MXPA00003023A - Nepovirus resistance in grapevine - Google Patents

Abstract

In general, the invention features a method for selecting a transgenic grapevine or grapevine component having increased resistance to a fanleaf disease, the method including the steps of:(a) trnsforming a grape plant cell with a grape nepovirus coat protein nucleic acid molecule or fragment thereof which is capable of being expressed in the plant cell;(b) regenerating a transgenic grapevine or grapevine component from the plant cell;and (c) selecting a transgenic grapevine or grapevine component which expresses, at a low level, the nucleic acid molecule or fragment thereof, wherein the low level expression increases the resistance of the transgenic grapevine or grapevine component to fanleaf disease. The invention also relates to an isolated nucleic acid molecule encoding a coat protein or fragment thereof of a"Geneva"isolate of a grapevinenepovirus virus.

Description

RESISTANCE TO NEPOVIRUS IN VID Background of the Invention This invention relates to disease resistance in vegetables. Grape leaf virus virus (GFLV) is a grape nepovirus, which is transmitted from plant to plant by the sword nematode, Xiphinema index. The grapevine leaf virus virus is the agent responsible for grapevine leaf disease, which occurs throughout the world. The disease is named after the fan-shaped leaf appearance of the leaves infected with the vine leaf fan virus. It is one of the most harmful and widespread diseases of the vine. Symptoms of vine leaf virus infection include morphology of abnormal shoot and discoloration of the leaves, producing a fan-like appearance (Agrios, Plant Pa thology, third edition, Academic Press, 1988, pp. 687- 688). In addition, the fruit production of the infected vines is low, producing small grape clusters that have abnormal fruit set and rot. Finally, the infected vines degenerate and die. The long-range propagation of the vine-fan virus is believed to be due to the use of infected planting material. Although it is believed that the range of the natural host is restricted to the grape, the grapevine virus of the grapevine is also transmissible to a wide range of herbaceous species by inoculation by rubbing sap. Chenopodium quinoa is a useful diagnostic species for the virus. In general, vine leaf virus isolates are antigenically uniform and diagnosis by Enzyme Linked Immunosorbent Test (ELISA) is a standard procedure. Current strategies to control vine leaf fan disease and other nepovirus-induced diseases in vineyards include nematode control (eg, soil fumigation and use of other pesticides), cultivate rhizomes resistant to nematode feeding , grow vines resistant to vine leaf virus, and plant certified vines without disease. SUMMARY OF THE INVENTION In general, the invention features a method for producing and selecting a vine or transgenic vine component that has increased resistance to leaf-blade disease. The method generally includes: (a) transforming a grape plant cell with a grape nepovirus coating protein nucleic acid molecule or fragment thereof (e.g., a protein nepovirus coat protein nucleic acid molecule). grape or fragment thereof having approximately 50 percent or more of sequence identification with SEQ ID NO: 1) that is capable of being expressed in the plant cell; (b) regenerating a vine or transgenic vine component of the plant cell; and (c) selecting a vine or transgenic vine component that expresses at low level, the nucleic acid molecule or fragment thereof, wherein low expression increases the resistance of the vine or transgenic vine component to the disease of the fan blade compared to plants that express the nucleic acid molecule at a high level. The low level expression of the grape nepovirus mRNA of the coat protein expressed in the transgenic plant itself is measured according to standard methods including, without limitation, a Northern blot analysis, linked enzyme immunosorbent assay, inoculation of transgenic plants with viruses and selection of resistant vines. In preferred embodiments, the nucleic acid molecule or fragments thereof is encoded by a transgene found in the transgenic vine. In other preferred embodiments, the nucleic acid molecule or fragment thereof is expressed as in a sense or antisense orientation. In other preferred embodiments, these grape nepovirus coating protein nucleic acid molecules or fragments thereof (a non-limiting example is non-translatable grape nepovirus viral coat protein mRNA having an ATG start codon of the start of the open reading frame with the rest of the mRNA that is outside the frame) is expressed in the vine or transgenic vine component. As discussed above, the invention also includes fragments of a grape nepovirus coating protein nucleic acid molecule that facilitates, when expressed at low levels, an increased resistance of a vine or transgenic vine component thereof, to a leaf disease of fan. Thus, the grape nepovirus coat protein nucleic acid sequences described herein or portions thereof can be expressed in a plant to facilitate resistance to disease. Sequences that mediate an increased resistance to leaf-blade disease are considered useful in the invention. As used herein, the term "fragment," as applied to sequences of nucleic acid molecules, 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. Fragments of the grape nepovirus nucleic acid molecule can be produced and subsequently integrated into any standard expression vector (e.g., those described herein) according to methods known to those skilled in the art. Preferably, the vine useful in the invention is a member of the genus Vi tis; and the component of the vine is a somatic embryo, a cutting, a rhizome, or a mother block. In still other preferred embodiments, the leaf-leaf disease is grapevine leaf disease caused by grape nepovirus. In still other preferred embodiments, the grape nepovirus is a grapevine leaf virus or an arabis mosaic virus. In another aspect, the invention features a vineyard that includes three or more transgenic vines or grapevine components each of which expresses, at low level, a nucleic acid molecule of grape nepovirus coating proteins or fragment thereof, wherein the expression at low level of the nucleic acid molecule or fragment thereof increases the resistance of the vines or transgenic vine components in the vineyard to the disease of the fan leaf. In yet another aspect, the invention features a substantially pure protein (e.g., a recombinant protein) that includes an amino acid sequence that has at least 97 percent amino acid identity with the amino acid sequence of the nepovirus coating protein of "Geneva" isolated grape shown in Figure 1 (SEQ ID NO: 2). In preferred embodiments, the protein includes the amino acid sequence of the grape nepovirus coating protein shown in Figure 1 (SEQ ID NO: 2). In still other preferred embodiments, the protein has the amino acid sequence of the grape nepovirus coating protein shown in Figure 1 (SEQ ID NO: 2) or fragments thereof. In still another aspect, the invention features an isolated nucleic acid molecule encoding a protein (e.g., a recombinant protein) that includes an amino acid sequence that has at least 97 percent amino acid identity with the amino acid sequence of the "Geneva" isolated grape nepovirus coating protein shown in Figure 1 (SEQ ID NO: 2). In preferred embodiments, the protein encoded by the nucleic acid molecule includes the amino acid sequence of SEQ ID NO: 2. In still other preferred modes, the protein encoded by the nucleic acid molecule has the amino acid sequence of SEQ ID NO: 2. NO: 2 or fragment of it. In another aspect, the invention features an isolated nucleic acid molecule (e.g., the DNA molecule) that encodes a grape nepovirus coating protein that specifically hybridizes to a nucleic acid molecule that includes the nucleic acid sequence of the Figure 1 (SEQ ID NO: l). Preferably, the hybridizing nucleic acid molecule specifically encodes a grape nepovirus sequence that mediates resistance when expressed at low levels in a grape plant cell to a fan leaf disease (e.g. the vine) The invention also characterizes an RNA transcript having a sequence complementary to any of the isolated nucleic acid molecules described above. In related aspects, the invention further features a cell (e.g., a prokaryotic cell or eukaryotic cell such as a mammalian