoter protein has a molecular weight of 13-15 kDa. Fragments of the aforementioned hypersensitive response promoting polypeptides or proteins are encompassed by the present invention. Suitable fragments can be produced by various means. First, subclones of the gene encoding a known promoter protein are produced by conventional molecular genetic manipulation, by subcloning gene fragments. The subclones are then expressed in vi tro or in vivo in bacterial cells to produce a smaller protein or a smaller peptide that can be tested for promoter activity, according to the procedure described below.

As an alternative, fragments of a promoter protein can be produced by digesting a full-length promoter protein with proteolytic enzymes such as chymotrypsin or proteinase A from Staphylococcus, or trypsin. It is likely that different proteolytic enzymes break the promoter proteins at different sites, based on the amino acid sequence of the promoter protein. Some of the fragments that result from proteolysis can be active promoters of resistance. In yet another procedure, based on the knowledge of the primary structure of the protein, the fragments of the promoter protein gene can be synthesized by using the PCR technique together with the specific groups of primers chosen, to represent particular portions. of the protein. These could then be cloned into an appropriate vector for the expression of a truncated protein or peptide. Chemical synthesis can also be used to make suitable fragments. Such synthesis is carried out using known amino acid sequences for the promoter that is produced. Alternatively, attaching a full-length promoter at high temperatures and high pressures will produce fragments. These fragments "8-aa-tg. they can then be separated by conventional procedures (e.g., chromatography, SDS-PAGE). Variants can be elaborated, for example by 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 can be conjugated to a signal sequence (or leader) at the N-terminus of the protein which co-translationally or post-translationally directs the transfer of the protein. The polypeptide can also be conjugated to a linker or to another sequence for ease of synthesis, purification, or identification of the polypeptide. The hypersensitive response promoter of the present invention is preferably in isolated form (for example separated from its host organism Xanthomonas campeetris) and more preferably produced in purified form (preferably at least about 60%, more preferably 80% pure) by conventional techniques. Typically, the hypersensitive response promoter 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 the growth medium. In the case of the non-secreted protein, to isolate the protein fragment, the host cell (e.g., E. coli) which possesses the recombinant plasmid is propagated, used by sonication, heat or chemical treatment, and the homogenate is centrifuged to eliminate bacterial waste. The supernatant is then subjected to heat treatment and the hypersensitive response promoter is separated by centrifugation. The supernatant fraction containing the hypersensitive response promoter is subjected to gel filtration on a dextran or polyacrylamide column of adequate size to separate the fragment. If necey, the protein fraction can be purified by ion exchange or HPLC high-performance liquid chromatography. The DNA molecule encoding the hypersensitive response promoter or polypeptide protein can be incorporated into cells using conventional recombinant DNA technology. In general, this involves the insertion of the DNA molecule into an expression system for which the DNA molecule is heterologous (for example not normally present). The heterologous DNA molecule is inserted into the expression system or vector in the sense orientation and in the correct reading structure. The vector contains the necey elements for the transcription and translation of the inserted protein coding sequences. U.S. Patent No. 4,237,224 to Cohen and Boyer, which is incorporated by reference herein, 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 into unicellular cultures including prokaryotic organisms and eukaryotic cells grown in tissue culture. Recombinant genes can also be introduced into viruses, such as vaccinia virus. Recombinant viruses can be generated by transfection of plasmids in cells infected with the virus. Suitable vectors include, but are not limited to, the following viral vectors such as the gtll system of the lambda vector, gt.WES.tB, Charon 4, and the plasmid vectors such as pBR322, pBR325, pACYC177, pACYC: .084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC10, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems" catalog (1993) by Stratagene, La Jolla, California, which it is incorporated by reference herein) the series pQE, pIH821, pGEX, pET (see FW j i Studier et al., "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes", Gene Expression Technology vol, 185 (1990), which is incorporated by reference herein), 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 incorporated by reference in the present. A variety of host-vector systems can be used to express the or sequences encoding the protein. 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 (for example vaccinia virus, adenovirus, etc.); insect cell systems infected with the virus (for example baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their resistance and specificities. Depending on the host vector system used, any of a number of suitable transcription and translation elements can be used. Different genetic signals and processing events control many levels of gene expression (eg, transcription of DNA and translation of messenger RNA (mRNA).) Transcription of DNA is dependent on the presence of a promoter that is a sequence. of DNA that directs the binding of RNA polymerase and thereby promotes mRNA synthesis.The DNA sequences of eukaryotic promoters differ from those of prokaryotic promoters.In addition, eukaryotic promoters and accompanying genetic signals can be recognized in or they may not work in a prokaryotic system, in addition, prokaryotic promoters are not recognized and do not work in prokaryotic cells, Similarly, the translation of mRNA in prokaryotes depends on the presence of adequate prokaryotic signals that differ from those of eukaryotes. Efficient translation of mRNA in prokaryotes requires a ribosome binding site called the Shine-Dalgarno sequence ("SD") on the mRNA. This t ~. * Sequence is a short nucleotide sequence of the mRNA that is located before the start codon, usually AUG, that codes for 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 the binding of the mRNA to the ribosomes, by duplication or duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lue. Methods in Enzymology. 