Patent Grant
6858426
The present invention relates to grapevine leafroll virus (type 2) proteins, DNA molecules encoding these proteins, and their uses.
The world's most widely grown fruit crop, the grape (Vitis sp.), is cultivated on all continents except Antarctica. However, major grape production centers are in European countries (including Italy, Spain, and France), which constitute about 70% of the world grape production (Mullins et al., Biology of the Grapevine, Cambridge, U.K.:University Press (1992)). The United States, with 300,000 hectares of grapevines, is the eighth largest grape grower in the world. Although grapes have many uses, a major portion of grape production (˜80%) is used for wine production. Unlike cereal crops, most of the world's vineyards are planted with traditional grapevine cultivars, which have been perpetuated for centuries by vegetative propagation. Several important grapevine virus and virus-like diseases, such as grapevine leafroll, corky bark, and Rupestris stem pitting, are transmitted and spread through the use of infected vegetatively propagated materials. Thus, propagation of certified, virus-free materials is one of the most important disease control measures. Traditional breeding for disease resistance is difficult due to the highly heterozygous nature and outcrossing behavior of grapevines, and due to polygenic patterns of inheritance. Moreover, introduction of a new cultivar may be prohibited by custom or law. Recent biotechnology developments have made possible the introduction of special traits, such as disease resistance, into an established cultivar without altering its horticultural characteristics.
Many plant pathogens, such as fungi, bacteria, phytoplasmas, viruses, and nematodes can infect grapes, and the resultant diseases can cause substantial losses in production (Pearson et al., Compendium of Grape Diseases, American Phytopathological Society Press (1988)). Among these, viral diseases constitute a major hindrance to profitable growing of grapevines. About 34 viruses have been isolated and characterized from grapevines. The major virus diseases are grouped into: (1) the grapevine degeneration caused by the fanleaf nepovirus, other European nepoviruses, and American nepoviruses, (2) the leafroll complex, and (3) rugose wood complex (Martelli, ed., Graft Transmissible Diseases of Grapevines, Handbook for Detection and Diagnosis, FAO, UN, Rome, Italy (1993)).
Of the major virus diseases, the grapevine leafroll complex is the most widely distributed throughout the world. According to Goheen (“Grape Leafroll, ” in Frazier et al., eds., Virus Diseases of Small Fruits and Grapevines (A Handbook), University of California, Division of Agricultural Sciences, Berkeley, Calif., USA, pp. 209-212 (1970) (“Goheen (1970)”), grapevine leafroll-like disease was described as early as the 1850s in German and French literature. However, the vital nature of the disease was first demonstrated by Scheu (Scheu, “Die Rollkrankheit des Rebstockes (Leafroll of grapevine),” D. D. Weinbau 14:222-358 (1935) (“Scheu (1935)”)). In 1946, Harmon and Snyder (Harmon et al., “Investigations on the Occurrence, Transmission, Spread and Effect of ‘White’ Fruit Colour in the Emperor Grape, ” Proc. Am. Soc. Hort. Sci. 74:190-194 (1946)) determined the viral nature of White Emperor disease in California. It was later proven by Goheen et al. (Goheen et al., “Leafroll (White Emperor Disease) of Grapes in California, Phytopathology, 48:51-54(1958), (“Goheen (1958)”)) that both leafroll and “White Emperor” diseases were the same, and only the name leafroll was retained.
Leafroll is a serious viral disease of grapes and occurs wherever grapes are grown. This wide distribution of the disease has come about through the propagation of diseased vines. It affects almost all cultivated and rootstock varieties of Vitis. Although the disease is not lethal, it causes yield losses and reduction of sugar content. Scheu estimated in 1936 that 80 per cent of all grapevines planted in Germany were infected (Scheu, Mein Winzerbuch, Berlin:Reichsnahrstand-Verlags (1936). In many California wine grape vineyards, the incidence of leafroll (based on a survey of field symptoms conducted in 1959) agrees with Scheu's initial observation in German vineyards (Goheen et al., “Studies of Grape Leafroll in California,” Amer. J. Enol. Vitic., 10:78-84 (1959)). The current situation on leafroll disease does not seem to be any better (Goheen, “Diseases Caused by Viruses and Viruslike Agents,” The American Phytopathological Society, St. Paul, Minn.:APS press, 1:47-54 (1988) (“Goheen (1988)”). Goheen also estimated that the disease causes an annual loss of about 5-20 per cent of the total grape production (Goheen (1970) and Goheen (1988)). The amount of sugar in individual berries of infected vines is only about ½ to ⅔ that of berries from noninfected vines (Goheen (1958)).