cell or yeast cell) that includes an isolated nucleic acid molecule of the invention. In preferred embodiments, the cell is a bacterium (e.g., E. coli or Agrobacterium tumefaciens) or is a plant cell (e.g., a grape plant cell of any of the cultures listed herein). This plant cell has resistance against a fan leaf disease (eg vine leaf fan disease). In still other related aspects, the invention further presents a vector (e.g., a plant expression vector) that includes an isolated nucleic acid molecule of the invention. In a preferred embodiment, the isolated nucleic acid molecule is operably linked to an expression control region that mediates the expression of a protein encoded by the nucleic acid molecule (e.g., a nucleic acid molecule (such as DNA) expressed as a transcript of sense-translatable mRNA, or non-translatable sense, or as a transcript of antisense mRNA). In still other aspects, the invention features a transgenic plant or plant component (e.g., a vine or vine component) that includes a nucleic acid molecule that encodes a protein (e.g., a recombinant protein) encoding a amino acid sequence having at least a 97 percent amino acid identity with the amino acid sequence of the "Geneva" isolated grape nepovirus coating protein shown in Figure 1 (SEQ ID NO: 2). In preferred embodiments, this transgenic plant or plant component includes a nucleic acid molecule of SEQ ID NO: 1. Furthermore, the fragments of this sequence can be made so that the nucleic acid molecule expresses a transcript of translatable sense RNA, non-translatable sense, or antisense. In still other preferred embodiments, the plant or plant component has the nucleotide sequence of SEQ ID NO: 1 or fragments thereof. These plants or plant components that include the nucleic acid molecules of the invention have an increased level of resistance against fan leaf disease caused by a grape nepovirus (e.g., vine leaf fan virus). The methods and sequences of the grapevine leaf virus described herein are useful for providing disease resistance or tolerance or both on a variety of vines (eg, Vi tis spp., Hybrids of Vi tis spp. all members of the subgenus Euvi tis and Muscadinia), including cultures of cuttings or rhizomes. Exemplary cuttings crops include, without limitation, what 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. Cultures of rhizomes which are useful in the invention include, without limitation, Vi tis rupestris Constantia, Vi tis rupestris St. George, Vi tis california, Vi tis girdiana, Vi tis rotundifolia, Vi tis rotundifolia Carlos, Richter 110 (Vitis berlandieri x rupestris), 101-14 Millarder et de Grasset. { Vi tis riparia x rupestris), Teleki 5C (Vi tis berlandieri x riparia), 3309 Courderc. { Vi tis riparia x rupestris), Riparia Gloire of Montpellier (Vitis riparia), 5BB Teleki (selection Kober, Vi tis berlandieri x riparia), S04 (Vi tis berlandieri x rupestris), 41B Millardet (Vitis vinifera x berlandieri), and 039- 16 (Vi tis vinifera x Muscadinia). The invention also features cuttings, rhizomes, somatic or zygotic embryos, cells, or seeds that are produced from any of the transgenic vine or grapevine components described herein. By "non-translatable" is meant a sequence of mRNA that is not translated into a protein. Examples of these non-translatable sequences include, without limitation, sequences that include a start ATG codon followed by an overlapping frame shift mutation and stop codon to prevent translation of the mRNA into a protein. The grape nepovirus coating protein genes expressing these non-translatable mRNA sequences can be constructed according to standard methods (eg, those described herein). By "low level expression" is meant a level of expression of the grape nepovirus coating protein gene in a transgenic plant that is greater than zero and that is sufficiently low to impart resistance to the disease of the fan blade. "High level expression" refers to the level of expression of the gene found in the transgenic plant that expresses a coat protein gene that is too high to confer resistance to the disease. Exemplary methods for analyzing low level expression of the grape nepovirus coating protein gene include, without limitation, Northern blot analysis for the detection of the mRNA transcript, as well as immunological techniques such as enzyme-linked immunosorbent assay for protein detection. By "substantially identical" is meant a nucleic acid molecule or protein that exhibits at least 97 percent or preferably 98 percent or more preferably 99 percent identity with a reference amino acid sequence (e.g., the amino acid sequence shown) in Figure 1; SEQ ID NO: 2) or the nucleic acid sequence (eg, the nucleic acid sequences shown in Figure 1; SEQ ID NO: 1). For proteins, the length of the comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and more preferably 35 amino acids or more. For nucleic acids the length of the comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and more preferably 110 nucleotides or more. Sequence identity, at amino acid or nucleic acid levels, is typically measured using sequence analysis software (eg, Sequence Analysis Software package from the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison , Wl 53705, BLAST, or the PILEUP / PRETTYBOX programs). This software compares identical or similar sequences assigning degrees of homology to various substitutions, deletions, - and / or other modifications. Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; usina, arginine; and phenylalanine, tyrosine. By a "substantially pure protein" is meant a grape nepovirus coating protein (e.g., the coat protein isolated from grape nepovirus Geneva, NY (Figure 1; SEQ ID NO: 2)) that has been separated from components which naturally accompany it. Typically, the protein is substantially pure is at least 60 weight percent, free of naturally occurring proteins and organic molecules with which it is naturally associated. Preferably, the preparation is at least 75 percent, more preferably at least 90 percent, and most preferably at least 99 percent by weight, of protein. A substantially pure protein (e.g., the coating protein of the grape nepovirus isolate "Geneva") can be obtained, for example, by extraction from a natural source (e.g., a plant infected with leaf fan virus). vid-CP such as C. Quinoa); by expressing a recombinant nucleic acid encoding a protein; or chemically synthesizing the protein. The purity can be measured by any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or by high performance liquid chromatography analysis. By "isolated nucleic acid molecule" is meant a nucleic acid molecule (e.g., DNA) that does not have the nucleic acids, which, in the naturally occurring genome of the organism from which the nucleic acid molecule is derived from the invention flanks the nucleic acid molecule. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into a plasmid or virus replicating autonomously; or in the genomic DNA of a prokaryote or eukaryote; or that it exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene that encodes additional protein sequences. By "specifically hybrid" is meant that a nucleic acid molecule that is capable of hybridizing to a nucleic acid sequence (eg, DNA) at least under stringent conditions and preferably under very stringent conditions. By "proteins" is meant a chain of amino acids, including polypeptides, regardless of length or post-translational modification (e.g., glycocylation or phosphorylation), including polypeptides. By "positioned for expression" is meant that the nucleic acid molecule (eg, DNA) is placed adjacent to a sequence that directs the transcription of the nucleic acid molecule (eg, a gene that expresses a non-translatable antisense sequence or Sense sequence not translatable). By "expression control region" is meant any minimum sequence sufficient for direct transcription. Included in the invention are the promoter and enhancer elements that are sufficient to render the expression of the promoter-dependent gene controllable for the expression of cell-specific, tissue or organ-specific gene, or elements that are inducible by external signals or agents (e.g. inducible elements by light, pathogens, wounds, stress or hormones; or constituent elements); these elements can be located in the 5 'or 3' regions of the original gene or overlap in a transgene construct. By "operably