68: 473 (1979), which is incorporated by reference herein. Promoters vary in their "strength or endurance" (for example their ability to promote transcription). For purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, therefore, expression of the gene. Depending on the system of the host cell used, any of a number of suitable promoters can be used. For example, when its bacteriophages or promoter plasmids such as the T7 phage promoter, the Jac promoter, the trp promoter, the recA promoter, the ribosomal RNA promoter, the PR and PL promoters of the lambda coliphage are cloned into E. coli. others, including but not limited to lacUV5, ompF, bla, Ipp, and the like, can be used to direct high levels of transcription of adjacent DNA segments. In addition, a hybrid promoter trp-_ZacUV5. { tac) or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques, can be used to provide transcription of the inserted gene. Bacterial host cell strains and expression vectors can be chosen, which inhibit the action of the promoter, unless specifically induced. In certain operations, the addition of specific inductors is necessary for the efficient transcription of the inserted DNA. For example, the operon Jac 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 transcription and translation of prokaryotic cell genes. These signals of transcription and translation initiation may vary in "strength" as measured by the amount of the specific messenger RNA of the gene and protein synthesized, respectively. The DNA expression vector, which contains a promoter, can also contain any combination of various "strong" start transcription and / or translation signals. For example, efficient translation in E. coli requires that an SD sequence of approximately 7-9 bases, 5 'to the start codon ("ATG"), provide a ribosome binding site. In this way, any combination of SD-ATG that can be used by the ribosomes of the host cell can be used. Such combinations include, but are not limited to, the SD-ATG combination from the ero gene or the N gene of the lambda coliphage, or from the E, D, C, B or A genes of tryptophan, from E. coli. In addition, any SD-ATG combination produced by recombinant DNA or other techniques involving the incorporation of synthetic nucleotides can also be used. Once the isolated DNA molecule encoding the peptide or hypersensitive response promoter 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 various transformation forms noted above, depending on the vector-host cell system. Suitable host cells include, but are not limited to, bacteria, viruses, yeast, mammalian cells, insect, plants and the like. The present invention also relates to methods for imparting disease resistance to plants, increasing the growth of plants, and / or effecting insect control for plants. These methods involve the application of the hypersensitive response promoter protein or polypeptide of the present invention in a non-infectious form to all or part of a plant or a seed of a plant under conditions effective for the promoter to impart resistance against the disease, increase growth and / or control insects. Alternatively, the protein or polypeptide promoter of the hypersensitive response can be applied to plants, such that the seeds recovered from such plants are capable of imparting resistance to the disease in plants, increasing the growth of the plant, and / or effecting the control of insects As an alternative to the application of a protein or polypeptide that promotes the hypersensitive response to plants or plant seeds, in order to impart resistance against the disease in plants, carry out the development of the plant and / or control the insects on plants or plants developed from the seeds, transgenic plants or seeds of transgenic plants can be used. When transgenic plants are used, this involves the provision of a transgenic plant transformed with a DNA molecule that codes for a polypeptide or response-promoting protein. - hyperpersible, whose fragment promotes a hypersensitive response, and the development of the plant under effective conditions to allow the DNA molecule to impart resistance against the disease to plants, increase the growth of the plant and / or control insects. Alternatively, a transgenic plant seed transformed with a DNA molecule encoding the hypersensitive response-promoting polypeptide or protein of the present invention can be provided and planted in the soil. A plant is then propagated from the seeded seed, under effective conditions to allow the DNA molecule to impart resistance against the disease to the plants, increase plant growth and / or control the insects. The embodiment of the present invention wherein the hypersensitive response promoter or polypeptide protein is applied to the plant or seed of the plant, can be carried out in a number of ways, including: 1) the application of the response promoter hypersensitive, isolated or 2) the application of bacteria that do not cause the disease and are transformed with a gene that codes for the promoter. In the latter embodiment, the promoter can be applied to plants or plant seeds by the application of bacteria containing the DNA molecule encoding the hypersensitive response-promoting polypeptide or protein. Such bacteria must be capable of secreting or exporting the promoter, so that the promoter can make contact with the plant cells or with the seeds of the plants. In these 5 modalities, the promoter is produced by the bacteria in plant or on the seeds, or just before the introduction of the bacteria to the plants or to the seeds of the plants. The methods of the present invention can be 10 used to treat a wide variety of plants or their seeds, to impart resistance against the disease, increase development and / or control insects. Suitable plants include dicotyledons and monocots. More particularly, the plants of 15 useful crops may include: alfalfa, rice, wheat, cebadar rye, cotton, sunflower, peanut, corn, potato, sweet potato, beans, peas, chicory, lettuce, chicory, cabbage, Brussels sprouts, beets, parsnips, turnip, cauliflower, broccoli, radish, spinach, onion, garlic, eggplant, 20 peppers, celery, carrots, zucchini or chayotes, pumpkin, courgettes, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugar cane. Examples of suitable plant-ornamentals are: Arabidopsis thaliana.