Symptoms of leafroll disease vary considerably depending upon the cultivar, environment, and time of the year. On red or dark-colored fruit varieties, the typical downward rolling and interveinal reddening of basal, mature leaves is the most prevalent in autumn; but not in spring or early summer. On light-colored fruit varieties however, symptoms are less conspicuous, usually downward rolling accompanied by interveinal chlorosis. Moreover, many infected rootstock cultivars do not develop symptoms. In these cases, the disease is usually diagnosed with a woody indicator indexing assay using Vitis vivifera cv. Carbernet Franc (Goheen (1988)).
Ever since Scheu demonstrated that leafroll was graft transmissible, a virus etiology has been suspected (Scheu (1935)). Several virus particle types have been isolated from leafroll diseased vines. These include potyvirus-like (Tanne et al., “Purification and Characterization of a Virus Associated with the Grapevine Leafroll Disease,” Phytopathology, 67:442-447 (1977)), isometric virus-like (Castellano et al., “Virus-like Particles and Ultrastructural Modifications in the Phloem of Leafroll-affected Grapevines,” Vitis, 2:23-39 (1983) (Castellano (1983)”) and Namba et al., A Small Spherical Virus Associated with the Ajinashika Disease of Koshu Grapevine, Ann. Phytopathol. Soc. Japan, 45:-70-73 (1979)), and closterovirus-like (Namba, “Grapevine Leafroll Virus, a Possible Member of Closteroviruses, Ann. Phytopathol, Soc. Japan, 45.497-502 (1979)) particles. In recent years, however, long flexuous closteroviruses ranging from 1,400 to 2,200 nm have been most consistently associated with leafroll disease (
Through the use of monoclonal antibodies, however, the original GLRaV II described in Gugerli (1984) has been shown to be an apparent mixture of at least two components, IIa and IIb (Gugerli et al., “Grapevine Leafroll Associated Virus II Analyzed by Monoclonal Antibodies,” 11th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine, Montreux, Switzerland, pp. 23-24 (1993) (“Gugerli (1993)”)). Recent investigation with comparative serological assays (Boscia (1995)) demonstrated that the IIb component of cv. Chasselas 8/22 is the same as the GLRaV-2 isolate from France (Zimmermann (1990)) which also include the isolates of grapevine corky bark associated closteroviruses from Italy (GCBaV-BA) (Boscia (1995)) and from the United States (GCBaV-NY) (Namba et al., “Purification and Properties of Closterovirus-like Particles Associated with Grapevine Corky Bark Disease,” Phytopathology, 81:964-970 (1991) (“Namba (1991)”)). The IIa component of cv. Chasselas 8/22 was given the provisional name of grapevine leafroll associated virus 6 (GLRaV-6). Furthermore, the antiserum to the CA-5 isolate of GLRaV-2 produced by Boscia et al. (Boscia et al., “Characterization of Grape Leafroll Associated Closterovirus (GLRaV) Serotype II and Comparison with GLRaV Serotype III, “Phytopathology, 80:117 (1990)) was show to contain antibodies to both GLRaV-2 and GLRaV-1, with a prevalence of the latter (Boscia (1995)).
Virions of GLRaV-2 are flexuous, filamentous particles about 1,400-1,800 nm in length (Gugerli et al., “L'enroulement de la Vigne: Mise en Evidence de Particles Virales et Development d'une Methode Immuno-enzymatique Pour le Diagnostic Rapide (Grapevine Leafroll: Presence of Virus Particles and Development of an Immuno-enzyme Method for Diagnosis and Detection),” Rev. Suisse Viticult, Arboricult, Horticult. 16:299-304 (1984)). A double-stranded RNA (dsRNA) of about 15 kb was consistently isolated from GLRaV-2 infected tissues (Goszczynski et al., Detection of Two Strains of Grapevine Leafroll-Associated Virus 2,” Vitis 35:133-35 (1996)). The coat protein of GLRaV-2 is ca 22˜26 kDa (Zimmermann et al., Characterization and Serological Detection of Four Closterovirus-like Particles Associated with Leafroll Disease on Grapevine,” J. Phytopathology 130:205-18 (1990); Gugerli and Ramel, Extended abstracts: “Grapevine Leafroll Associated Virus II Analyzed by Monoclonal Antibodies,” 11th ICVG at Montreux, Switzerland, Gugerli, ed., Federal Agricultural Research Station of Changins, CH-1260 Nyon, Switzerland, p 23-24 (1993); Boscia et al., “Nomenclature of Grapevine Leafroll-Associated Putative Closteroviruses,” Vitis 34-171-75 (1995)), which is considerably smaller than other GLRaVs (35˜43 kDa) (Zee et al., “Cytopathology of Leafroll-Diseased Grapevines and the Purification and Serology of Associated Closterovirus Like Particles,” Phytopathology 77:1427-34 (1987); Hu et al., “Characterization of Closterovirus-Like Particles Associated with Grapevine Leafroll Disease,” J. of Phytopathology 128:1-14(1990); Ling et al., “The Coat Protein Gene of Grapevine Leafroll Associated Closterovirus-3Cloning, Nucleotide Sequencing and Expression in Transgenic Plants,” Arch. of Virology 142:1101-16(1997)). Although GLRaV-2 has been classified as a member of the genus Closterovirus based on particle morphology and cytopathology (Martelli, Circular of ICTV-Plant Virus Subcommittee Study Group on Closterolike Viruses” (1996)), its molecular and biochemical properties are not well characterized.