linked" is meant that a gene or regulatory sequence (s) is connected in a manner that allows expression of the gene when the appropriate molecules (e.g., transcriptional activating proteins) are linked to the (s) regulatory sequence (s). By "plant cell" is meant any self-propagating cell bounded by a semipermeable membrane and containing a plastid. A plant cell, as used herein, is obtained from, without limitation, seeds, suspension cultures, embryos, meristematic regions, callous tissue, protoplasts, leaves, roots, shoots, somatic and zygotic embryos, as well as any part of a reproductive or vegetative tissue or organ. ~ "Plant component" means a part, segment, or organ obtained from an intact plant or plant cell. Exemplary plant components include, without limitation, somatic embryos, leaves, fruits, cuttings and rhizomes. By "vineyard" is meant a plot that includes three or more transgenic vine components or vines that are selected for low level expression of a grape nepovirus coating protein nucleic acid molecule or fragment thereof. By "transgenic" is meant any cell that includes a nucleic acid molecule (eg, a DNA sequence) that is artifically inserted into a cell and becomes part of the organism's genome (either integrated or extrachromosomal for example). , a viral expression construct that includes a subgenomic promoter) that develops from that cell. As used herein, transgenic organisms are generally transgenic vine or grapevine components and the nucleic acid molecule (eg, a transgene) is artifically inserted into the plastid nuclear compartments of the plant cell. Preferably, this transgenic vine or transgenic vine component expresses at least a translatable sense sense, non-translatable sense, or antisense of grape nepovirus (eg, an antisense sequence of grapevine virus of the CP-vine). "Transgene" means any piece of nucleic acid molecule (eg, DNA) that is artifically inserted into a cell, and becomes part of the organism (integrated into the genome or maintained extrachromosomally) that develops from that cell. This transgene may include a gene that is partially or entirely heterologous (ie, foreign) of the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. By "antisense nucleic acid sequence" is meant a nucleotide sequence that is complementary to the transcribed RNA. In general, this antisense sequence will usually have at least 15 nucleotides, preferably about 15 to 200 nucleotides, and more preferably 200 to 2000 nucleotides in length. The antisense sequence may be complementary to all or a portion of the nucleotide sequence of transcribed RNA (eg, a grape nepovirus sequence such as the antisense constructs of grapevine-CP virus described herein). or in any of the grape leaf virus sequences described in Brant et al., Arch. Virol., 140: 157-164, 1995, Margis et al., J. Gen. Virol. 74: 1919-1926, 1993; Fuchs et al, J. Gen. Virol 955-962, 1989, Serghini et al, J. Gen. Virol 71: 1433-1441, 1990, Bardonnet et al., Plan t Cell Reports 13: 357-360, 1994; Krastanova et al., Plant Cell Rep. 14: 550-554, 1995; Ritzenthaler et al., J. Gen. Virol. 72: 2357-2365, 1991; Mauro et al., Plant Science: 112: 97-106, 1995; and Sánchez et al, Nucleic Acids Res.19: 5440, 1992), and, as appreciated by those skilled in the art, the particular site or sites to which the antisense sequence is linked as well as the length of the antisense sequence will vary, depending on the degree of inhibition desired and the uniqueness of the sequence. antisense Preferably, a transcriptional construct expressing a grape nepovirus sequence (e.g., an antisense nucleotide sequence of grapevirus-CP fan leaf virus) includes, in the direction of transcription, an expression control region, the sequence encoding the antisense RNA in the sense chain, and a transcription termination region. Sequences of antisense grape nepoviruses (eg, grapevine leaf virus sequences) can be constructed and expressed as described herein or as described, for example, in van der Krol et al., Gene 72 : 45, 1988; Rodermel et al., Cell 55: 673, 1988; Mol et al., FEBS Let t. 268: 427, 1990; Weigel and Nilsson, Na ture 377: 495, 1995; Cheung et al., Cell 82: 383, 1995); and U.S. Patent No. 5,107,065. "Increased resistance to leaf-blade disease" means a higher level of resistance to leaf-blade disease (e.g., any disease caused by grape nepovirus such as the one caused by the vine leaf virus virus, arabis mosaic virus, and the like) in the transgenic vine (or vine or cell component or seed thereof) than the level of resistance to a control vine ( for example, a non-transgenic vine). In preferred embodiments, the level of resistance in the 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 disease resistance of the fan blade is 50 percent greater, 60 percent greater, and more preferably up to 75 percent or 90 percent greater than a control vine; with up to 100 percent resistance compared to a control vine is preferred more. The level of resistance is measured using conventional methods. For example, the level of resistance to leaf-blade disease can be determined by comparing aspects and physical characteristics (e.g., plant height and weight, or comparing the symptoms of the disease, e.g., development of delayed injury, size of lesion reduced, the leaf withers and curls, mottled and leaf necrosis, stem deformity, number of internodes, mosaic rings in leaves, and discoloration of cells) of transgenic vines. The ineffectiveness of the grape nepovirus (eg, a grapevine leaf virus virus or an Arabis mosaic virus) can also be monitored using, for example, standard enzyme linked immunosorbent assay. As discussed above, it has been discovered that low level expression of a grape nepovirus translatable sense coat protein gene, as well as an antisense sequence, provides the transgenic vines with resistance against the disease caused by a grape nepovirus. . In accordance with the above, the invention provides several important advances and advantages for wine growers. For example, by selecting the transgenic vines expressing low levels of a coat protein gene from recombinant grape nepovirus and thus having increased resistance against infection with grape nepovirus, the invention provides an effective and economical element to protect against the disease. Leaf fan of the vine and other diseases induced by grape nepovirus. This protection reduces or minimizes the need for traditional chemical practices (eg, soil fumigation) typically used by vine growers to control the spread of grape nepovirus and provides protection against these disease causing pathogens. In addition, because grape plants expressing these grape nepovirus sequences are less vulnerable to grape nepovirus infection and fan leaf disease, the invention also provides greater production efficiency as well as quality improvement. , color, flavor, and yield of grapes. In addition, because the invention reduces the need for chemical protection against the pathogens of the vine, the invention also benefits the environment in which the vineyards are planted. The invention can also be used in combination with cultivated rhizomes that have resistance to nematodes that carry the soil. 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 First, the drawings will be described. Drawings Figure 1 is a schematic illustration showing the nucleotide (SEQ ID NO: 1) and the deduced amino acid sequence (SEQ ID NO: 2) of the coating protein of a Geneva N.Y grape nepovirus isolate. Figure 2 is a schematic illustration showing the maps of the expression vectors of the plant containing different viral gene constructs. Figure 3 shows the results of experiments analyzing the expression levels of the vine leaf blade coat protein in the transgenic 3309C and Gloire. A description of the production of transgenic vines resistant to the disease comes next. Transgenic grape plants expressing either envelope protein genes of grapevine leaf virus viruses with sequences of either translatable sense, non-translatable sense, or antisense (GFLV-CP) were regenerated from derived embryogenic callus cultures anther of rhizomes (3309 Courder ("3309C"), Riparia Gloire ("Gloire"), Teleki 5C ("5C"), 110 Richter ("110R"), S04, and MGT 101-14 ("101-14" )). Unexpectedly, transgenic plants expressing low levels of a recombinant vine leaf blade protein gene were found to be resistant to leaf-blade disease.