Saintpaulia, petunia, pelargonium, red shepherdess, chrysanthemum, carnation and zinia. With respect to the use of the hypersensitive response promoter protein or polypeptide of the present invention in imparting resistance against the disease, absolute immunity against infection may not be conferred, but the severity of the disease is reduced and the development of the symptoms It is retarded. The number of lesions, the size of the lesions, and the degree of sporulation of fungal pathogens are all diminished. This method of imparting resistance against the disease has the potential to treat previously untreatable diseases, the treatment of diseases systemically that can not be treated separately, due to cost, and to avoid the use of infectious agents or environmentally harmful materials. The method for imparting pathogen resistance to plants according to the present invention is useful in imparting resistance to a wide variety of pathogens, including viruses, bacteria and fungi. Resistance, among others, to the following viruses can be achieved by the method of the present invention; Tobacco mosaic virus and tomato mosaic virus. Resistance, among others, to the following bacteria can be imparted to plants according to the present invention: Pseudomonas solanacearum, Pseudomonas syrinqae pv. tabaci and Xanthomonas campestris pv. pelargonii. The plants can be made resistant, inter alia, to the following fungi by the use of the present invention: Fusarium um oxysporum and Phytoph thora infestans. With respect to the use of the promotor protein or polypeptide of the hypersensitive response of the present invention, to improve or increase the development of the 10 plants, various ways of increasing or promoting the growth of plants can be achieved. This can happen as early as when the plant begins to develop from the seeds or later in the life of a plant. For example, him Development according to the present invention encompasses higher yield, increased amount of seeds produced, increased percentage of germinated seeds, increased size of the plant, greater biomass, more and more large fruits, earlier coloration of the fruit, and maturation 20 earliest of the fruit and the plant. As a result, the present invention provides significant economic benefits for growers. For example, early germination and early maturation allow crops to be grown in areas where 25 short development stations could otherwise exclude its development on that site. The increased percentage of germination of the seeds results in improved resistances in the harvests and more efficient use of the seeds. Higher yield, increased size, and increased biomass production allow greater income generation from a given portion of land. Yet 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 the insects from coming into contact with the plants to which the hypersensitive response promoter has been applied, preventing direct damage by the insect to the plants by means of the damage by feeding, causing insects to move away from such plants, killing insects near such plants, interfering with the feeding of insect larvae on such plants, preventing insects from colonizing host plants, preventing colonizing insects from releasing phytotoxins, etc. The present invention also prevents subsequent damage by disease to plants, which results from infection by the insect. The present invention is effective against a wide variety of insects. The European corn borer is a major pest of maize (sweet and tooth corn) but also feeds more than 200 plant species including beans, sesame beans and crescent beans and beans Edible soybeans, peppers, potatoes, tomatoes , plus many species of herbs. Pests of additional insect larvae that feed on many plants that plant a wide variety of plant crops include the following: beet devastating worm, cabbage measuring worm, corn ear worm, devastating autumn worm, diamond back moth, cabbage root larva, onion larva, corn seed larva, stripping worm (melon worm), pepper larva, tomato see me and larvae. Collectively, this group of insect pests represents the economically most important group of pests for the production of vegetables worldwide. The method of the present invention which involves the application of the hypersensitive response promoter or polypeptide, can be carried out through a variety of procedures when all or part of the plant is treated, including leaves, stems, roots, propagules (for example, cuttings), etc. This may involve (but does not need) the infiltration of the polypeptide or promoter protein of the hypersensitive response within the plant. The methods of - &jaajjaa suitable application include spraying at high or low pressure, injection and blade abrasion next to when the application of the promoter takes place. When plant seeds or propagules thereof are treated (e.g., cuttings, according to the embodiment of the present invention, the hypersensitive response promoter protein or polypeptide according to the present invention), it can be applied by means of low or high pressure spray, coating, immersion or injection. Other suitable methods of application may be considered by those skilled in the art, with the proviso that they are capable of effecting contact of the promoter with the plant cells or the seed of the plant. Once treated with the hypersensitive response promoter of the present invention, the seeds can be planted in natural or artificial soil and grown using conventional procedures to produce plants. After the plants have been propagated from the treated seeds according to the present invention, the plants can be treated with one or more applications of the hypersensitive response promoter or protein or polypeptide to impart disease resistance to the plants , to increase the growth of the plants, and / or to control the insects on the plants. The hypersensitive response promoting polypeptide or protein, according to the present invention, can be applied to plants or plant seeds, alone or in admixture with other materials.

