MXPA00003651A - Bacterial resistance in grapevine - Google Patents

Abstract

The invention relates to a transgenic grapevine or transgenic grapevine component transformed with a i(vir) gene or an anti-pathogenic fragment thereof, wherein expresssion of the i(vir) gene or the anti-pathogenic fragment thereof in the transgenic grapevine or transgenic grapevine component provides resistance to a plant bacterial pathogen (e.g., i(Agrobacterium vitis)).

Description

BACTERIAL RESISTANCE IN VID Background of the Invention This invention relates to resistance to disease in plants. Grapes are the most widely cultivated fruit crop in the world and are difficult to breed by conventional methods (Mullins et al., BioTechnology 8: 1041-1045, 1990). Considerable effort has been developed directed towards the production of transgenic grape crops. Recently, the transformation of grape crops has been successful using embryogenic callus or suspension cultures for transformation. Transgenic plants have been recovered from rhizome crops Rupestris St. George (Vi tis rupestris, Mullins et al., BioTechnology 8: 1041-1045, 1990), Richter 110 (V. rupestris x V. berlandieri, LeGall et al., Plant Science 102: 161-170, 1994; Krastanova et al., Plant Cell Reports 14: 550-554, 1995), 41B (V. berlandieri x V. rupestris, Mauro et al., Plant Science 112: 97-106, 1995), as well as Chancellor cuttings cultures (Vitis spp., Kikkert et al. , Plant Cell Reports 15: 311-316, 1996), seedless Thompson (V. vinifera, Scorza et al., J. Amer. Soc. Hort. Sci. 121: 616-619, 1996), and Superior without seed. { V. Vinifera, Perl et al., Plant Science 104: 193-200, 1996). Many of the genes introduced into these grape crops have been vine-coated protein genes, including grapevine leaf virus (Mauro et al., 1995; Krastanova et al., Plant Cell Reports 14: 550-554, 1995), grapevine chromium mosaic virus (LeGall et al., Plant Science 102: 161-170, 1994), and tomato ring spot virus (Scorza et al., J. Amer. Soc. Hort.Sci. : 616-619, 1996). Additionally, transformation with a gene encoding a lytic peptide, Shiva-1, has been reported (Scorza et al., J. Amer. Soc. Hort, Sci. 121: 616-619, 1995). The use of these techniques and others provides the basis for overlapping disease resistance in grapes; for example, develop transgenic grape plants that are resistant to infestation by pests (eg, insects and nematodes) and attack by pathogenic microorganisms (eg, fungi, bacteria and viruses). A disease induced by microbes found in grapes is the disease of the crown gall. This is caused by Agrobacterium spp. , a gram negative bacteria that lives in the earth. Persistent for long periods of time in the waste of plants found in the earth. This bacterium has one of the widest host ranges of any plant pathogen. This bacterium induces galls or plant tumors in the roots, crowns, trunks, and stems of plants. Agrobacterium causes crown gall by copying and transferring a segment of its tumor-inducing plasmid (Ti), termed as the transfer DNA (T-DNA), to the plant cell where it is integrated into the genome of the plant cell. The transfer of the transfer DNA into the nucleus of the plant cell depends on the expression of several virulence genes (vir) that are also localized in the Ti plasmid. A virulence protein, VirD2, is a site-specific endonuclease which, when aided by the VirD1 protein, recognizes and nickers the "base chain" of the left and right T-DNA border sequences (Stachel et al., EMBO J. 6: 857-863, 1987). The VirD2 protein becomes covalently bound to the 5 'end of the nicked chain (Ward and Barnes, Science 242: 927-930, 1988). The single-stranded T-DNA (DNA-Tss) is exported to the plant cell (Tinland et al, Proc.Nat.Acid.Sci.91: 8000-8004, 1994, Yusibov et al., Proc. Na ti. Sci. 91: 2994-2998, 1994). A current hypothesis for the transport of T-DNA to the nucleus of the plant cell is that the Tss-DNA is coated by molecules of the VirE2 protein, which has been shown to bind to the Tss-DNA. The coated T-DNA, called the T complex, is thought to be exported to the plant via a pore consisting of VirB proteins. T complexes can be imported into the nucleus using a plant-protein pathway; involving the transport of proteins that have nuclear localization signals to the nucleus. Both VirD2 and VirE2 are required for the optimal transference of T-DNA to the nucleus of the plant cell. The T complex therefore travels from Agrobacterium to the plant cell where the T-DNA is integrated into the plant genome. The T-DNA of Agrobacterium encodes several enzymes involved in the biosynthetic pathways of auxin and cytokinin. The infected cells overproduce plant hormones auxin and cytokinin, leading to rapid and uncontrolled cell division, and the formation of galls or plant tumors. The formation of galls interferes with the flow of water and nutrients in the plant. Infected plants typically become unproductive and are more susceptible to adverse environmental conditions. The disease is especially destructive in fruit crops such as grapes. The gall of the grape crown is caused almost exclusively by A. vi tis and to a lesser degree by A. tumefaciens. The bacterium infects the vine at sites of lesions on the trunk, and leads to the formation of galls. Bacteria survive in the xylem of the vine, and are disseminated by infected propagation materials. Agrobacterium infections are particularly harmful to young vines during the establishment of the vineyard; gills that grow rapidly are capable of infecting young vines in a single season (Agrios, Plant Pathology, 3rd edition, Academic Press, 1998). Infected vines have reduced yield and reduced productivity. SUMMARY OF THE INVENTION In general, the invention features a method for providing resistance to bacterial pathogen in a plant (for example, a vine such as Vitis). The method generally involves the steps of: (a) transforming plant cells with a virulence gene (vir) or an antipathogenic fragment thereof; (b) regenating the plant cells to provide a differentiated plant; and (c) selecting a transformed plant expressing the vir gene or the antipathogenic fragment thereof, wherein the expression of the vir gene or the antipathogenic fragment thereof provides resistance to the bacterial pathogen of the plant. In general, the processing and transfer of T-DNA from the bacterium to the plant are mediated by gene products encoded by the vir region residing in the tumor-inducing plasmids (Ti) or (Ri) Agrobacterium root inducers. Exemplary vir genes useful in the invention include, without limitation, virE2 and virD2. In the preferred embodiments of the invention, the vir gene or antipathogenic fragment thereof is integrated into the genome of the plant. Preferably, the vir gene is virE2, virD2, or both; or it is mutated (eg, it is a fragment of the antipathogenic vir gene such as one that encodes a deletion of a Vir protein such as virE2 suppression B, vir2 suppression C, or virE2 deletion E). Vir sequences that mediate an increased resistance to gall gall disease are considered useful in the invention. As used herein, the term "fragment", applied to nucleic acid molecule sequences, means at least 5 contiguous nucleotides, preferably at least 10 contiguous nucleotides, more preferably at least 20 to 30 contiguous nucleotides, and more preferably at least 40 to 80 or more contiguous nucleotides. Natural or synthetic fragments of a vir gene (eg, virE2 or virD2) can be produced and subsequently integrated into a standard plant expression vector (eg, those described herein) according to methods known to experts in the technique. The ability of these vir gene fragments (eg, a vir gene