The examples provided below are for the purpose of illustrating the invention, and should not be considered as limiting. Results Start of Embryogenic Callus and Embryogenesis The callus was started apart from grape crops: Gloire, 3309C. 5C, 110R, and 101-14 in MSE medium (infra). Anthers of groups of flowers of five rhizomes began to swell after a week in culture. After four weeks, a smooth, gelatinous, bright yellow callus developed. At this time, some embryos of Gloire 3309C, and 110R were visible in the tissue of the calluses. After eight weeks, all the calluses were transferred to HMG medium (infra) to allow the embryos to develop further. For the eighth week in HMG medium, many clusters of embryos were induced from the callus tissue. Plant Regeneration After culturing for eight to sixteen weeks in HMG medium, clusters of embryos, and hypocotyls were found to develop from the calluses. At the same time, secondary embryos were continuously produced from the primary embryos. The clusters of embryos were then transferred to the MGC medium (infra) to increase the size of the embryo and the growth rate. However, fewer embryos were produced in MGC medium compared to the HMG medium for all rhizome cultures that were examined. The development of embryos in culture 110R was found to depend on the use of both media; the HMG medium was required to induce many small secondary embryos and the MGC medium was necessary to simulate the growth of the hypocotyl. The hypocotyls were subsequently transferred to woody plant medium (Lloyd and McCown, infra) and shoots appeared in one or two months. The plants were generally induced at a frequency of thirty to sixty-six in the middle of a woody plant. The resulting plants were transplanted to the ground and kept in the greenhouse. The Gloire plants, 5C, 110R, 101-14, and 3309C exhibited normal morphology. Maintenance of Somatic Embryogenesis A continuous supply of embryogenic callus was produced using an embryo cycle method; pieces of hypocotyl induced embryogenic calluses in MSE medium in two or three months. These calluses were docile for transformation because they developed many uniform embryos. The 5C embryos required MSE medium culture for three months, followed by culture in HMG medium for two or three months to induce embryo formation. The length of time required for embryo cycling (embryogenic callus to hypocotyl and back to embryogenic callus) varied for different cultures; Riparia Gloire required two to three months, 3309C and 101-14 required five to ten months, and 5C required six to seven months. Transformation Using standard molecular biology techniques, a nucleotide sequence encoding a coat protein gene from a grape nepovirus isolate, Geneva, NY was isolated and characterized. The nucleic acid sequence (SEQ ID NO: 1) and the deduced amino acid sequence (SEQ ID NO: 2) of the isolated coat protein gene (Geneva) are shown in Figure 1. These sequences were also compared with three isolated different from the vine leaf virus of France (Serghini et al., J. Gen. Virol. 71: 1433-1442, 1990), California (Sánchez et al., Nucleic Acids Res. 19 5440, 1991), and Austria (Brandt et al, Aren, Virol. 140: 157-164, 1995) using a Prettybox program. The percent identity of the amino acid sequences of the French, Californian, and Austrian isolates with the "Geneva" isolate was 96.4 percent, 95.0 percent, and 95.4 percent, respectively. Three different gene constructs (translatable sense, nontranslatable sense, and antisense) were used to transform grape (Figure 2). Small somatic embryos and embryogenic stems of the five rhizomes Gloire, 3309C, 110R, 5C, and 101-14 were cocultivated with the strain C58Z707 of A. t umefa -ciens breeding binary vectors carrying the coat protein gene of the isolate "Geneva " After cocultivation, somatic embryos were transferred to HMG or MSE with cefotaxime, carbenicillin, and kanamycin to select embryos and transgenic plants. The transgenic plants were generated in this way. Analysis of Transgenic Plants In an experiment with sense-translational gene expression constructs and vid-CP fan-leaf virus β-glucuronidase (FLcpST + GUS, Figure 2), the putative transgenic plants of Gloire and 110R were tested to determine the GUS activity, the NPTII gene, and the expression of the fan-leaf virus gene of the vid-CP by immunosorbent assay of bound enzyme. By polymerase chain reaction analysis, approximately 93 percent of the Gloire plants were found to have the vine-CP fan-leaf virus gene of the expected size, although these plants were negative for leaf virus expression. fan of the vine by immunosorbent enzyme linked test. These results indicated that expression of the vine-CP fan-leaf virus gene in the Gloire transgenic was too low to be detected using enzyme-linked immunosorbent assay. In contrast, culture 3309C was transformed with leaf-blade virus from the CP-sense-translational construct into a vector without GUS. We analyzed the coat protein expression of putative transgenic plants, and found that 97.7 percent of plants were positive by enzyme-linked immunosorbent assay. Among those plants, 37.7 percent showed low expression (0.1 <OD405> 0.5), 30.7 percent showed medium expression (0.5 <OD405> 1.0), and 31.0 percent had high expression (OD405> 1.0). The non-transformed control plants were negative (OD405 < 0.020). In another series of experiments, the enzyme linked immunosorbent assay results also revealed different levels of expression of the vine leaf virus virus coat protein gene gene in 3309C and Gloire transgenic plants. The expression of the low coat protein gene was observed in 61 percent and 53 percent of the transformed 3309C and Gloire, respectively. The expression of the middle coat protein gene was found in 26 percent and 22 percent, respectively. The expression of the high coat protein gene was found in 13 percent and 25 percent, respectively, of the transformed 3309C and Gloire (Figure 3). Protection against the Abanxco Leaf Virus Virus Infection The transgenic plants were tested for their resistance to the vine leaf virus virus as follows. The plants were inoculated with the vine leaf fan virus by heterograft to C. quinoa infected with grape leaf virus or by grafting to cultures infected with non-transgenic vine leaf virus according to standard methods. The plants were maintained for several months after the inoculation and then they were valued to determine the resistance to the disease. Disease resistance was assessed by standard enzyme linked immunosorbent assay. The results of these experiments are shown in Tables I-V (below). In particular, as shown in Table III, it was found that a transgenic line expressing the antisense expression constructs of GFLVcpAntiS (Figure 2) resisted infection by grapevine leaf virus.