Alternatively, the promoter can be applied separately to the plants with other materials that are applied at different times. A composition suitable for treating plants or plant seeds according to the embodiment of the present invention, contains the hypersensitive response promoting polypeptide or protein, in a carrier. Suitable carriers include water, aqueous solutions, suspensions or dry powders. In this modality, the composition contains more than 500 nM fragment. Although not required, this composition may contain additional additives including fertilizers, insecticides, fungicides, nematicides, and mixtures thereof. Suitable fertilizers include (NH4) 2N03. An example of a suitable insecticide is malathion. Useful fungicides include Captan. Other suitable additives include buffers, wetting agents, coating agents, and abrasive agents. These materials can be used to facilitate the process of the present invention. In addition, the fragment that promotes the hypersensitive response 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, which involves the use of transgenic plants and transgenic seeds, the hypersensitive response promoter need not be applied topically to plants or seeds. Rather, transgenic plants transformed with a DNA molecule encoding such a promoter are produced according to procedures well known in the art. The vector described above can be directly microinjected into plant cells by the use of micropipettes to mechanically transfer the recombinant DNA. Crossway, Mol. Gen. Genetics, 202: 179-85 (1985), which is incorporated by reference herein. The genetic material can be transferred to the plant cell using polyethylene glycol. Krens et al., Nature, 296: 72-74 (1982) which is incorporated by reference herein.

Another method for transforming plant cells with a gene that imparts resistance against 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 the propulsion of inert or biologically active particles to the cells. This technique is described in U.S. Patent Nos. 4,945,050, 5,036,006 and 5,100,792, all to Sanford et al. Which are incorporated by reference herein. In general, this method involves the propulsion of inert or biologically active particles in the cells under effective conditions to penetrate the outer surface of the cell, and to be incorporated into the interior thereof. When inert particles are used, 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 brought to the cell by the awakening of the particle. Biologically active particles (eg, dehydrated bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells.

Another method for the introduction is the fusion of protoplasts with other entities, either mini-cells, cells, lysosomes, or other fusible bodies with a lipid surface. Fraley et al. Proc. Nati Acad. Sci 5 USA. 79: 1859-62 (1982), which is incorporated by reference herein. The DNA molecule can also be introduced into plant cells by electroporation. Fromm et al., Proc. Nati Acad. Sci. USA, 82: 5824 10 (1985), which is incorporated by reference herein. In this technique, the plant protoplasts are subjected to electroporesis in the presence of plasmids containing the expression cassette. The high field strength electrical impulses reversibly permeabilize the 15 biomeiabranas, allowing the introduction of the plasma. Plant protoplasts subjected to eletroporesis reform the cell wall, divide it and regenerate it. Another method for the introduction of the 20 DNA molecule to 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, transformed plant cells are developed to form 25 shoots or roots, and are further developed in & ^ áat ^? Jtíii ^^ ¡^^^ 'hi! ^ í:' i plants. In general, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on the regeneration medium without antibiotics at 25-28 ° C. Agrobacterium is a representative genus of the Gram negative family of Rhizobiaceas. Their species are responsible for diseases of the gall bladder. { A. tvmefaciens) and of the hairy root (A. rhizogenes). Plant cells in crown gallbladder tumors and hairy roots are induced to produce amino acid derivatives known as opinas, which are catabolized only by bacteria. The bacterial genes responsible for the expression of opines are a convenient source of control elements for chimeric expression cassettes. In addition, the test for the presence of opines can be used to identify the transformed tissue. The heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid is transmitted to the plant cells in the Agrobacterium infection and is stably integrated into the genome of the plant. J. Shell. Science. 237-1176-83 (1987), which is incorporated by reference herein.