encoding deletion B, deletion C, deletion D, or deletion E of the virE2 gene) to confer resistance, when expressed in a plant, to a bacterial plant pathogen it can be tested according to standard methods (for example, those described here). Fragments that confer resistance to a plant bacterial pathogen are known as "antipathogenic fragments." In the preferred embodiments, the plant is a vine or a vine component (e.g., a somatic embryo, cuttings, or rhizome); the bacterial pathogen is Agrobacterium vitis or Agrobacterium tumefaciens; and resistance to Agrobacterium vitis or Agrobacterium tumefaciens reduces the formation of crown galls or the growth of these bacteria in the infected plant. In yet other preferred embodiments, the vir gene (or an antipathogenic fragment thereof) is from a plasmid that induces tumor (Ti) or induces root (Ri) of Agrobacterium (eg, Agrobacterium vitis, Agrobacterium tumefaciens, Agrobacterium rhizogenes). Examples of these Ti plasmids, without limitation, include nopalin-type plasmids, vitopin, octopine, octopi-na / cucumopin, leucinopin / agropin, succinamopin, or agropin. These vir gene sequences or antipathogenic fragments are obtained according to standard methods known in the art. The methods described herein are useful for providing resistance or tolerance to the disease or both in a variety of vines (eg, Vi tis spp., Hybrids of Vitis spp., And all members of the subgenus Euvitis and Muscadinia) to pathogens bacterial (for example, Agrobacterium vi tis or A. tumefaciens), including cuttings and rhizome cultures. Exemplary cut cultures include, without limitation, what are known as table grapes or raisins and those used in the production of juice and wine such as Cabernet Franc, Cabernet Sauvig-non, Chardonnay (for example, CH 01, CH 02). , CH Dijon), Merlot, Pinot Noir (PN, PN Dijon), Semillon, White Riesling, Lambrusco, Thompson Seedless, Autumn Seedless, Niagrara Seedless, and Seval Blanc. Rhizome cultures that are useful in the invention include, without limitation, Vitis rupestris Constantia, Vitis rupestris St. George, Vi tis california, Vitis girdiana, Vitis rotundifolia, Vitis rotundifolia Carlos, Richter 110. { Vitis berlandieri x rupestris; ? 110R "), 101-14 Millarder et de Grasset (Vitis riparia x rupestris;? 101-14Mgt"), Teleki 5C (Vi tis berlandieri x riparia), Courderc 3309. { Vi tis riparia x rupestris; "C3309"), Riparia Gloire of Montpellier (Vitis riparia), 5BB Teleki (selection Kober, Vitis berlandieri x riparia), S04 (Vi tis berlandieri x rupestris), 41B Millardet (Vitis vinifera x berlandieri), Ramsey. { Vitis champinii), K5140 (Vitis champinii x Vitis riparia) and 039-16. { Vitis vinifera x Muscadinia). The invention also features cuttings, rhizomes, somatic or zygotic embryos, cells, or seeds that are produced from any transgenic vine or vine components described herein. For example, the invention features a transgenic plant transformed with a nucleic acid molecule encoding a Vir protein or an antipathogenic fragment thereof, wherein the expression of the nucleic acid molecule provides resistance to a bacterial pathogen (e.g., A vi tis or A. tumefaciens). . The invention also includes a grape cell that has been transformed with a nucleic acid molecule (e.g., a virE2 deletion B transgene construct that is placed for expression by operably linking the transgene to a control region of plant expression) which provides resistance to a bacterial pathogen. These grape cells are then used to generate rhizomes, cuttings, somatic embryos, or seeds using methods that are known in the art (for example, those described herein). By "placed for expression" it is meant that the DNA molecule is placed adjacent to a DNA sequence that directs the transcription of the sequence. By "expression control region" is meant any sequence sufficient to direct transcription. Included in the invention are the promoter and enhancer elements which are sufficient to render the expression of the gene dependent on the promoter controllable for the expression of the gene specific for cell, tissue, or organ, or elements that are inducible by signals or external agents (e.g. , elements inducible by light, pathogens, injuries, stress or hormones; or constituent elements); these elements can be located in the 5 'or 3' regions of the original gene or overlap in the transgenic construct. By "operably linked" it is meant that a gene and a regulatory sequence (s) are connected in a manner that allows expression of the gene when suitable molecules (e.g., transcriptional activating proteins) bind to the or the regulatory sequences. By "plant cell" is meant any self-propagating cell linked by a semipermeable membrane and containing a plastid. A plant cell, as used herein, is obtained from, without limitation, seeds, suspension cultures, embryos, meristemic regions, callus tissue, protoplasts, leaves, roots, shoots, somatic and zygotic embryos, as well as any part of a reproductive or vegetative tissue or organ. By "plant component" is meant a part, segmented, or organ obtained from an intact plant or plant cell. Exemplary plant components include, without limitation, somatic embryos, leaves, fruits, cuttings, and rhizomes. By "transgenic" is meant any cell that includes a DNA sequence that is artifically inserted into a cell and becomes part of the organism's genome (integrated or extrachromosomal) which develops from that cell. As used herein, transgenic organisms are generally transgenic vines or vine components and the DNA (e.g., a transgene) is artificially inserted into nuclear or plastid compartments of the plant cell. Preferably, this vine or transgenic vine component expresses at least one vir nucleic acid sequence (eg, a vir gene or an antipathogenic fragment thereof such as virE2 deletion B of A. vitis strain CG450). In other preferred embodiments, a transgenic plant may express more than one vir sequence or a combination of vir sequences (eg, a vir nucleic acid sequence derived from different Ti plasmids such as the Ti or Ri plasmids nopaline type, vitopin, octopine , octopi-na / cucumopina, leucinopina / agropina, succinamopina, or agropina). By "transgene" is meant any piece of DNA that is artificially inserted into a cell, and becomes part of the organism (integrated in the genome or maintained extrachromosomal) that develops from that cell. This transgene may include a gene that is partially or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. "Resistance to a plant bacterial pathogen" means a higher level of resistance to a pathogen (for example, Agrobacterium vitis or A. tumefaciens) on a transgenic vine (or component of vine or cell, seed, or somatic embryo thereof) that the level of resistance relative to a vine control (for example, a non-transgenic vine). In preferred embodiments, the level of resistance to a pathogen in a transgenic vine is at least 5 to 10 percent (and preferably 20 percent, 30 percent, or 40 percent) greater than the resistance of a control vine. . In other preferred embodiments, the level of resistance to the pathogen is 50 percent greater, 60 percent greater, and more preferably up to 75 percent or 90 percent greater than a control vine; most preferred up to 100 percent strength compared to a control vine. The level of resistance is measured using conventional methods. For example, the resistance level of Agrobacterium can be determined by comparing aspects and physical characteristics (e.g., height and weight of the plant, or comparing symptoms of disease, e.g., delayed gall formation, reduced gill formation, or reduced deformity. in the stems) of transgenic vines or by