In the closterovirus group, several viruses have recently been sequences. The partial or complete genome sequences of beet yellows virus (BYV) (Agranovsky et al. “Nucleotide Sequence of the 3′-Terminal Half of Beet Yellows Closterovirus RNA Genome Unique Arrangement of Eight Virus Genes,” J. General Virology 72:15-24 (1991), Agranovsky et al., “Beet Yellows Closterovirus: Complete Genome Structure and Identification of a Papain-like Thiol Protease,” Virology 198:311-24 (1994)), beet yellow stunt virus (BYSV) (Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221:199-207 (1996)), citrus tristeza virus (CTV) (Pappu et al., “Nucleotide Sequence and Organization of Eight 3′ Open Reading Frames of the Citrus Tristeza Closterovirus Genome,” Virology 199:35-46 (1994); Karasev et al., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,” Virology 208:511-20 (1995)), lettuce infectious yellows virus (LIYV) (Klaassen et al., “Partial Characterization of the Lettuce Infectious Yellows Virus Genomic RNAs, Identification of the Coat Protein Gene and Comparison of its Amino Acid Sequence With Those of Other Filamentous RNA Plant Viruses,” J. General Virology 75:1525-33 (1994); Klaassen et al., “Genome Structure and Phylogenetic Analysis of Lettuce Infectious Yellows Virus, a Whitefly-Transmitted, Bipartite Closterovirus,” Virology 208:99-110 (1995)), little cherry virus (LChV) (Keim and Jelkmann, “Genome Analysis of the 3′-Terminal Part of the Little Cherry Disease Associated dsRNA Reveals a Monopartite Clostero-Like Virus,” Arch. Virology 141:1437-51 (1996); Jelkmann et al., “Complete Genome Structure and Phylogenetic Analysis of Little Cherry Virus, a Mealybug-Transmissible Closterovirus,” J. General Virology 78:2067-71 (1997)), and GLRaV-3 (Ling et al., “Nucleotide Sequence of the 3′ Terminal Two-Thirds of the Grapevine Leafroll Associated Virus-3-Genome Reveals a Typical Monopartite Closterovirus,” J. Gen. Virology 79(5):1289-1301 (1998)) revealed several common features of the closteroviruses, including the presence of HSP70 chaperone heat shock protein and a duplicate of the coat protein gene (Agranovsky “Principles of Molecular Organization, Expression, and Evolution of Closteroviruses: Over the Barriers,” Adv. in Virus Res. 47:119-218 (1996); Dolja et al. “Molecular Biology and Evolution of Closteroviruses: Sophisticated Build-up of Large RNA Genomes,” Annual Rev. Photopathology 32:261-85 (1994); Boyko et al., “Coat Protein Gene Duplication in a Filamentous RNA Virus of Plants,” Proc. Nat. Acad. Sci. USA 89:9156-60 (1992)). Characterization of the genome organization of GLRaVs would provide molecular information on the serologically distinct closteroviruses that cause similar leafroll symptoms in grapevine.