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, 1934, and Vol III (1986), which are incorporated by reference herein. 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 in general it is first provided a suspension of transformed protoplasts or a petri dish containing transformed explants. The callus tissue is formed and the shoots can be induced from the callus and subsequently rooted. Alternatively, the formation of the embryo 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 species such as corn and alfalfa. Efficient regeneration will depend on the medium, the genotype, and the history of the crop. If these three variables are controlled, then regeneration is usually reproducible and repeatable. After the expression cassette is stably incorporated into the transgenic plants, it can be transferred to other plants by cross-breeding 10 sexual. Any of a number of standard breeding techniques can be used, depending on the species that is to be crossed. Once transgenic plants of this type are produced, the plants themselves can be 15 cultured according to conventional procedures, with the presence of the gene encoding the hypersensitive response promoter, resulting in resistance against disease, increased growth of the plant, and / or control of insects on the 20 floor. Alternatively, transgenic seeds or propagules (for example, cuttings) are recovered from the transgenic plants. The seeds can then be planted in the soil and grown using conventional procedures to produce plants 25 transgenic. Transgenic plants are propagated to g »s ^! ¡Maa¡ & Fafae ^ a» * | i ^^^. from planted transgenic seeds, under effective conditions to impart resistance against the disease to the plants, to improve or increase the growth of the plant and / or to control the insects. While not wishing to be bound by any theory, such resistance against disease, increased growth, and / or control of insects may be mediated by RNA, or may result from expression of the polypeptide or protein fragment. When transgenic plants and plant seeds are used according to the present invention, they can also be treated with the same materials that are used to treat plants and seeds, to which a hypersensitive response promoter is applied. according to the present invention. These other materials, including the hypersensitive response promoter according to the present invention, can be applied to other transgenic plants and plant seeds by the above-indicated procedures, including high or low pressure spray, injection, coating and immersion. Similarly, after the plants have been propagated from the seeds of transgenic plants, the plants can be treated with one or more applications of the fragment of a response promoter.

Hypersensitive, according to the present invention, to impart resistance against the disease, increase development and / or control insects. Such plants can also be treated with conventional agents for the treatment of plants (for example, insecticides, fertilizers, etc.).

EXAMPLES 10 Example 1. Crop Development For the purpose of this study, Xanthomonas campestris pelargonii (Xcp) was grown on Luria agar plates. From these plates, the 15 colonies to inoculate seed cultures Xcp. Planting cultures were developed in 250 ml buffered flasks containing 50 ml of 50% Luria broth. Seed crops were developed at approximately 21 ° 0., while stirring at 250 rpm, 20 until an optical density (? 620) of 0.5 to 0.8 was reached. To inoculate the minimum medium, the sowing cultures were centrifuged in sterile, 500 ml cetrifuge bottles for 10 minutes at 4 ° C and 10,000 rpm. 25 The supernatant was discarded and the button or concentrate The resulting cell was resuspended in minimal medium already prepared, in such a way that no Luria broth was introduced in the culture of the minimum medium. A 1:10 ratio of the seed culture to the minimum medium was consistently used to inoculate the minimum medium. In other words, the cell button formed a 50 ml seed culture that was used to inoculate 500 ml of minimal medium. The minimum medium culture was developed in a 2.8-liter Fernbach flask containing 500 ml of medium, 10 approximately at 27 ° C which was stirred at 250 rpm until an optical density (? 620) of 1.7 to 2.0 was reached. After the production of the culture flask had been optimized, the fermentation was transferred to a 10 liter MicroFern Fermenter (New 15 Brunsw: .ck Scientific, Edison, New Jersey, USA). For the fermentation of 10 liters, the proportion of the seed culture to the minimum medium was maintained, as described above. The fermentation was run at approximately 27 ° C with an initial pH of 6.0 and a final pH of 5.8. He The vessel was stirred at 400 rpm with 0.8 to 1.0 volumes of the air vessel, per minute. One liter of a stock solution of the minimum medium contained 39.2 g of dipotassium acid phosphate, 71.5 g of potassium diacid phosphate, 10.0 g of ammonium sulfate, 25 3.5 g of magnesium chloride, 1.0 g of sodium chloride, and ttfflS -i8fe ^ & 34.23 g of sucrose with a final pH of 6.0 to 6.2, which was prepared after mixing thoroughly. The reserve was sterilized by filtration and kept at 4 ° C.

Example 2 - Preparation of the Cell-Free Promoter The first step in the purification of the hypersensitive response promoter of Xcp was the development of a cell-free promoter preparation ("CFEP"). The production of CFEP involves four steps as described below. 1. Initial centrifugation In the initial centrifugation step, the minimum medium cell culture was divided into 370 ml aliquots and centrifuged in 500 ml centrifuge bottles for 10 minutes at 4 ° C and 8,000 rpm. The resulting cell button was resuspended at a 1:10 weight to volume ratio using the lysis buffer. The lysis buffer consisted of 10 mM sodium chloride and Tris-HCl at pH 8.0. The resuspension of the cellular button was achieved through the vortexing of the individual centrifuge bottles. In the case of the 10-liter fermentation, a homogenizer was used. 2. Sonication The resuspended button was then sonicated in aliquots of 50 ml at a setting of 5 for 3 minutes (with the 5 tip of the horn, VirTris, VirSonic). While sonicéiba, the container containing the solution was submerged in a bath of ice water. The resulting sonication was kept on ice until all the solution was sonic. The setting used for sonication 10 was the maximum setting suggested by the manufacturer for the tip used. 3. Heat treatment The sonicate was placed on a preheated stir plate and brought to a rotary boil. The rotary boil was held for 5 minutes. After 5 minutes of heat treatment, the solution was immediately placed in an ice-water bath and 20 cooled to about 10 ° C.