comparing the survival population of pathogens in the plant (for example, by measuring the systemic population of Agrobacterium in the transgenic and control plants.) As discussed above, it has been found that the expression of a sequence of viric nucleic acid, virE2 suppression B, provides transgenic vines with resistance against crown gill disease caused by the bacterium Agrobacterium In accordance with the foregoing, because there are no viable alternatives to control Agrobacterium in grapes, the invention provides several important advances and advantages for wine growers, for example, demonstrating that the sequence is effective against the development of crown gill disease, the invention provides an effective and economical means for protection against the disease. This protection reduces or minimizes the need for traditional practices, for example, chemical treatments, which are typically used by vine growers to control the spread of the Agrobacterium and provide protection against the pathogen that causes the disease in vineyards. In addition, because grape plants expressing vir nucleic acid sequences or antipathogenic fragments are less vulnerable to Agrobacterium (eg, A. vitis and A. tumefaciens) and thus to crown gall disease, the invention further provides increased production efficiency, as well as improvements in quality, color, flavor and yield of the grapes. In addition, because the invention reduces the need for chemical protection against the pathogens of the vine, it benefits the environment where the vineyards are planted.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Detailed Description The drawings will be described first. Drawings Figure 1 is a schematic illustration showing the partial map of the T-DNA region containing the deletion B virE2 transgene under the control of the CaMV 35S online promoter. Figure 2 is a bar graph showing the transformation of 101-Mgt with the virE2 gene suppression B generated from strain A6 of Agrobacterium tumefaciens which resulted in several transgenic lines having resistance as compared to control plants. Overview A genetic study was carried out using Agrobacterium tumefaciens and A. vitis to identify Vir protein products (eg, a deletion mutant of the virE2 gene) that are useful for increasing resistance to pathogenic infection in grape plants. VirE2 deletion mutant transgene B constructs (Citovsky et al., Science 256: 1802-1806, 1992) was generated using the C58 strain of A. tumefaciens (carrying a nopalin-type Ti plasmid), strain A6 A. tumefaciens that carries a Ti plasmid type octopina), and the CG450 strain of A. vitis (which carries a plasmid Ti vitopina). The viral E2 suppression B gene encodes a VirE2 protein that lacks 215 carboxy-terminal amino acids. This truncating suppresses the binding domain of a single strand of T-DNA (DNA-Tss) of the VirE2 protein. Grape rhizomes (for example, Courderc 3309. {Vi tis riparia x rupestris, - "C3309"), 101-14 Millarder et de Grasset (Vitis riparia x rupestris; "101-14 Mgt"), and Richter 110 (Vi tis berlandieri x rupestris; "110R")), as well as tobacco (for example, Nicotiana benthamiana), were then transformed with a transgene expressing the deletion B of the 2 gene ( Figure 1) . The transgenic plants expressing these transgenic constructs were generated and the resistance to crown gall disease was subsequently evaluated. The following examples are provided for the purpose of illustrating the invention, and should not be considered as limiting. Construction of Transrén 2 Suppression B and Strains of Acrrobac-ter ± -um Constructs of the 2 deletion B gene were generated as follows. The 2 deletion B genes were 2 mutant genes, which lacked the region encoding the ssDNA binding motif (Citovsky et al., Science 256: 1802-1805, 1992). These genes were obtained from strain C58 of A. tumefaciens and strain A6 of A. tumefaciens, which reared nopalin and octopine type Ti plasmids, respectively. The vírE2 deletion B gene was also generated from the CG450 strain of A. vitis which carries a vitropin-like Ti plasmid. The 2 deletion B gene was amplified from strain C58 of A. tumefaciens and of strain CG450 DNA of A. vitis using the first set 5'nop (5 '-TACTTACCATGGATCCGAAGGCCGAAGGC; SEQ ID NO: l), which is identical to the 5 'coding region of the nopaline 2 gene, and 3' nop (5 '-TCTTGACCATGGCTATCGATTCTCGCCGGCGGAACTC; SEQ ID NO: 2), hybridizing to nucleotide positions 1000-1020 of the same gene. The p2 mutant deletion B type octopine was amplified from strain A6 of A. tumefaciens using primers of 5 'oct oligomers (identical to the 5' region of the octopine type 2 gene) and 3 'oct (complementary to the positions of nucleotides 927-951 of the translation initiation codon). The amplification by polymerase chain reaction (PCR) of the 2 deletion B gene of different Ti plasmids was carried out as follows. The 2 deletion B gene was amplified using 0.5 μg of each of the oligomer primers according to manufacturer's instructions (Perkin-Elmer Cetus). The polymerase chain reaction cycle was 1 minute at 92 ° C (denaturation), 1 minute 50 ° C (annealing), and 2 minutes at 72 ° C (polymerization). The samples were loaded directly and separated on a 1.2 percent agarose gel. The separated deletion B 2 fragments were extracted from the gel, precipitated in ethanol, and dissolved in 20 μl of distilled water. The gel-isolated mutant gene fragment was digested with Ncol restriction enzyme and directly cloned into the digested plant expression vector Ncol pEPT8. The expression of the 2 coding sequences was thus controlled by a double CaMV 35S promoter fused to the 5 'untranslated forward sequence of the alfalfa mosaic s (AIMV) of the expression vector pEPT8. The expression cassette was separated from the construct with HindIII, and ligated into the transformation vector of the plant pBIN19 that had been cut with the same enzymes. The resulting transformation vectors, pBIN19-EPT8-2-C58 (Figure 1), pBIN19-EPT8-v? RE2-A6, and pBIN19-EPT8-2-CG450 were transferred to the C58sZ707 strain of A. tumefaciens by electroincorporation according to with standard methods. Grape and Tobacco Transformation with ylr? 2 Transgene Constructs Suppression B Embryogenic calli from C3309, 101-14 Mgt, and 110R were transformed with 2 deletion B transgene constructs using an Agrobacterium co-cultivation method as follows. Embryogenic calluses capable of generating somatic embryos were developed from anthers grown according to standard methods. Briefly, the anthers of the rhizome clones C3309, 101-14 Mgt, and 110R were used to initiate callus cultures by the method of Rajasekaran and Mullins (J. Exp. Bot.30: 399-407, 1979). Buttons were harvested before anthesis from plants grown in the field during spring and early summer, were removed from the bouquets, and surface sterilized in 70 percent EtOH for 1 to 2 minutes. The buttons were transferred to 1 percent sodium hypochlorite for 15 minutes, then rinsed three times in sterile double distilled water. The translucent yellow anthers separated aseptically from the flower buds. The anthers were isolated under sterile conditions and plated at a density of 40 or 50 anthers per petri dish in the initiation medium. The anthers were grown at 28 ° C in the dark. The embryogenic callus developed in 30 days. The overnight cultures of A. tumefaciens containing the virE2 suppression B transgene construct used for transformation were cultured in LB medium at 28 ° C in a shaking incubator. The bacteria were centrifuged for 5 minutes at 3000 rpm and resuspended in MS liquid medium (OD 0.4 - 0.5 to A600nm). The corms with globular or heart-shaped