TABLE I Evaluation by ELISA of Transgenic 110 of Richter Heteroin? Etated with C. Quinoa ELISA ELISA ELISA ELISA (%) Transgenic lines 31/4/97 27/5/97 6/8/97 12/2/97 infected # 2 0/2 1/2 1/4 1/4 25.0 # 3 0/6 1/6 4/10 4/10 40.0 # 6 0/2 NT 1/2 1/2 50.0 # 8 0/2 2/2 2/5 NT 40.0 # 21 0/8 NT 8/8 8/8 100.0 # 41 NT 2/8 5/8 NT 62.0 # 45 - - 2/2 NT 100.0 # 50 - 3/4 4/4 4/4 100.0 # 84 - 4/6 5/6 5/6 83.0 Control - 6/8 14/16 14/16 88.0 FLcpST + GUS (Figure 2); NT: Not tested TABLE II Evaluation by ELISA of Goire Riparia Heteroinserted Transgenic with C. Quinoa infected with GFLV Lineas 2 months 4-5 months Total (%) transgenic 1 infected # 5 0/3 0/10 0/10 0 # 6 0/3 0/3 0/3 0 # 7 0/5 2/7 2/7 29.0 # 9 0/5 1/8 1/8 13.0 # 10 1/16 4/14 4/14 29.0 # 12 0 0 0 0 0 0 0 0 0 0 3/8 3/8 0 # 14 0/7 0/7 0/7 0 # 15 0/4 11/15 11/15 73.0 # 16 0/5 0/9 0/9 0 # 19 NT 0/4 0/4 0 # 20 2/12 2/12 2/12 17.0 # 21 0/11 3/14 3/14 14.0 # 22 0/1 0/4 0/4 0 # B-6 NT 0/8 0/8 0 # B-17 NT 6/13 6/13 46.0 # B-20 0/7 0/6 0/6 0 # B-24 NT 0/3 0/3 0 # B-42 0/4 or 0/4 0/4 0 Control 23/26 23/26 23/26 88.0 FLcpST + GUS (Figure 2) TABLE III ELISA Evaluation of TG-101-14 Heteroinserted Transsynicolated with C. Infected Quinoa ELISA ELISA ELISA (%) ELISA Lines Transsexual1 27/3/97 4/31/97 7/27/97 6/8/97 T18, FL (5--1) l1 0/7 NT 0/7 0/7 0 T18, FL (1--1) 12 0/8 1/8 2/8 4/8 50.0 T18, FL (1--1) 22 3/4 3/4 3/4 3/4 75.0 T18, FL (1--1) 32 2/5 3/5 4/5 4/5 80.0 T18, FL (1--1) E2 0/4 1/4 3/4 4/4 100.0 T18, FL (B - 8) 2 0 0 // 22 1/2 2/2 2/2 100.0 T18, FL (A - 3) * N NTT 0/4 3/4 4/4 100.0 T18, FL (K - i) 2 N NTT 2/8 2/8 2/8 25.0 CONTROL - 11/16 23/29 79 FLcpAntiS (Figure 2 LcpST (Figure 2) TABLE IV Misroinierto In Vitro Transgenic L. / grape GFLV No. of plants ELISA ELISA ELISA in GH 11/27/96 1/30/97 4/24/97 Riparia 0/2 0/2 0/2 # 16 / Cabernet sauv. Riparia 1/5 1 / -5 5/5 # 21 / Cabernet sauv. Riparia 2/5 2/5 4/5 # 23 / Cabernet sauv. Riparia 0/4 or 4 # 19 / Cabernet sauv. Riparia 0/4 or 4 B-2 / Cabernet sauv. Riparia 1/4 2/4 3/4 B-13 / Cabernet sauv. Riparia 1/2 1/2 2/2 B-17 / Cabernet sauv. Riparia 0/2 0/2 0/2 B-67 / Cabernet sauv. Riparia, hg / Cab 4/5 4/5 4/4 TABLE V Microinierto In Vitro L. Transgenic / grape GFLV No. of plants ELISA ELISA ELISA in GH 11/27/96 1/30/97 4/24/97 Richter 45 / Rupestris 3 2/3 2/3 2/3 Richter 26 / Rupestris 6 0/6 0/6 0/6 Richter 56 / Rupestris 2 0/2 0/2 0/2 Richter 11 / Rupestris 3 0/3 0/3 0/3 Richter 75 / Rupestris 6 2/3 2/3 2/4 Rupestris / Richter 56 2 0/2 0/2 0/2 Rupestris / Richter 75 6 2/3 2/3 2/5 Rupestris / Richter 4 4 0/3 0/2 0/2 Richter ht / Rup 4 3/4 3/4 4/4 Materials and Methods The results described above were carried out using the following materials and methods. Plant Materials The crops of rhizomes Couderc 3309 ("3309C") (V. Riparia x Rupestris), Riparia Gloire ("Gloire") (V. Riparia), Teleki 5C ("5C"), (V. Berlandieri x V. Riparia), MGT 101-14 ("101-14") (V. Riparia x V. Rupestris) and 110 Richter ("110R") (V. Rupestris x V. Berlandieri) were used in the experiments described above. Callus cultures were initiated from anthers using the methods of Rajasekaram and Mullins (J. Exp. 30: 399-407, 1979). Flower buttons 3309C, 5C, 110R and 101-14 were collected from a vineyard at the Geneva, Geneva, N.Y. Gloire dormant reeds were harvested from the same vineyard and stored in moist pearlite in plastic bags at 4 degrees Celsius. Two to five sections of nodes were rooted in pots with perlite in the greenhouse; flower buds developed in four weeks. Flower buds were harvested before the anthesis of vineyards grown in the field. The buttons were removed from the clusters and surface sterilized in 70 percent ETOH for one or two minutes. The buttons were transferred to 1 percent sodium hypochlorite for 15 minutes, then blocked three times in sterile double distilled water. The anthers were aseptically separated from the flower buds at the same time that a stereo microscope was used. To determine which state was most favorable for callus induction, the pollen was crushed under a microscope under a cover with a drop of acetocarmine to observe the cytological stage according to standard methods. Means Four different solid media were used to produce embryos and regenerate plants. The four media used were the following. (1) Initiation medium. This medium was a modified MS medium (Murashige and Skoog, Physiol, Plan t.15: 473-497, 1962) and is known as an MSE (Mozsar and Sule, Vi tis 33: 245-246, 1994). (2) Means of differentiation. This medium is known as HMG medium as described by Mozsar and Sule (Vi tis 33: 245-246, 1994); (3) Regeneration medium. This medium is known as the MGC medium. It consists of MS salts of total strength modified with 20 grams / liter of sucrose, 4.6 grams / liter of glycerol, 1 gram / liter hydrolyzed casein and 0.8 percent noble agar; and (4) root medium. This medium (pH 5.8) is woody plant medium (Lloyd and McCown, Proc. In tl. Plan t Prop. Soc. 30: 421-427, 1981) supplemented with 0.1 milligram / liter BA, 3 grams / liter of activated charcoal and 1.5 percent sucrose. Somatic Embryogenesis and Regeneration The anthers were isolated under sterile conditions and plated at a density of forty to fifty anthers by a Petri dish of 9 centimeters in diameter and cultured at 28 degrees centigrade in the dark. Calluses were induced in MSE. After sixty days, embryos were induced and then transferred to HMG medium without hormone for differentiation. Embryos in the torpedo stage were transformed from HMG medium to MGC to promote germination of the embryo. The cultures were kept in the dark at 26-28 degrees Celsius and transferred to fresh medium at intervals of three to four weeks. The hypocotyls (elongated embryos) were transferred to the rooting medium in baby food jars (five to eight embryos per bottle). The embryos were grown at 25 degrees centigrade with a daily photoperiod of sixteen hours to induce shoot and root formation. After the root development, the plants were transplanted to the ground and placed in the greenhouse. - Maintenance and Propagation of Somatic Embryos The hypocotyls of elongated embryos that were developed in HMG or MGC medium were cut into pieces of 3-4 millimeters and placed in MSE medium to promote the development of secondary embryogenic calli. The secondary embryogenic calli were then transferred to HMG for their differentiation and development of the new hypocotyls. These secondary hypocotyls from the HMG medium were transferred to MSE medium to obtain a third cycle of embryogenic calli and hypocotyls. The fourth and fifth cycles of embryogenic callus were obtained in a similar manner. Alternatively, the embryogenic calli developed from anther were propagated in MSE medium to produce enough young embryos for transformation. All embryo cultures were transferred at twenty to thirty day intervals to fresh medium for maintenance. Genes and Vectors Three genetic constructs were used to genetically transform grapes in this study (Figure 2). Strain C58Z707 of A. Tumefaciens that contained either the binary plasmid calli or pGA482G were used to transform the grape plants with the fan-leaf virus of the vid-cp. The coat protein gene of a vine leaf virus virus designated as CF57 isolate of "Geneva" NY was cloned and sequenced according to standard methods. The vine leaf nepovirus coating protein is produced by post-translational processing of the polyprotein by proteinase encoded by the virus. The coat protein gene, which is located in the 3 'half of the RNA2 genome, does not contain an ATG start codon. Oligonucleotide primers containing the Ncol site were thus used to introduce the translatable initiation codon into genetic construct. Two sets of primers designed