. Final Centrifugal Before the final centrifugation, the cooled solution was brought back to its original volume with Te ^^^ jgjtj ^ deionized water, replacing the lost volume to evaporation during boiling. The solution was then centrifuged in 50 ml centrifuge bottles for 30 minutes at 4 ° C and at 15,000 rpm. The resulting supernatant from each bottle was combined and frozen in a freezer at -80 ° C. A sample of 1 ml was also saved and used for the hypersensitive response activity test.

Example 3 - Protein Verification To determine whether the hypersensitive response promoter was of course a protein, protease digestions were performed with CFEP. CFEP, prepared as previously described, was inoculated with protease K at a concentration of 1 mg / ml. After an incubation of 1.5 hours at 37 ° C, the CFEP incoculated with protease together with the positive control (CFEP alone) and the negative control (protease K at 2 mg / ml in lysis buffer) were infiltrated in tobacco plants to test the promoter of the hypersensitive response. The positive control showed hypersensitive response necrosis, whereas the negative control showed no signs of testing. The CEFP inoculated with protease K also showed no signs of hypersensitive response, indicating that the promoter of the response • * * * * * ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ hypersensitive Xcp was sensitive to protease digestion, and of course it is a protein.This experiment was repeated several times with different batches of CFEP, each time with the same results.

Example 4 - Chromatographic Purification The production of the CFEP of the hypersensitive response promoter was the first step in the purification scheme for the promoter of the hypersensitive response of Xcp. In addition, the purification of the promoter consisted of four chromatographic steps. Anion exchange, cation exchange, various types of affinity chromatography, hydrophobic interaction, and reverse phase chromatography media were all analyzed for their usefulness in the purification of the hypersensitive response promoter. All chromatography experiments were conducted with FPLC and the FPLC detector (Pharmacia Biotech, Upsala, Sweden). The final purification scheme used consisted mainly of the chromatography medium that was linked to the hypersensitive response promoter Xcp based on the hydrophobic characteristics of the hypersensitive response promoter. 1. Butyl-Sepharose The CFEP was first linked to a medium strength hydrophobic interaction chromatography medium. The CFEP was adjusted to 600 mM NaCl and loaded onto a rapid flow column of Butyl-Sepharose (Pharmacia Biotech, Uppsala, Sweden). The column was eluted with a gradient of 75-100% buffer B. Buffer A contained 600 mM sodium chloride, 20 mM Tris-HCl, pH 8, and buffer B contained 10 mM Tris-HCl at pH 8 At 85% gradient B, buffer B was exchanged with deionized water. 2. Monkey S The fractions that were judged to have the highest concentration of the hypersensitive response promoter (determined by performing serial dilution of the hypersensitive response promoter, with active fractions) from the Butyl-Sepharose column were pooled together and charged to a strong cation exchanger, Mono S (Mono Column S 10/10 Pharmacia Biotech). Before loading, the combined reactions were adjusted to 20 mM NaCl, 20 mM Tris-HCl at pH 5.5. Buffer A was 20 mM NaCl, 20 mM Tris-HCl at pH 5.5. Buffer B contained 1 M NaCl, 20 mM Tris-HCl at pH 5.5. The sample was loaded, followed by a wash with 0% B buffer and then washed with 100% B buffer. The hypersensitive response promoter did not bind to Mono S medium, but, at pH 5.5, many of the contaminants in the sample did bind. In this way, it served as a non-binding chromatography. Immediately after collecting the flow through the column (the promoter fraction of the hypersensitive response) the pH of the solution was adjusted to 8.0. 3. Phenyl-Sepharose (low) The active fraction from the Mono column S was then loaded onto a low-substitution Fast Flow column of phenyl-Sepharose (a weak hydrophobic interaction medium, Pharmacia biotech, Uppsala, Sweden). Buffer A contained 1M NaCl, 20mM Tris-HCl at pH 8 and buffer B contained 10mM Tris-HCl at pH 8.0. As previously mentioned, at 85% of B, buffer B was changed to deionized water. The promoter fraction of the hypersensitive response was eluted as the gradient reached 100% B. The fractions that were then used in the next purification step contained the highest concentration of the hypersensitive response promoter. Since the active fraction eluted in deionized water, it was necessary to stabilize the fraction by adjusting it to 20 mM NaCl and 20 mM Tris-HCl, at pH 8.0. 4. Reverse Phase Chromatography The final chromatographic step in the purification of the hypersensitive response promoter used a ProRPC 5/5 reverse phase column (Pharmacia Biotech, Uppsala, Sweden). The sample was adjusted to 15% acetonitrile (HPLC grade) and 0.1% TFA (HPLC grade) and loaded at a flow rate of 0.7 ml / min. After a 5 ml wash, a gradient of 15-50% B was run over 58 ml at a flow rate of 0.7 ml / min. Fractions of 1 ml were collected for the full volume of the gradient. The hypersensitive response promoter Xcp eluted approximately 25% of B. Fractions containing the hypersensitive response promoter were determined by visual identification on silver-stained SDS-PAGE gels. After visual identification, the fractions that were relatively pure and contained high concentrations of promoter were combined and dialysed in previously mentioned lysis buffer to remove acetonitrile and TFA. The dialyzed fraction was then concentrated to approximately 1 hundredth using Centricon 3,000 MWCO (Amicon, Beverly, MA).