embryos were immersed in the bacterial suspension for 15 minutes, dried with a blotter, and transferred to HGM medium with acetosyringone (100 μM). The embryogenic callus tissue was cocultivated with the bacteria for 48 hours in the dark at 28 ° C. Then, the plant material was washed in MS liquid plus cefotaxime (300 μg / ml) and carbenicillin (200 μg / ml) 2-3 times. The material was transferred to HMG medium with the same antibiotics for 1-2 weeks. After 2 weeks, the embryogenic calli were transferred to HMG medium containing either 20 or 40 mg / L of kanamycin and 300 mg / L of cefotaxime, plus 200 mg / L of carbenicillin to select the transgenic embryos - After growth in means of selection for 3 to 4 months, the embryos were transferred to HMG, MGC, or MSE without kanamycin. After about 4 months, all the materials were transferred to medium without antibiotics. After the development of hypocotyls, the embryos were transferred to root formation medium without antibiotics. The untransformed calli were cultured in the same medium with or without kanamycin as a control to verify the efficiency of the antibiotic selection and the regeneration capacity of the material. Tobacco Transformation The tobacco plants, N. tabacum and N. benthamiana, were transformed using a leaf disc transformation method (Horsch et al., Science 227: 1229, 1985). The binary vector pBI? 19-EPT8-virE2-C58 and pBI? -EPT8-virE2-A6 was the same as that used in the transformation of the grape (Figure 1). The shoots were regenerated from pieces of leaf, separated from the original explant and allowed to root in MS medium with kanamycin. The tobacco plants were allowed to self-fertilize and the Rl generation was used in resistance tests. Characterization of Transgenic Plants The transgenic plants were recovered and verified by conventional PTII-ELISA (enzyme-linked immunosorbent assay). Transgenic plants C3309, 101-14 MGt, and 110R and transgenic tobacco lines were recovered. The AD? Total was extracted as described below from young leaves of selected plants and tested for the presence of the transgene by conventional polymerase chain reaction (PCR). The product of the 1,020 kb polymerase chain reaction corresponding to the deletion B virE2 transgene was observed in most of the transgenic plants. Selected C3309 transgenic plants were further analyzed for gene copy number by Southern blot hybridization using the 1.0 kb virE2 deletion B gene as a probe. Both susceptible and resistant plants had relatively few copies (1 to 4) of the transgene. The mRNA level of virE2 deletion B gene was examined by Northern spotting analysis as described below. It was found that transgenic resistant plants express higher levels of virE2 mRNA suppression than susceptible plants. For these aforementioned tests, plant DNA was isolated as described by Krastanova et al. { Plant Cell Reports 14: 550-554, 1995). Polymerase chain reaction reactions were carried out on the genomic DNA of the total plant as described above, using an annealing temperature of 45 ° C instead of 50 ° C. For Southern blot hybridization, 20 micrograms of DNA were digested with HindIII, PstI, and Kpnl. Total genomic DNA was run in TBE gels with 1 percent agarose and dried on a Nytran N filter (Schleicher and Schuell, Keene, NH) according to the manufacturer's instructions. The staining was dried in an oven at 80 ° C for one hour, then prehybridized and hybridized as described in Sambrook et al. { Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y. 1989). The probe labeled dCTP32P was constructed using the RadPrime DNA labeling system from Gibco BRL (Gaithersburg, MD). RNA was extracted from the transgenic plants by the same method as the DNA extraction, the total nucleic acid was precipitated overnight at -20 ° C with 2M LiCl to select the total RNA. The RNA (20 μg) was run in denaturing formaldehyde gels as described by Sambrook et al. (Supra), and dried with Nytran-N, and prehybridized and hybridized using the same probe as for the Southern blots. Resistance to Disease The resistance of the transgenic lines of grape plants to crown gall disease was tested as follows. The internodes of the grape stem were inoculated as described by Pu and Goodman. { Physiological and Molecular Plant Pathology 41: 241-254, 1992). The inoculum used in these experiments was either A. tumefaciens strain C58, or A. tumefaciens strain A6. The bacteria were cultured at 28 ° C for 48 hours in PDA medium, with antibiotics if appropriate. For inoculation, the bacterium was resuspended in sterile distilled water (A600nm = 0.1), which is approximately 1 X 108 cfu / ml for C58. Five μL of inoculum or a dilution thereof was applied to cut grape internodes and plants were observed to determine tumor formation at 7, 14, and 21 days after inoculation (dpi). Non-transgenic controls inoculated with A. tumefaciens strain C58 or A6 consistently exhibited tumorigenesis. In susceptible plants, gall formation was occasionally seen 7 days after inoculation, but generally appeared around the tenth day after inoculation, and was easily observed around 14 days after inoculation. The primary transformants were tested and some of the primary transformants were found to inhibit gill formation. Some of the inoculated buds became tumorigenic. The plants were classified as resistant if the galls were suppressed by 50 percent more compared to those of the non-transformed shoots. The lines of C3309, 101-Mgt, and 110R that have resistance to crown gills have been generated. Exemplary results showing the resistance to crown gall formation of the transgenic 101-14 Mgt transgene (expressing the transgene of virE2 deletion B that was generated from A. tumefaciens strain A6) after inoculation with A. tumefaciens strain A6 presented in Figure 2. In addition, the rhizome of C3309 grape that expressed that the virEn2 B deletion of A. tumefaciens strain C58 showed that approximately 45 percent of these plants had significant resistance to crown gall disease (Table 1 , later) .

Table 1 Evaluation of transgenic lines of C3309 grape rhizome expressing the viral deletion BirE2 transgene for crown gall resistance by inoculation with A. tumefaciens strain C58 L a n e a Plant No. Gill Resistance No. crown 1 223, 224, 225, susceptible 228, 243 2 240 resistant 3 218, 227 susceptible 3 169, 222 resistant 4 239 susceptible 5 245 resistant 6 234, 241 susceptible 11 235 susceptible 15 226 resistant 16 171, 220 resistant Fifteen to twenty shoot sections of each plant were inoculated in vitro with a bacterial suspension of A. tumefaciens strain C58 (approximately 107 cfu / ml). One plant was considered resistant if less than 40 percent of shoot inoculation developed galls six days after inoculation. One hundred percent of outbreaks of non-transgenic control plants developed galls. Tobacco resistance to crown gill disease was tested as follows. Transgenic tobacco plants were grown to a size of 6 centimeters. These plants were then inoculated with 10 μl of a suspension in overnight culture of Agrobacterium strains CG49 or K306. The plants were checked for tumor formation two to three weeks after inoculation. Tobacco lines expressing the virE2 suppression B genes generated from A. tumefaciens strain C58 (bearing a Ti plasmid type nopaline) and from A. tumefaciens strain Aβ (bearing a Ti plasmid type octopine) were also valued for resistance to crown gall disease These plants were inoculated with A. vitis strain CG49 (bearing a Ti plasmid type nopalina) or the strain A. vitis K306 (which carries a Ti plasmid type octopina). Exemplary results of these experiments are shown in Table 2 (below).