to flank the coat protein gene for amplification by polymerase chain reaction were used according to standard methods. The primer set (P2: cstcaaTCTAGACCATGGTGAGAGGATTAGCTGGTAGAGGAG (SEQIDNO: 3) and KSL95-10: ctgtaCCATGGTCTTTTAAAGTCAGATACC (SEQ ID NO: 4)) was used to generate a translatable construct. To overlap a nontranslatable construction of sense, we introduced an additional nucleotide (T) only three nucleotides downstream of the translation initiation codon (ATG) to make a mutation of frame motion, as well as to create a stop codon. This was carried out using the KSL95-10 (SEQ ID NO: 4) and KSL96-15 (acgttaCCATGGTGTAGAGGATTAGCTGGTAGA; SEQ ID NO: 5) primers. (The lowercase letters are nonsense sequences used for effective restriction digestion.) - The underlined areas are restriction sites which were used for efficient cloning.The bold letters represent the stop codon that was used to overlap a construction not transferable of meaning). The resulting amplified polymerase chain reaction products were treated with the restriction enzyme, Ncol, and cloned into the plant expression vector pEPT8. The sense or antisense orientation was determined using standard restriction mapping and polymerase chain reaction analysis using the specific primer of the S-promoter 35 S (KSL 9 6-12: agtgctCTCGAGCAATTGAGACTTTTCAACAA; SEQ ID NO: 6) and transgene primers. The expression cassette containing the transgene and the plant transcription elements, 35S enhancers, 35S promoter, alfalfa mosaic virus RNA4 5 'untranslated sequence and the 35S terminator was subsequently cloned into the plant transformation vector pGA482G. Transformation The transformation protocols were modified from those described by Scorza and Cordts, (Plant Cell Rep. 14: 589-592, 1995, Krastanova et al., Plan t Cell Rep. 24: 550-554, 1995). The overnight cultures of the Agrobacterium strain C58Z707 were cultured in LB medium at 28 degrees centigrade in shaker incubator. The bacteria were centrifuged for five minutes at 5000 rpm (or 3000 rpm) and resuspended in liquid medium MS (OD600 = 0.4-1.0). The callus with globular or heart-shaped embryos was immersed in the bacterial suspension for fifteen to thirty minutes, dried by staining, and transferred to HMG medium with or without acetosyringone (100 uM). The embryogenic calluses were cocultivated with the bacteria for forty-eight hours in the dark at 28 degrees centigrade.

Then, the plant material was washed in MS liquid plus cefotaxime (300 mg / ml) and carbenicillin (200 mg / ml) two or three times. The material was then transferred to HMG medium containing either 20 or 40 milligrams / liter of kanamycin, 300 milligrams / liter of cefotaxime, and 200 milligrams / liter of carbenicillin to select transgenic embryos. Alternatively, after forty-eight hours of co-cultivation with Agrobacterium, the embryogenic calli were transferred onto initiation MSE medium containing 25 milligrams / liter of kanamycin plus the same antibiotics listed above. All plant material was incubated continuously in the dark at 28 degrees Celsius. After growth in a selection medium for three months, the embryos were transferred to HMG or MGC without kanamycin for the development of hypocotyls. The embryos were then transferred to root medium without antibiotics. The untransformed calli were cultured in the same media with and without kanamycin to verify the efficiency of kanamycin selection and the ability of the plant to regenerate in the presence of the antibiotic. Analysis of Transgenic Plants Transgenic plants were analyzed using standard assays in the GUS assay (Jefferson, Plant Mol. Biol. Rep. 5: 387-405, 1987), enzyme linked immunosorbent assay for the detection of NPTII (Cabanes-Bastos and collaborators, Gene 11: 69-176, 1989), linked enzyme immunosorbent assay for the detection of cp (Clark et al, J. Gen. Virol. 34: 475-483), and polymerase chain reaction and Southern analysis ( Ausubel et al., Infra). Isolation of Other Grape Nepovirus-CP Genes Any Grape Nepovirus isolate (eg, vine leaf fan virus) can serve as the source of nucleic acid for the molecular cloning of a protein coat protein gene. grape nepovirus (CP). For example, the isolation of a fan-leaf virus gene from vid-CP involves the isolation of DNA sequences that encode a protein that exhibits structures, properties, or activities associated with CP. Based on the nucleotide and amino acid sequences of the vid-CP fan-leaf virus described herein (Figure 1; SEQ ID NOS: 1 and 2), the isolation of the coding sequences of the fan-leaf virus of additional vid-CP is made possible using standard strategies and standard techniques well known in the art. In a particular example, the grapevine CP virus sequences described herein may be used, in conjunction with conventional analysis methods with nucleic acid hybridization analysis. These hybridization techniques and methods of analysis 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. (Supra); Berger and Kimmel (supra); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. In a particular example, the entire part of the nucleotide sequence of the "Geneva" isolate (described herein) can be used as a probe to select a DNA library of the fan-leaf virus from the recombinant vine to determine genes that have sequence identity with the "Geneva" isolated coat protein gene. 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 the coat protein gene of the "Geneva" isolate can easily design oligonucleotide probes specific to vine-CP virus, including regenerated oligonucleotide probes of grapevirus leaf-CP virus (ie, a mixture of all possible coding sequences for a given amino acid sequence). These oligonucleotides can be based on the sequence of the DNA strand and any "appropriate" portion of the grapevine virus sequence of the CP-vine (Figure 1).; SEQ ID NOS: 1 and 2). General methods for designing and preparing these probes are provided, for example, in Ausubel et al., 1996, Current Protocole 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 fan-leaf virus gene from the vid-CP, either through its use as probes capable of hybridization for "sequences complementary to the vine-leaf fan virus of the CP-vine or as primers for various amplification techniques, for example, cloning strategies with polymerase chain reaction (PCR) If desired, a combination of different oligonucleotide probes can be used for the selection of a recombinant DNA library. can detectably label using methods known in the art and used for probe filter replicates from a recombinant DNA library.Reinformative DNA libraries are prepared according to methods well known in the art, for example, as described in US Pat. Ausubel et al., (Supra), or can be obtained from commercial sources.Another sources of fan leaf virus sequences of the vid-CP include those described in Brandt et al., Arch. Virol, 140: 157-164, 1995; Margis et al., J. Gen. Virol, 71: 1433-1441, 1990 Bardonnet et al., Plan t Cell Rep. 13: 357-360, 1994; Krastanova et al., Plan t Cell Rep. 14: 550-554, 1995; Ritzenthaler et al., J. Gen Virol, 72: 2357-23T65, 1991; Mauro et al., Plan t Science; 112: 97-106, 1995; and Sánchez et al., Nuclei c. Acids Res. 19: 5440, 1992. As soon as the fan-leaf virus sequences of the vid-CP are identified, it is cloned according to standard methods and used for the construction of plant expression vectors as described herein. Construction of Plant Transgenes More preferably, a grape nepovirus coating protein (e.g., a vine-CP fan leaf virus) is expressed as a transcription of mRNA translatable in the sense or non-translatable in sense or as a transcript of antisense mRNA by a stably transfected grape cell line or by a vine or transgenic vine component. Several suitable vectors for either stable or extrachromosomal transgenic plant cells, or for the establishment of transgenic plants are available to the public; these vectors are described in Weissbach and Weissbach (Methods for Plant Molecular Biology, Academic Press, 1989) and Gelvin et al., (Plant Molecular Biology Manual, Kluwer, Academic Publishers, 1990. Methods for constructing these cell lines are described in, for example. , Weissbach and Weissbach (supra), and Gelvin et al., (Supra) Example of useful vectors for the expression of transgenes in vines are also described in Scorza et al., (Plan t Cell Rep. 