Example 5 - Electroelution of the Xcp Hipersensitive Response Promoter The final concentrate was then run on a PAGE-SDS mini-gel of 18% acrylamide (the gel was emptied into an empty Novex gel cassette and run with a Novex X-Cell apparatus, San Diego, CA). The gel was stained and destained with normal staining techniques with Coomassie blue. Due to the extreme overload of the gel, it was possible to observe and trim the band that correlated with the hypersensitive response promoter. The cut gel was then loaded into an Elutrap device (Schleicher &; Schuell, Keene, New Hampshire). The protein present on the cut was eluted from the gel, and into a collection chamber. A non-SDS tank buffer was used in the Elutrap apparatus to produce a relatively free sample of SDS. A portion of the resulting fraction was then run on a 16% acrylamide PAGE-SDS mini gel (apparatus and Novex gel). The gel was stained with silver in order to determine the purity of the sample. In addition, a portion of the eluted fraction is > - ^^, ^ r ^ ^ ^ used to make dilutions of the hypersensitive response promoter, to determine the relative concentration of the hypersensitive response promoter present in the final sample.

Example 6 - Visualization of the Hipersensitive Response Promoter Perhaps the most significant and problematic feature found during the isolation of the hypersensitive response promoter Xcp was its abnormal staining characteristics. PAGE-SDS gels that had been stained using normal techniques with Coomassie blue showed the band of the hypersensitive response promoter only when the gel was extremely overloaded. Even under overloaded conditions, the promoter stained very faintly and to some degree temporarily. The complete fading of the gel usually resulted in shading of the hypersensitive response promoter band. Only with silver staining techniques was it possible to visualize the promoter at a concentration apparently lower. The band, depending on the concentration of the hypersensitive response promoter present in the sample, appeared either as a negatively stained band with relatively different limits (at higher concentration) or as a slightly colored band, resembling almost a shadow with borders fuzzy, not distinguished. Silver-stained SDS-PAGE gels were used to visualize the hypersensitive response promoter in all cases, except when the gel had to be used for electroelution with Elutrap or transfer of the protein onto a PVDF membrane . As in the case of the Blue stain of 10 Coomassie of the PAGE-SDS gels, Coomassie Blue staining of the PVDF membranes (used to immobilize the protein for N-terminal sequencing) resulted in very faint staining if intensely overloaded. Normal load volumes gave as 15 result a membrane apparently clean without apparent protein bands.

Example 7 - N-terminal Sequence of the Xcp Hipersensitive Response Promoter 20 After the purification of the promoter had been achieved it was then possible to learn the N-terminal sequence of the protein. The promoter protein of the hypersensitive response of Xcp, purified, was run 25 on 3DS-PAGE. From this gel, the protein was transferred to a PVDF membrane via a Trans-Blot (Brio-Rad, Hercules CA). The membrane containing the protein was subjected to an automated Edmans degradation process, followed by high pressure liquid chromatography for the detection and identification of the individual amino acids. The first ten amino acids of the promoter of the hypersensitive response of Xcp were determined as: 10 Met Asp Gly lie Gly Asn His Phe Ser Asn 1 5 10 (SEQ ID NO.1) Example 8 - Resistance to the Disease Induced by the 15 Promoter, in Tobacco and Tomato The promoter of the hypersensitive response of Xanthomonas campestris pv. pelargonii was sprayed on tobacco plants at a concentration of approximately 5 20 ppm. Three days after the treatment, the plants were inoculated with tobacco mosaic virus (TMV) at a concentration of approximately 2 ppm. Four days after the inoculation, the plants treated with the promoter showed more than a 65% reduction in the 25 number of lesions compared to untreated control plants. In addition to resistance to TMV the promoter of the rrH lffflripif 1 1 • hypersensitive response also induced resistance to tomato bacterial wilt.