Table 2 Exemplary resistance data of N. transgenic benthamiana expressing the virE2 transcript B suppression of A. tumefaciens strain C58 (bearing a nopaline type Ti plasmid) and A. tumefaciens strain A6 (bearing a Ti plasmid type octopine) inoculated with A. vitis strains CG49 (carrying a nopalin-type Ti plasmid) and A. vitis strain K306 (which carries an octopine type Ti plasmid). Plant expression CG49 CG49-1 K306 K306-1 Susceptibility virE2 1 Oct, ++++ 1.0 0 0.25 0.25 resistant 2 Oct, ++ 0.25 0 0.25 0.25 resistant 3 Oct, + 2.5 2.5 1.0 1.0 susceptible ß Oct, ++++ 1.0 0.5 0.5 0.5 resistant Oct, +++ 2.0 2.0 2.0 0.75 susceptible Oct 11, +++ 2.0 0.5 0.75 0.25 octopine-resistant plasmid type 30? Op, + 0.75 .75 1.0 0.5 resistant 31? Op, +++ 2.5 2.5 3.0 3.0 susceptible 32? Op, ++++ 2.5 2.0 3.0 3.0 susceptible 34? Op, + 0 0.5 0.5 0.25 resistant Expression of virE2 was determined by Northern blotting and the + number indicates the strength of the signal. The plants were inoculated with a pathogen concentration of approximately 107 cfu / ml and approximately 106 cfu / ml. 0 = no galls or swelling at the site of inoculation. 0.5 = small gall defined at the inoculation site; and 3 = very large gall at the site of inoculation. All non-transgenic control plants developed gills with scores of 2 to 3. The tobacco lines expressing the octopine transgene virE2 deletion B of A. tumefaciens strain Aß or the nopaline transgene virE2 deletion B of A. tumefaciens strain C58 showed approximately a 50 percent reduction in crown gill disease when inoculated with A. vitis strain CG49 or A. vi tis strain K306 (Table 2, supra). Gene Isolation of Anti-Pathogenic Fragments of the Same Any Ri or Ti plasmid (eg nopalin type plasmids, vitopin, octopine, octopine / cucumopin, leucinopine / agropin, succinamopin, or agropin) can serve as the source of acid nucleic acid for the molecular cloning of a vir gene or an antipathogenic fragment thereof. For example, the isolation of a vir gene (eg, vir2 or virD2) involves the isolation of the DNA sequence encoding a protein that exhibits structures, properties, or activities associated with vir. Based on the nucleotide and amino acid sequences described for virE2 (see, for example, GenBank accession numbers 2773266, 138480, 138481, 95134, 77949, 737146, 39124, 154801, and 154727) and virO2 (see, for example, GenBank numbers Access 138464, 138463, 138465, 95129, 95128, 77931, 95077, 737141, 39000, 154829, and 154796), the isolation of the vir coding sequences is made possible using standard strategies and techniques that are well known in the art. In another particular example, the vir sequences described herein may be used, in conjunction with conventional nucleic acid hybridization analysis methods. These hybridization techniques and selection procedures are well known to those skilled in the art and are described, for example, in Benton and Davis, Science 196: 180, 1977; Grunstein and Hogness, Proc. Na ti. Acad. Sci. , USA 72: 3961, 1975; Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience, New York; Berger and Kimmel, Guide to Molecular Cloning Techniques, 1987, Academic Press, New York; and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989. In a particular example, all or part of the nucleotide sequence of virE2 or virD2 (described by Citovsky, infra) can be used. as a probe for selecting a recombinant Ti plasmid DNA library for genes having sequence identity with the genes vrE2 or vi D2. Hybridization sequences are detected by plaque or colony hybridization according to standard methods, for example, those described below. Alternatively, using all or a portion of the amino acid sequence of a vir gene and the genetic code one can easily design vir-specific oligonucleotide probes, including vir regenerated oligonucleotide probes (i.e., a mixture of all possible sequences). of coding for a given amino acid sequence). These oligonucleotides can be based on sequences of some DNA strand and any suitable portion of the vir sequence. General methods for designing and preparing these probes are provided, for example, in Ausubel et al., 1996, Current Protocols in Molecular Biology, Wiley Interscience, New York, and Berger and Kimmel, Guide to Molecular Cloning Techniques, 1987, Academic Press, New York These oligonucleotides are useful for the isolation of the vir gene, either through their use as probes capable of hybridizing to complementary sequences vir or as primers for different amplification techniques, for example, cloning strategies of polymerase chain reaction (eg example, the methods described herein). If desired, a combination of different oligonucleotide probes can be used for the selection of a recombinant DNA library. Oligonucleotides can be detectably labeled using methods known in the art and used to test filter replicates from a recombinant DNA library. Recombinant DNA libraries are prepared according to methods well known in the art, for example, as described in Ausubel et al., (Supra), or can be obtained from commercial sources. As soon as a vir nucleotide sequence is identified, it is cloned and manipulated according to standard methods and used for the construction of plant expression vectors as described herein. Construction of Plant Transgenes In addition to transforming grape plants with a transgene construct of the virE2 deletion B gene, resistance to infection by Agrobacterium can also be achieved by transforming these plants with the original virE2 type gene or other virE2 mutant genes, for example, deletion C (reducing the binding activity with DNA-Tss polypeptide and NSE1, for example, by deletion of amino acids 228-244 of the virE2 gene) or deletion D (reducing the polypeptide binding activity of ssTDNA and NSE2). An example of a virE2 deletion C mutation involves the production of transgene encoding a VirE2 protein lacking amino acids 228-244 (Citovsky et al., Science 256: 1802-1806, 1992). An example of a mutation of virE2 deletion D involves the production of a transgene encoding a VirE2 protein lacking amino acids 296-310 (Citovsky et al., Supra). Grape plants can also be transformed with original type vírD2 or virD2 mutant genes to confer resistance to tumorigenicity by Agrobacterium. Mutations in the virD2 gene will also reduce the nuclear transport of DNA-Tss from Agrobacterium (for example, mutations that inhibit the importation of the T-DNA strand into the nucleus of the plant, which increase the activity of the nuclear localization signal , or that avoid the binding of the VirD2 protein in the T chain).