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 Rep. 15: 311-316, 1995), Krastanova et al., (Plant Cell Rep. 1: 550-554, 1995), Scorza et al., (Plan t Cell Rep. 14: 589-592, 1994), Scorza et al., (J. Amer. Soc. Hort. Sci. 121: 616-619, 1996), Martinelli et al. ores, (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, a nucleic acid molecule that expresses a grape nepovirus RNA translatable in sense, non-translatable in sense, or antisense) 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 (e.g., one that confers inducible or constitutive, pathogen or wound induced, environmentally or developmentally regulated, or cell-specific or tissue-specific expression), a transcription initiation initiation site, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal. As soon as the desired grape nepovirus coating protein nucleic acid molecule is obtained as described above, it can be manipulated in a variety of ways known in the art. For example, a DNA sequence of the vine-CP virus of the invention can, if desired, be combined with other DNA sequences in a variety of ways. The DNA sequence of the vine-CP virus can be used with all or part of the gene sequences normally associated with the vine-CP fan-leaf virus. In its component parts, the DNA sequence encoding the vina-CP fan-leaf virus is combined into a DNA construct having a transcription initiation control region capable of promoting transcription in a host grape cell. . In general, the constructs involved functional regulatory regions in plants that provide the modified production of a grapevirus leaf-CP virus as discussed herein. For example, the non-translatable sense sequence for a grapevirus CP-leaf virus or fragment thereof will be joined at its 5 'end to a transcriptional initiation regulatory region, eg, such as a sequence naturally found in the upstream region 5 '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 the DNA sequence encoding the vid-CP fan-leaf virus or any convenient transcription termination region from a different gene source (e.g., 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. The plant expression constructs having the vine-CP fan-leaf virus as the DNA sequence of interest for expression (either in antisense orientation or translatable with sense or non-translatable with sense of mRNA production) can be employ with a wide variety of vines. Such genetically overlapping plants are useful for a variety of industrial and agricultural applications. Importantly, this invention is applicable to all vines or grapevine components, and will be readily 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 grapevine virus of the vid-CP translatable sense, or non-translatable sense or antisense. An example of a plant promoter useful according to 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., Na ture 313: 810, 1985). The CaMV promoter is also very active in monocotyledons (see, for example, Dekeyser et al., Plan t 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. Nati, Acad. Sci., U.S.A. 84: 4870, 1987, and Fang et al., Plan t 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 fan-leaf virus sequence of the vid-CP 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, for example, Callis et al., Plant Physiol, 88: 965, 1988; Takahashi and Komeda, Mol. Gen. Genet. 219: 365, 1989, and Takahashi et al., Plan t J. 2: 751, 1992), expression of light-regulated genes (e.g., the rbcS-3A pea described by Kuhlemeier et al., Plant Cell 1: 471, 1989 the rbcS maize promoter described by Schaffner and Sheen, Plan t 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., Plan t Cell 1: 969, 1989, HVA1 and HVA22, and ABA-inducible rd29A promoters described for the pars il and Arabidopsis by Straub et al., Plant Cell 6: 617, 1994 and Shen et al., Plant Cell 1: 295, 1995; and the expression of wound-induced genes (for example, from a wunl described by Siebertz et al., Plan t Cell 1: 961, 1989), expression of organ-specific genes (for example, of the tuber specific storage protein gene). described by Roshal et al, EMBO J. 6: 1155, 1987; the 23-kDa maize gene described by Schernthaner et al., EMBO J. 1: 1249, 1988; or the French β-phaseolin gene described by Bustos et al. , Plan t Cell 1: 839, 1989), or pathogen-inducible promoters (eg, PR-1, prp-1, or β-1,3 glucanase, the inducible wirla promoter of wheat fungus, and inducible promoters by nematode, TobRB7-5A and Hmg-1, of tobacco and parsley, respectively). Plant expression vectors may also optionally include RNA processing signals, for example, introns, which have to be shown to be important for efficient RNA synthesis and accumulation (Callis and collaborators, '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 the vine-CP virus leaf sequence 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 contains a dominant selectable marker gene used to identify cells that have been transformed. Selectable genes useful for plant systems include genes that encode antibiotic resistance genes, 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 a broad herbicidal spectrum BASTA® (Hoechest AG, Frankfurt, Germany). In addition, if desired, the construction of plant expression may contain a modified or fully synthetic vine-CP virus sequence 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 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) the 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; and Lichtenstein, C.P., and Draper, J. In: DNA Cloning, Vol. II, D.M. Glover, ed, Oxford, IRI Press, 1985)), (2) the particle delivery system (see, for example, Gordon-Kamm et al., Plant Cell 2: 603 (1990)); or Sanford et al., U.S. Patent Nos. 4,945,050, 5,036,006, and 5,100,792), (3) microinjection protocols (see, e.g., 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, 16: 835, 1988), (5) liposome-mediated DNA uptake. (see, for example, Freeman et al., Plant Cell Physiol, 25: 1353, 1984), (6) electroporation protocols (see, for example, Gelvin et al., supra; Dekeyser et al., supra; Fromm et al., Na Ture 319: 791, 1986; Sheen Plant Cell 2: 1021, 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 for transforming grapes are found in Scorza et al. (Plan t Cell Reports 14: 589-5_92, 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 Rep. 15: 311-316, 1995), Krastanova et al., (Plant Cell Rep. 1: 550-554, 1995), Scorza et al., (Plant Cell Rep. 