Example 9 - Increase in Growth Induced by the Promoter of the Hypersensitive Response in Tomato To demonstrate that the promoter of the hypersensitive response of Xanthomonas campestris pv. perlagonii can increase the growth of the plant, tomato seeds were soaked in a promoter solution of approximately 5 ppm for more than 4 hours. The seeds soaked in a solution but without the promoter protein were used as an untreated control. The treated and untreated seeds were planted in 20 cm (eight inches) pots with artificial soil, 20 seeds per macetci. Twenty days after sowing, the size of the tomato seedlings was measured. The plants derived from the seeds treated with the promoter were 10% higher than those generated from the untreated seeds. A promoter of the hypersensitive response, protein, has been purified almost to homogeneity from the pathogen for plants Xanthomonas campestris perlagonii. The protein has a molecular weight of approximately 14 kDa and is heat tolerant. The hypersensitive response promoter exhibits unusual staining characteristics when stained with normal staining techniques with Coomassie blue and silver staining. The promoter of the hypersensitive response of Xanthomonas campestris perlargonii is a member of the family of hypersensitive response promoter proteins due to its shared characteristics with other proteins of this family. These characteristics include biochemical characteristics, the ability to promote a hypersensitive response, and, from prinary experiments, the ability to induce resistance against disease and increased plant growth. Although preferred embodiments have been described and detailed herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention, and is considered to be so. both that they are within the invention as defined in the following claims.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

LIST OF SEQUENCES < 110 > Eden Bioscience Corporation < 120 > PROMOTER OF THE HYPERSENSITIVE RESPONSE OF XANTHOMONAS CAMPES TRIS < 130 > 21829/52 < 140 > < 141 > < 150 > 60/103, 124 < 151 > 1998-10-05 < 160 > 1 < 170 > Patentin Ver. 2.0 < 210 > 1 < 212 > PRT < 213 > Xanthomonas campestris < 400 > 1 Met Asp Gly Lie Gln Asn His Phe Ser Asn 1 5 10 d &

Claims (12)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A protein or polypeptide promoter of the hypersensitive response, of Xanthomonas campestris, isolated.

2. A protein or polypeptide promoter of the hypersensitive response, isolated, according to claim 1, characterized in that the protein or polypeptide has a molecular weight of 13-15 kDa.

3. A protein or polypeptide promoter of the hypersensitive response, isolated, according to claim 1, characterized in that the protein or polypeptide has an amino acid sequence of SEQ. ID. No. 1.

4. A method for imparting disease resistance to plants, characterized in that the method comprises: the application of a hypersensitive response promoter protein or polypeptide according to claim 1 in a non-infectious form to a plant or seed of plant under effective conditions to impart resistance for the disease.

5. A method according to claim 4, characterized in that the plants are treated during said application.

6. A method according to claim 4, wherein the seeds of plants are treated during application, the method is further characterized because it comprises: the sowing of seeds treated with the hypersensitive response promoter, in natural or artificial soil , and the propagation of plants from the seeds planted in the soil.

7. A method for increasing plant growth, characterized in that it comprises: the application of a protein or polypeptide promoter of the hypersensitive response according to claim 1, in a non-infectious form, to a plant or plant seed, under effective conditions to increase the growth of the plant.

8. A method according to claim 7, characterized in that the plants are treated during said application. = -8- * £ i * - ** $. t. s ". ±. ^ ^ * "> a -Sia * a-í-ta

9. A method according to claim 7, wherein the seeds of plants are treated during application, the method is characterized in that it further comprises: sowing the seeds treated with the promoter of the hypersensitive response, in natural or artificial soil, and the propagation of the plants from the seeds planted in the soil.

10. A method for the control of insects for plants, characterized in that it comprises: the application of the protein or polypeptide promoter of the hypersensitive response according to claim 1, in a non-infectious form to a 15 plant or plant seed, under effective conditions to control insects.

11. A method according to claim 10, characterized in that the plants are treated during said application.

12. A method according to claim 10, wherein the seeds of plants are treated during application, the method is characterized in that it comprises: the sowing of the seeds treated with the hypersensitive response promoter, in natural or artificial soil, and the propagation of the plants from the seeds planted in the soil. ? ^^ í, ^ t. SUMMARY OF THE INVENTION The present invention is directed to a protein or polypeptide promoter of the hypersensitive response, of Xanthomonas campestris, isolated. The proteins or polypeptides that promote the hypersensitive response, according to the present invention, and the isolated DNA molecules that code for them, have the following activities: imparting resistance against diseases to plants, increasing plant growth and / or control of insects on plants. This can be accomplished by applying the hypersensitive response promoter in a non-infectious manner, to plants or plant seeds under conditions effective to impart resistance against the disease, to increase the growth of the plant, and / or to control insects on the plants or plants developed from said plant seeds. Alternatively, transdermal plants or seeds of transgenic plants transformed with a DNA molecule encoding the promoter can be provided, and the transgenic plants or plants resulting from the seeds of transgenic plants are developed under conditions effective to impart resistance against the disease, to increase the 6 / - growth of the plant and / or to control insects on the plants or plants developed from the seeds of plants. - ^^^ jy ^^^