When the DNA sequences encoding the desired original type or the mutant vir gene are obtained, the sequences are inserted into a plant transformation vector suitable for the transformation of the grape plant. Several suitable vectors for stable or extra-ossomal transfection of plant cells or for the establishment of transgenic plants are available to the public; these vectors are described in Pouwels et al. { supra), Weissbach and Weiss-bach (supra), and Gelvin et al. { supra). Methods for constructing these cell lines are described in, for example, Weissbach and Weissbach (supra), and Gelvin et al., (Supra). Example of vectors useful for the expression of transgenes in grapevine are also described in Scorza et al. (Plant Cell Reports 14: 589-592, 1995), Baribault et al. (J. Expt Bot. 41: 1045-1049, 1990), Mullins and collaborators. { BioTechnology 8: 1041-1045, 1990), Nakano et al. (J. Expt. Bot. 45: 649-656, 1994), Kikkert et al. { Plant Cell Reports 15: 311-316, 1995), Krastanova et al. { Plant Cell Reports 1: 550-554, 1995), Scorza et al. { Plant Cell Reports 14: 589-592, 1994), Scorza et al. (J. Amer. Soc. Hort. Sci. 121: 616-619, 1996), Martinelli et al. { Theor. Appl. Genet 88: 621-628, 1994), and Legall et al. (Plant Sci. 102: 161-170, 1994). Typically, plant expression vectors include (1) a cloned gene (eg, an original or mutated vir gene) under the control of transcription of the 5 'and 3' expression control sequences; and (2) a dominant selectable marker. These plant expression vectors may also contain, if desired, a promoter regulatory region (eg, one that confers inducible or constitutive, pathogen-induced, or wound expression, environmentally or developmentally, or cell-specific or tissue-specific) , a transcription start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal. In its component parts, a DNA sequence encoding a wild type or mutated vir gene is combined in a DNA construct having a transcription initiation control region capable of promoting transcription in a host grape cell. In general, constructs will generally include functional regulatory regions in plants which provide the modified production of a wild type or mutated Vir protein as discussed herein. The sequence, mutated sequence, or fragment thereof will be joined at its 5 'end to a transcriptional initiation regulatory region, for example, such as a sequence naturally found in the 5' upstream region of the structural gene of the plant. Numerous transcription initiation regions are available that provide constitutive or inducible regulation. For applications where development, cell, tissue, hormonal, or environmental expression is desired, the appropriate upstream 5 'non-coding regions of other genes, eg, of genes regulated during meristem development, are obtained. of the seed, the development of the embryo, the development of the leaf, the development of the stem or development of the tendril. The regulatory transcription termination regions can also be provided in DNA constructs of this invention. The transcription termination regions can be provided by any convenient transcription termination region from a conventional gene source (eg, NOS or 35S CaMV terminators). The transcription termination region will preferably contain at least 1-3 kb of the 3 'sequence for the structural gene from which the termination region is derived. Plant expression constructs having the vir gene as the DNA sequence of interest for expression can be employed with a wide variety of vines. Such genetically overlapping plants are useful for a variety of industrial and agricultural applications. In an important way, this invention is applicable to all vines or grapevine components, and will be easily applicable to any new or improved transformation or grape regeneration methods. Expression constructs include at least one promoter operably linked to at least one sequence of the original or mutated vir type. An example of a plant promoter useful in accordance with the invention is a caulimovirus promoter, for example, a cauliflower mosaic virus (CaMV) promoter. These promoters confer high levels of expression in most plant tissues, and the activity of these promoters does not depend on virally encoded proteins. Cauliflower mosaic virus is a source for both the 35S and 19S promoters. In most transgenic plant tissues, the CaMV 35S promoter is a strong promoter (see, for example, Odell et al., Nature 313: 810, 1985). The CaMV promoter is also very active in monocotyledons (see, for example, Dekeyser et al., Plant Cell 2: 591, 1990; Terada and Shimamoto, Mol. Gen Genet 220: 389, 1990). Moreover, the activity of this promoter may increase more (ie, between 2 and 10 times) by the duplication of the CaMV 35S promoter (see for example, Kay et al., Science 236: 1299, 1987; Ow et al., Proc. Na ti, Acad Sci., USA 84: 4870, 1987, and Fang et al., Plant Cell 1: 141, 1989, and McPherson and Kay, U.S. Patent No. 5,378,142). Other useful plant promoters include, without limitation, the nopaline synthase promoter (NOS) (An et al., Plant Physiol., 88: 547, 1988), the octopine synthase promoter (Fromm et al., Plant Cell 1: 977, 1989 ), the rice actin promoter (Wu and McElroy, W091 / 09948), the cyclase promoter (Chappell et al., W096 / 36697), and the cassava vein mosaic virus promoter (Verdaguer et al., Plant Mol. Biol. 31: 1129-1139, 1996). Still other exemplary promoters useful in the invention include, without limitation, cornelin mottled yellow virus promoter, sugarcane badna virus promoter, rice tungro bacilliform virus promoter, maize striped virus element, and virus promoter dwarf of wheat. For certain applications, it may be desirable to produce the original or mutated vir sequence in a suitable tissue, at an appropriate level, or at an appropriate development time. For this purpose, there is a classification of gene promoters, each with its own distinctive characteristics immersed in its regulatory sequences, which show to be regulated in response to inducible signals such as the environment, hormones, and / or developmental markers. These include, without limitation, promoters of genes that are responsible for the expression of heat-regulated genes (see, eg, Callis et al., Plant Physiol, 88: 965, 1988; Takahashi and Komeda, Mol. Gen. Genet. 219: 365, 1989; and Takahashi et al., Plant J. 2: 751, 1992), gene expression regulated by light (e.g., the rbcS-3A pea described by Kuhlemeier et al., Plant Cell 1: 471, 1989 the corn rbcS promoter described by Scháffner and Sheen, Plant Cell 3: 997, 1991, the chlorophyll a / b binding protein gene found in the pea described by Simpson et al., EMBO J. 4: 2723, 1985; the Arabssu promoter, or the rice rbs promoter), the expression of hormone-regulated genes (eg, the sequences responding to the abscisic acid (ABA) of the wheat Em gene described by Marcotte et al., Plant Cell 1: 969, 1989, HVA1 and HVA22, and ABA-inducible rd29A promoters described for fattening a and Arabidopsis by Straub et al., Plant Cell 6: 617, 1994 and Shen et al., Plant Cell 1: 295, 1995; and gene expression induced by lesions (eg, from a wunl described by Siebertz et al., Plant Cell 1: 961, 1989), organ-specific gene expression (e.g., of the tuber specific storage protein gene described by Roshal et al, EMBO J. 6: 1155, 1987; the zein gene of 23-kDa corn described by Schernthaner et al., EMBO J. 1: 1249, 1988; or the β-phaseolin gene of French bean described by Bustos et al., Plant Cell 1: 839, 1989), or pathogen-inducible promoters (e.g., PR-1, prp-1, or β-1,3 glucanase, the wirla promoter inducible by wheat fungus, and the promoters inducible by nematode, TobRB7-5A and Hmg-1, of tobacco and parsley, respectively). Plant expression vectors may optionally also include RNA processing signals, eg, introns, which have been shown to be important for efficient RNA synthesis and accumulation (Callis et al., Genes and Dev. 1: 1183, 1987). . The location of the RNA cleavage sequences can