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 new methods are available to transform cultures or other host cells, it can also be applied directly. Suitable plants for use in the practice of the invention include, but are not limited to, vines (eg, Vitis spp, hybrids of Vi tis spp, and all members of the subgenus Euvitis and Muscadinia), including cultures of cuttings or of rhizomes. Exemplary cuttings crops include, without limitation, those which 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 cultures that can be used include those commonly referred to as table grapes or raisins, such as Alden, Almeria, Anab-E-Sh.ahi, 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, Fiesta, Fíame seedless, Fíame Tokay, Gasconade, Gold, Himrod, Hunisa, Hussiene, Isabella, Italy, July Muscat, Khandahar, Katta, Kourgane, Kishmishi, Loóse Perlette, Malaga, Monukka, Muscat of Alexandria, Muscat Fíame, 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 Pilas, 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 Gioa, Red Veltliner, Refosco, Rkatsiteli, Royalty, Rubbed, Ruby Cabernet, Saint-Emilion, Saint Macaire, Savior, Sangiovese, Sauvignon Blanc, Sauvignon Gray, Sauvignon Vert, Scarlet, Seibel 5279, Seibel 9110, Seibel 13053, Semillon, Servant, Shiraz, Souzao, Sultana Crimson, Sylvaner, Tannat, Teroldico, Madeira Ink, Cao Red, Touriga, Traminer, Trebbiano Toscano, Trousseau, Valdepeñas, Viognier, Walschries-ling, White Riesling, and Zinfandel . Cultures of rhizomes which are useful in the invention include, without limitation, Vi tis rupestris Constantia, Vi tis rupestris St. George, Vitis california, Vitis girdiana, Vitis rotundifolia, Vi tis rotundifolia Carlos Richter 110 (Vitis berlandieri x rupestris), 101 -14 Millarder et de Grasset (Vi tis riparia x rupestris), Teleki 5C (Vitis berlandieri x riparia), 3309 Courderc (Vi tis riparia x rupestris), Riparia Gloire de Montpellier (Vitis riparia), 5BB Teleki (Selection Kober, Vitis berlandieri x riparia), S04 (Vi tis berlandieri x rupestris), 41B Millardet (Vi tis vinifera x berlandieri), and 039-16 (Vi tis 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), Harmony, Kober 5BB, LN33, Millardet &; from Grasset 41B, Millardet & from Grasset 420A, Millardet & from 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 rupestris Constantia, Vi tis california , and Vi tis girdiana. In general, the transfer and expression of transgenes in plant cells, including grape plants, are now routine practices for those skilled in the art, and have become important tools for carrying out studies of plant gene expression and for producing plant varieties improved agricultural or commercial interest. Transgenic Vine Regeneration 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. In a particular example, a construction of non-translatable sense v-CP grape leaf virus sequences (e.g., a sense translatable or non-translatable sense protein gene, or an antisense construct) (for example, example, a vine-CP virus leaf sequence sequence in the sense orientation having a non-ATG reading frame that includes a stop codon after the start codon) under the control of the CaMV 35S promoter and the terminator nopaline synthase and carrying a selectable marker (eg, resistance to kanamycin) is transformed into Agrobacterium. The transformation of a vine with a vector containing Agrobacterium is carried out as described by Scorza and Cordts. 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 transgenic 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 Northern blotting assays and nuclear exit assays (see, for example, Ausubel et al., Supra). Plants with positive RNA are analyzed for resistance to vine leaf virus infection using the methods described above. Transformed vines expressing a non-translatable sense vine virus sequence of the vine having resistance to fan leaf disease relative to controlling plants are taken to be 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 (9)

1. CLAIMS 1. A method for selecting a transgenic vine or transgenic vine component that has increased resistance to a leaf fan disease, said method comprising: (a) transforming a cell from the vine plant with a nucleic acid molecule of grape nepovirus coating protein or fragment thereof that is capable of being expressed in said plant cell; (b) regenerating a transgenic vine or transgenic vine component of the plant cell; and (c) selecting a transgenic vine or transgenic vine component that expresses, at a low level, said nucleic acid molecule or fragment thereof, wherein said low level expression increases the resistance of the transgenic vine or transgenic vine component. to said leaf fan disease. The method of claim 1, wherein said nucleic acid molecule or fragment thereof is encoded by a transgene integrated into a transgenic vine genome. 3. The method of claim 1, wherein said nucleic acid molecule or fragment thereof is expressed in a sense orientation. 4. The method of claim 3, wherein said nucleic acid molecule or fragment thereof is expressed as a non-translatable, sense mRNA molecule. 5. The method of claim 1, wherein said nucleic acid molecule or fragment thereof is expressed in an anti-sense orientation. 6. The method of claim 1, wherein said vine is a member of the genus Vitis. I. The method of claim 1, wherein said vine component is a somatic embryo. - ~~ 8. The method of claim 1, wherein said vine component is a shoot. The method of claim 1, wherein said vine component is a root material. The method of claim 1, wherein said grape nepovirus coating protein nucleic acid molecule is from a vine leaf fan virus. II. The method of claim 1, further comprising a nucleic acid molecule of grape nepovirus coating protein or fragment thereof having about 50% or more sequence identity with SEQ ID NO: 1. 12. A protein substantially pure protein comprising an amino acid sequence having at least 97% amino acid identity with the amino acid sequence of the "Geneva" isolated grape nepovirus coating protein of SEQ ID NO: 2. 13. The substantially pure protein of claim 12, wherein said protein comprises the amino acid sequence of the grape nepovirus coating protein of SEQ ID NO: 2. 14. The substantially pure protein of claim 12, wherein said protein has the amino acid sequence of the grape nepovirus coating protein of SEQ ID NO: 2 or a fragment thereof. 15. An isolated nucleic acid molecule, which encodes a recombinant protein comprising an amino acid sequence having at least 97% amino acid identity with the amino acid sequence of the "Geneva" isolated grape nepovirus coating protein of the SEQ ID NO: 2. The isolated nucleic acid molecule of claim 15, wherein said protein comprises the amino acid sequence of SEQ ID NO: 2. 17. The isolated nucleic acid molecule of claim 15, wherein said protein has the amino acid sequence of SEQ ID NO: 2 or a fragment thereof. 18. The isolated nucleic acid molecule of claim 15, wherein said nucleic acid molecule is operably linked to an expression control region. 19. A vector comprising an isolated nucleic acid molecule encoding a recombinant protein comprising an amino acid sequence having at least 97% amino acid identity with the amino acid sequence of the "Geneva" isolated grape nepovirus coating protein. of SEQ ID NO: 2. 20. The vector of claim 19, wherein said nucleic acid molecule is positioned for expression of a sense translatable RNA transcript. 21. The vector of claim 19, wherein said nucleic acid molecule is positioned for expression of a transcription of non-translatable sense RNA. 22. The vector of claim 19, wherein said nucleic acid molecule is positioned for expression of an anti-sense RNA sequence. 23. A cell transformed with the vector of claim 19. 24. A transgenic plant or transgenic plant component, comprising the vector of claim 19. 25. The transgenic plant or transgenic plant component of claim 24, wherein said Nucleic acid molecule is expressed in said plant as a sense translatable RNA transcript. 26. The transgenic plant or transgenic plant component of claim 24, wherein said nucleic acid molecule is expressed in said plant as a sense non-translational RNA transcript. 27. The transgenic plant or transgenic plant component of claim 24, wherein said nucleic acid molecule is expressed in said plant as an anti-sense RNA transcript.