Several shorter closteroviruses (particle length 800 nm long) have also been isolated from grapevines. One of these, called grapevine virus A (“GVA”) has also been found associated, though inconsistently, with the leafroll disease (Agran et al., “Occurrence of Grapevine Virus A (GVA) and Other Closteroviruses in Tunisian Grapevines Affected by Leafroll Disease,” Vitis, 29:43-48 (1990), Conti, et al., “Closterovirus Associated with Leafroll and Stem Pitting in Grapevine,” Phytopathol. Mediterr., 24:110-113 (1985), and Conti et al., “A Closterovirus from a Stem-pitting-diseased Grapevine,” Phytopathology, 70:394-399 (1980)). The etiology of GVA is not really known; however, it appears to be more consistently associated with rugose wood serau lato (Rosciglione at al., “Maladies de l'enroulement et du bois strié de la vigne: analyse microscopique et sérologique Leafroll and Stem Pitting of Grapevine: Microscopical and Serological Analysis),” Rev. Suisse Vitic Arboric. Hortic., 18:207-211 (1986) (“Rosciglione (1986)”), and Zimmermann (1990)). Moreover, another short closterovirus (800 nm long) named grapevine virus B (“GVB”) has been isolated and characterized from corky bark-affected vines (Boscia et al., “Properties of a Filamentous Virus Isolated from Grapevines Affected by Corky Bark,” Arch. Virol., 130:109-120(1993) and Namba(1991)).
As suggested by Martelli I, leafroll symptoms may be induced by more than one virus or they may be simply a general plant physiological response to invasion by an array of phloem-inhabiting viruses. Evidence accumulated in the last 15 years strongly favors the idea that grapevine leafroll is induced by one (or a complex) of long closteroviruses (particle length 1,400 to 2,200 nm).
Grapevine leafroll is transmitted primarily by contaminated scions and rootstocks. However, under field conditions, several species of mealybugs have been shown to be the vector of leafroll (Engelbrecht et al., “Transmission of Grapevine Leafroll Disease and Associated Closteroviruses by the Vine Mealybug Planococcus-ficus,” Phytophylactica, 22:341-346 (1990), Rosciglione, et al., “Transmission of Grapevine Leafroll Disease and an Associated Closterovirus to Healthy Grapevine by the Mealybug Planococcus ficus,” (Abstract), Phytoparasitica, 17:63-63 (1989), and Tanne, “Evidence for the Transmission by Mealybugs to Healthy Grapevines of a Closter-like Particle Associated with Grapevine Leafroll Disease,” Phytoparasitica, 16:288 (1988)). Natural spread of leafroll by insect vectors is rapid in various parts of the world. In New Zealand, observations of three vineyards showed that the number of infected vines nearly doubled in a single year (Jordan et al., “Spread of Grapevine Leafroll and its Associated Virus in New Zealand Vineyards,” 11th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine, Montreux, Switzerland, pp. 113-114 (1993)). One vineyard became 90% infected 5 years after GLRaV-3 was first observed. Prevalence of leafroll worldwide may increase as chemical control of mealybugs becomes more difficult due to the unavailability of effective insecticides.
In view of the serious risk grapevine leafroll virus poses to vineyards and the absence of an effective treatment of it, the need to prevent this affliction continues to exist. The present invention is directed to overcoming this deficiency in the art.
The present invention relates to an isolated protein or polypeptide, corresponding to a protein or polypeptide of a grapevine leafroll virus (type 2). The encoding RNA and DNA molecules, in either isolated form or incorporated in an expression system, a host cell, a transgenic Vitis or citrus scion or rootstock cultivar, or a transgenic Nicotiana plant or beet plant are also disclosed.
Another aspect of the present invention relates to a method of imparting grapevine leafroll virus (type 2) resistance to Vitis scion or rootstock cultivars or Nicotiana plants by transforming them with a DNA molecule encoding the protein or polypeptide corresponding to a protein or polypeptide of a grapevine leafroll virus (type 2). Other aspects of the present invention relate to a method of imparting beet yellows virus resistance to beet plants and a method of imparting tristeza virus resistance to citrus scion or rootstock cultivars, both by transforming the plants or cultivars with a DNA molecule encoding the protein or polypeptide corresponding to a protein or polypeptide of a grapevine leafroll virus (type 2).
The present invention also relates to an antibody or binding portion thereof or probe which recognizes the protein or polypeptide.
Grapevine leafroll virus resistant transgenic variants of the current commercial grape cultivars and rootstocks allows for more complete control of the virus, while retaining the varietal characteristics of specific cultivars. Furthermore, these variants permit control of GLRaV-2 transmitted either by contaminated scions or rootstocks or by a presently uncharacterized insect vector. With respect to the latter mode of transmission, the present invention circumvents increased restriction of pesticide use which has made chemical control of insect infestation increasingly difficult. In this manner, the interests of the environment and the economics of grape cultivation and wine making are all furthered by the present invention.