dramatically influence the level of transgene expression in plants. In view of this fact, an intron can be placed upstream or downstream of a sequence of a vir gene (or fragment thereof) in the transgene to modulate the levels of gene expression. In addition to the above mentioned 5 'regulatory control sequences, expression vectors can also include regulatory control regions that are generally present in the 3' regions of the plant genes (Thornburg et al., Proc. Na ti. Acad. Sci. U. S.A. 84: 744, 1987; An et al., Plant Cell 1: 115, 1989). For example, the 3 'terminator region can be included in the expression vector to increase the stability of the mRNA. A region of terminator can be derived from the PI-II terminator region of the potato. In addition, other commonly used terminators are derived from the octopine or nopaline synthase signals. The plant expression vector typically contains a dominant selectable marker gene used to identify cells that have been transformed. Selectable genes useful for plant systems include genes that encode genes with resistance to antibiotics, for example, those encoding resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin, or spectinomycin. The genes required for photosynthesis can also be used as selectable markers in strains deficient in photosynthesis. Finally, the genes that code for herbicide resistance can be used as selectable markers; useful herbicide resistance genes include the bar gene encoding the enzyme phosphinothricin acetyltransferase and conferring resistance to the broad spectrum herbicide BASTA® (Hoechest AG, Frankfurt, Germany). In addition, if desired, the construction of the plant expression may contain a modified or fully synthetic vir transcript sequence (or fragment thereof) that has been changed to increase the performance of the gene in plants. It should be readily apparent to a person skilled in the art of molecular biology, especially in the field of plant molecular biology, that the level of gene expression depends, not only on the combination of promoters, on the processing signals of RNA, and terminator elements, but also how these elements are used to increase the levels of expression of the selectable marker gene. Vine Transformation and Regeneration After the construction of the plant expression vector, several standard methods are available for the introduction of the vector into a plant host, thereby generating a transgenic plant. These methods include (1) transformation mediated by Agrobacterium (A. tumefaciens or A. rhizogenes) (see, for example, Lichtenstein and Fuller in: Genetic Engineering, vol 6, PWJ Rigby, ed. London, Academic Press, 1987; Lichtenstein, CP, and Draper, J., in: DNA Cloning, Vol. II, DM Glover, ed, Oxford, IRI Press, 1985)), (2) the particle delivery system (see, for example, Gordon- Kamm et al., Plant Cell 2: 603 (1990), or BioRad Technical Bulletin 1687, supra), (3) microinjection protocols (see, for example, Green et al., Supra), (4) polyethylene glycol (PEG) procedures. (see, for example, Draper et al., Plant Cell Physiol, 23: 451, 1982, or for example, Zhang and Wu, Theor, Appl. Genet, 76: 835, 1988), (5) AD absorption? liposome mediated (see, for example, Freeman et al., Plant Cell Physiol, 25: 1353, 1984), (6) electroincorporation protocols (see, eg, Gelvin et al., supra; Dekeyser et al., supra; Fromm et al. contributors, Na ture 319: 791, 1986; Sheen Plant Cell 2: 1027, 1990; or Jang and Sheen Plant Cell 6: 1665, 1994), and (7) the vortex method (see, for example, Kindle supra). The transformation method is not critical to the invention. Any method that provides efficient transformation can be employed. Some exemplary methods to transform grapes are. find in Scorza and collaborators. { Plant Cell Reports 14: 589-592, 1995), Baribault et al. (J. Expt. Bot. 41: 1045-1049, 1990), Mullins et al. (Biotechnology 8: 1041-1045, 1990), Nakano et al. (J. Expt Bot. 45: 649-656, 199.4), Kikkert et al. (Plant Cell Reports 15: 311-316, 1996), Krastanova et al. { Plant Cell Reports 1: 550-554, 1995), Scorza et al. (Plant Cell Reports 14: 589-592, 1994), Scorza et al. (J. Amer. Soc. Hort. Sci. 121: 616-619, 1996), Martinelli et al. (Theor. Appl. Genet, 88: 621-628, 1994), and Legall et al. . { Plant Sci. 102: 161-170, 1994). As newer methods are available to transform grapes these can be applied directly. Suitable plants for use in the practice of the invention include, but are not limited to, vines (e.g., Vi tis spp, Vitis spp hybrids, and all members of the subgenus Euvitis and Muscadinia), including cuttings cultures or of rhizomes. Exemplary cuttings crops include, without limitation, those that are known as table grapes or raisins and those used in wine production such as Cabernet Franc, Cabernet Sauvignon, Chardonnay (for example, CH 01, CH 02, CH Dijon) , Merlot, Pinot Noir (PN, PN Dijon), Semillon, White Riesling, Lambrusco, Thompson Seedless, Autumn Seedless, Niagrara Seedless, and Seval Blanc. Other cuttings crops that can be used include those commonly known as table grapes or raisins, such as Alden, Almeria, Anab-E-Shahi, Autumn Black, Beauty Seedless, Black Corinth, Black Damascus, Black Malvoisie, Black Prince, Blackrose , Bronx Seedless, Burgrave, Calmeria, Campbell Early, Canner, Cardinal, Catawba, Christmas, Concord, Dattier, Delight, Diamond, Dizmar, Duchess, Early Muscat, Emerald Seedless, Emperor, Exotic, Ferdinand de Lesseps, Feast, Flame seedless, Flame Tokay, Gasconade, Gold, Himrod, Hunisa, Hussiene, Isabella, Italy, July Muscat, Khandahar, Katta, Kourgane, Kishmishi, Loo Perlette, Malaga, Monukka, Muscat of Alexandria, Muscat Flame, Muscat Hamburg, New York Muscat, Niabell , Niagara, Olivette blanche, Ontario, Pierce, Queen, Red Malaga, Ribier, Rish Baba, Romulus, Ruby Seedless, Schuyler, Seneca, Suavis (IP 365), Thompson without seed, and Thomuscat. They also include those used in the production of wine, such as Aleatico, Alicante Bouschet, Aligote, Alvarelhao, Aramon, Baco blanc (22A), Burger, Cabernet franc, Cabernet, Sauvignon, Calzin, Carignane, Charbonne, Chardonnay, Chasselas dore, Chenin blanc, Clairette blanche, Early Burgundy, Emerald Riesling, Feher Szagos, Fernao Pires, Flora, French Colombard, Freesia, Furmint, Gamay, Gewurztraminer, Grand noir, Gray Riesling, Green Hungarian, Green Veltliner, Grenache, Cricket, Helena, Inzolia, Lagrein, Lambrusco de Salamino, Malbec, Malvasia bianca, Mataro, Melon, Merlot, Meunier, Mission, Montua de Piles, Muscadelle du Bordelais, Muscat blanc, Muscat Ottonel, Muscat Saint-Vallier, Nebbiolo, Nebbiolo fino, Nebbiolo Lampia, Orange Muscat , Palomino, Pedro Ximenes, Petit Bouschet, Petite Sirah, Peverella, Pinot noir, Pinot Saint-George, Primitivo di Gio, Red Veltliner, Refosco, Rkatsiteli, Royalty, Rubired, Ruby Cabernet, Saint-Emilion, Saint Macaire, Salvador, Sangiovese , Sauvignon blanc, Sauvignon gray, Sauvignon vert, Scarlet, Seibel 5279, Seibel 9110, Seibel 13053, Semillon, Servant, Shiraz, Souzao, Sultana Crimson, Sylvaner, Tannat, Teroldico, Ink Madeira, Red wine, Touriga, Traminer, Trebbiano Toscano, Trousseau , Valdepeñas, Viognier, Walsch-riesling, White Riesling, and Zinfandel. Rhizome cultures that are useful in the invention include, without limitation, Vitis rupestris Constantia, Vitis rupestris St. George, Vitis california, Vitis girdiana, Vitis rotundifolia, Vitis rotundifolia Carlos, Richter 110. { Vitis berlandieri x rupestris), 101-14 Millarder et de Grasset (Vitis riparia x rupestris), Teleki 5C. { Vitis berlandieri x riparia), 3309 Courderc. { Vitis riparia x rupestris), Riparia Gloire de Montpellier. { Vitis riparia), 5BB Teleki (Selection Kober, Vitis berlandieri x riparia), S04. { Vitis berlandieri x rupestris), 41B Millardet. { Vitis vinifera x berlandieri), and 039-16 (Vitis vinifera x Muscadinia). Additional rhizome cultures that can be used include Couderc 1202, Couderc 1613, Couderc 1616, Couderc 3309, Dog Ridge, Foex 33EM, Freedom, Ganzin 1 (A x R # 1)E.

, Har-mony, Kober 5BB, LN33, Millardet &; from Grasset 41B, Millardet & from Grasset 420A, Millardet & of Grasset 101-14, Oppenheim 4 (S04), Paulsen 775, Paulsen 1045, Paulsen 1103, Richter 99, Richter 110, Riparia Gloire, Ruggeri 225, Saint-George, Salt Creek, Teleki 5A, Vitis rüpestris Constantia, Vitis california, and Vitis girdiana. In general, transfer and expression of transgenes in plant cells are now routine practices for those skilled in the art, and have become important tools for carrying out gene expression studies in plants and for producing improved plant varieties of agricultural interest. or commercial Plant cells transformed with plant expression vectors can be regenerated, for example, from single cells, callus tissue, or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs of almost any plant can be grown successfully to regenerate an entire plant; these techniques are described, for example, in Vasil supra; Green et al., Supra; Weissbach and Weissbach, supra; and Gelvin et al., supra. Exemplary methods for regenerating grape plants of transformed material are found in Scorza et al. (Plant Cell Reports 14: 589-592, 1995), Baribault et al. { J. Expt. Bot. 41: 1045-1049, 1990), Mullins et al. { BioTechnology 8: 1041-1045, 1990), Nakano et al. (J. Expt. Bot. 45: 649-656, 1994), Kikkert et al. { Plant Cell Reports 15: 311-316, 1996), Krastanova et al. (Plant Cell Reports 1: 550-554, 1995), Scorza et al. (Plant Cell Reports 14: 589-592, 1994), Scorza et al. (J. Amer. Soc. Hort. Sci. 121: 616-619, 1996), Martinelli et al., (Theor Appl Genet. 88: 621-628, 1994), and Legall et al., (Plant Sci. 102: 161-170, 1994).

In a particular example, a cloned vir transgene construct (e.g., mutation vi_rE2, virD2, virE2 deletion B, virE2 deletion C or virE2 deletion E, based on the virE2 coding sequences of a Ti plasmid type nopaline, vitopin, octopine , octopine / cucumopin, leucinopine / agropin, succinamopin, or agropin or based on coding sequences residing in Ri plasmids) under the control of the CaMV 35S promoter and the nopaline synthase terminator and carrying a selectable marker (e.g. resistance to kanamycin) is transformed into Agrobacterium. The transformation of a vine with a vector containing Agrobacterium is carried out as described by Scorza et al. (J. Amer. Soc. Hort, Sci. 121: 616-619, 1996). The putative transformants are selected after a few weeks in tissue culture medium containing kanamycin. The kanamycin resistant plant material is then placed in plant tissue culture medium without hormones for root initiation. The transgenic plants expressing the selectable marker are selected for transmission of the transgene DNA by standard detection techniques as described above. Each transgenic positive plant and its transgenic progeny are unique compared to other transgenic plants established with the same transgene. The integration of the transgene DNA into the plant genomic DNA is in most cases random, and the integration site can profoundly affect the levels and the tissue and the development patterns of the expression of the transgene. Consequently, several transgenic lines are selected for each transgene to identify and select plants with the most suitable expression profiles. The transgenic lines are evaluated to determine the levels of transgene expression. The expression at the RNA level is initially determined to identify and quantify plants with positive expression. Standard techniques for RNA analysis are employed and include amplification assays by polymerase chain reaction using oligonucleotide primers designed to amplify only transgene RNA templates and solution hybridization assays using probes specific for transgene (see, for example , Ausubel et al., Supra). Plants with positive RNA are analyzed for resistance to Agrobacterium infection and crown gall formation using the methods described above. Transformed vines expressing a virE2 gene (or virD2 gene) or fragment thereof and having resistance to crown gall disease relative to control plants is considered useful in the invention. All publications and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each separate publication or patent application specifically and individually was indicated as incorporated by reference.

Claims (6)

1. CLAIMS 1. A method to provide resistance to a bacterial plant pathogen that infects a vine or vine component, said method comprising the steps of: (a) transforming plant cells of the vine with a vir gene or an anti-pathogenic fragment of the same that is expressed in said grapevine cells; (b) regenerating a transgenic vine or transgenic vine component from said vine plant cells; and (c) selecting a transgenic vine or transgenic vine component expressing said vir gene or said antipathogenic fragment thereof, wherein the expression of said vir gene or said anti-pathogenic fragment provides resistance to said bacterial plant pathogen. The method of claim 1, wherein said vir gene or said anti-pathogenic fragment thereof is integrated into the genome of the transgenic vine or transgenic vine component. 3. The method of claim 1, wherein said vir gene is virE2. 4. The method of claim 1, wherein said vir gene is virD2. The method of claim 1, wherein said anti-pathogenic gene fragment is virE2 suppression B. 6. The method of claim 1, wherein said transgenic vine or transgenic vine component is a member of the genus Vitis. I. The method of claim 1, wherein said transgenic vine component is a somatic embryo, a shoot, or a root material. 8. The method of claim 1, wherein said bacterial pathogen is Agrobacterium. 9. The method of claim 8, wherein said Agrobacterium is Agrobacterium vi tis. The method of claim 8, wherein said Agrobacterium is Agrobacterium tumefaciens. II. The method of claim 1, wherein the expression of said vir gene or anti-pathogenic fragment thereof reduces the formation of crown gill in said transgenic vine or transgenic vine component. The method of claim 1, wherein said vir gene or a pathogenic fragment thereof is of a Ti plasmid. 13. A transgenic vine or transgenic vine component transformed with a vir gene or an anti-pathogenic fragment thereof, wherein the expression of said vir gene or said anti-pathogenic fragment thereof in said transgenic vine or transgenic vine component provides resistance to a bacterial plant pathogen. 14. The transgenic vine or transgenic vine component of claim 13, wherein said vir gene or antipathogenic fragment thereof is integrated into the genome of the transgenic vine or transgenic vine component. 15. The transgenic vine or transgenic vine component of claim 13, wherein said vir gene is virE2. 16. The transgenic vine or transgenic vine component of claim 13, wherein said vir gene is vi D2. 17. The transgenic vine or transgenic vine component of claim 3, wherein said anti-pathogenic gene fragment is virE2 suppression B. 18. The transgenic vine or transgenic vine component of claim 13, wherein said vine or vine component. It is a member of the genus Vitis. 19. The transgenic vine or transgenic vine component of claim 13, wherein said vine component is a somatic embryo, a shoot, or a root material. 20. The transgenic vine or transgenic vine component of claim 13, wherein said bacterial pathogen is Agrobacterium. 21. The transgenic vine or transgenic vine component of claim 20, wherein said Agrobacterium is Agrobacterium vitis. 22. The transgenic vine or transgenic vine component of claim 20, wherein said Agrobacterium is Agrobacterium tumefaciens. 23. The transgenic vine or transgenic vine component of claim 13, wherein the expression of said vir gene or anti-pathogenic fragment thereof reduces the formation of crown gill in said transgenic vine or vine component. The transgenic vine or transgenic vine component of claim 13, wherein said vir gene or antipathogenic fragment thereof is of a Ti plasmid.