The present invention relates to isolated DNA molecules encoding for the proteins or polypeptides of a grapevine leafroll virus (type 2). A substantial portion of the grapevine leafroll virus (type-2) (“GLRaV-2”) genome has been sequenced. Within the genome are a plurality of open reading frames (“ORFs”) and a 3′ untranscribed region (“UTR”), each containing DNA molecules in accordance with the present invention. The DNA molecule which constitutes a substantial portion of the GLRaV-2 genome comprises the nucleotide sequence corresponding to SEQ. ID. No. 1 as follows:
Another DNA molecule of the present invention (GLRaV-2 ORF1a) includes nucleotides 4-7923 of SEQ. ID. No. 1 and is believed to code for a large, grapevine leafroll virus polyprotein containing the conserved domains characteristic of two papain-like proteases, a methyltransferase, and a helicase. This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 2 as follows:
The large polyprotein (papain-like proteases, methyltransferase, and helicase) has an amino acid sequence corresponding to SEQ. ID. No. 3 as follows:
and has a molecular weight of about 290 to 300 kDa, preferably 294 kDa.
Another such DNA molecule (GLRaV-2 ORF1b) includes nucleotides 7922-9301 of SEQ. ID. No. 1 and codes for a grapevine leafroll virus RNA-dependent RNA polymerase (RdRP). This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 4 as follows:
The RNA-dependent RNA polymerase has an amino acid sequence corresponding to SEQ. ID. No. 5 as follows:
and a molecular weight from about 50 to about 54 kDa, preferably about 52 kDa.
Another such DNA molecule (GLRaV-2 ORF2) includes nucleotides 9365-9535 of SEQ. ID. No. 1 and codes for a small, grapevine leafroll virus hydrophobic protein or polypeptide. This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 6 as follows:
The small hydrophobic protein or polypeptide has an amino acid sequence corresponding to SEQ. ID. No. 7 as follows:
and a molecular weight from about 5 to about 7 kDa, preferably about 6 kDa.
Another such DNA molecule (GLRaV-2 ORF3) includes nucleotides 9551-11350 of SEQ. ID. No. 1 and encodes for a grapevine leafroll virus heat shock 70 protein. This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 8 as follows:
The heat shock 70 protein is believed to function as a chaperone protein and has an amino acid sequence corresponding to SEQ. ID. No. 9 as follows:
and a molecular weight from about 63 to about 67 kDa, preferably about 65 kDa.
Another such DNA molecule (GLRaV-2 ORF4) includes nucleotides 11277-12932 of SEQ. ID. No. 1 and codes for a putative grapevine leafroll virus heat shock 90 protein. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 10 as follows:
The heat shock 90 protein has an amino acid sequence corresponding to SEQ. ID. No. 11 as follows:
and a molecular weight from about 61 to about 65 kDa, preferably about 63 kDa.
Yet another DNA molecule of the present invention (GLRaV-2 ORF5) includes nucleotides 12844-13515 of SEQ. ID. No. 1 and codes for a diverged coat protein. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 12 as follows:
The diverged coat protein has an amino acid sequence corresponding to SEQ. ID. No. 13 as follows:
and a molecular weight from about 23 to about 27 kDa, preferably about 25 kDa.
Another such DNA molecule (GLRaV-2 ORF6) includes nucleotides 13584-14180 of SEQ. ID. No. 1 and codes for a grapevine leafroll virus coat protein. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No.14 as follows:
The coat protein has an amino acid sequence corresponding to SEQ. ID. No. 15 as follows:
and a molecular weight from about 20 to about 24 kDa, preferably about 22 kDa.
Another such DNA molecule (GLRaV-2 ORF7) includes nucleotides 14180-14665 of SEQ. ID. No. 1 and codes for a second undefined grapevine leafroll virus protein or polypeptide. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 16 as follows:
The second undefined grapevine leafroll virus protein or polypeptide has a deduced amino acid sequence corresponding to SEQ. ID. No. 17 as follows:
and a molecular weight from about 17 to about 21 kDa, preferably about 19 kDa.
Yet another such DNA molecule (GLRaV-2 ORF8) includes nucleotides 14667-15284 of SEQ. ID. No. 1 and codes for a third undefined grapevine leafroll virus protein or polypeptide. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 18 as follows:
The third undefined protein or polypeptide has a deduced amino acid sequence corresponding to SEQ. ED. No. 19 as follows:
and a molecular weight from about 22 to about 26 kDa, preferably about 24 kDa.
Another DNA molecule of the present invention (GLRaV-2 3′ UTR) includes nucleotides 15285-15500 of SEQ. ID. No. 1 and comprises a nucleotide sequence corresponding to SEQ. ID. No. 23 as follows:
Also encompassed by the present invention are fragments of the DNA molecules of the present invention. Suitable fragments capable of imparting grapevine leafroll resistance to grape plants are constructed by using appropriate restriction sites, revealed by inspection of the DNA molecule's sequence, to: (i) insert an interposon (Felley et al., “Interposon Mutagenesis of Soil and Water Bacteria: a Family of DNA Fragments Designed for in vitro Insertion Mutagenesis of Gram-negative Bacteria,” Gene, 52:147-15 (1987), which is hereby incorporated by reference) such that truncated forms of the grapevine leafroll virus coat polypeptide or protein, that lack various amounts of the C-terminus, can be produced or (ii) delete various internal portions of the protein. Alternatively, the sequence can be used to amplify any portion of the coding region, such that it can be cloned into a vector supplying both transcription and translation start signals.
Suitable DNA molecules are those that hybridize to a DNA molecule comprising a nucleotide sequence of at least 15 continuous bases of SEQ. ID. No. 1 under stringent conditions characterized by a hybridization buffer comprising 0.9M sodium citrate (“SSC”) buffer at a temperature of 37° C. and remaining bound when subject to washing with SSC buffer at 37° C.; and preferably in a hybridization buffer comprising 20% formamide in 0.9M saline/0.9M SSC buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2×SSC buffer at 42° C.
Variants may also (or alternatively) be modified by, for example, the deletion or addition of nucleotides that have minimal influence on the properties, secondary structure and hydropathic nature of the encoded polypeptide. For example, the nucleotides encoding a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The nucleotide sequence may also be altered so that the encoded polypeptide is conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide.
The protein or polypeptide of the present invention is preferably produced in purified form preferably at least about 80%, more preferably 90%, pure, )by conventional techniques. Typically, the protein or polypeptide of the present invention is isolated by lysing and sonication. After washing, the lysate pellet is resuspended in buffer containing, Tris-HCl. During dialysis, a precipitate forms from this protein solution. The solution is centrifuged, and the pellet is washed and resuspended in the buffer containing, Tris-HCl. Proteins are resolved by electrophoresis through an SDS 12% polyacrylamide gel.
The DNA molecule encoding the grapevine leafroll virus (type 2) protein or polypeptide of the present invention can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture.
Recombinant genes may also be introduced into viruses, such as vaccinia virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK+/− or KS+/− (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla. Calif., which is hereby incorporated by reference), pQE, pIH21, pGEX, pET series (see Studier et. al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technology, Vol. 185 (1990), which is hereby incorporated by reference), and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1982), which is hereby incorporated by reference.
A variety of host vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms, such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria or transformed via particle bombardment (i.e. biolistics). The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
Different genetic signals sand processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (“mRNA”) translation).
Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promoters differ from those of procaryotic promoters. Furthermore, eucaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eucaryotic cells.
Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes tie amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology, 68-473 (1979), which is hereby incorporated by reference.
Promoters vary in their “strength” (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters maybe used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter other E. coli promoter produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA.
For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthiobeta-D-galactoside). A variety of other operons, such trp, pro, etc., are under different controls.
Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
Once the isolated DNA molecules encoding the various grapevine leafroll virus (type 2) proteins or polypeptides, as described above, have been cloned into an expression system, they are ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.
The present invention also relates to RNA molecules which encode the various grapevine leafroll virus (type 2) proteins or polypeptides described above. The transcripts can be synthesized using the host cells of the present invention by any of the conventional techniques. The mRNA can be translated either in vitro or in vivo. Cell-free systems typically include wheat-germ or reticulocyte extracts. In vivo translation can be effected, for example, by microinjection into frog oocytes.
One aspect of the present invention involves using one or more of the above DNA molecules encoding the various proteins or polypeptides of a grapevine leafroll virus (type 2) to transform grape plants in order to impart grapevine leafroll resistance to the plants. The mechanism by which resistance is imparted is not known. In one hypothetical mechanism, the transformed plant can express a protein or polypeptide of grapevine leafroll virus (type 2), and, when the transformed plant is inoculated by a grapevine leafroll virus, such as GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-4, GLRaV-5, or GLRaV-6, or combinations of these, the expressed protein or polypeptide prevents translation of the viral DNA.
In this aspect of the present invention the subject DNA molecule incorporated in the plant can be constitutively expressed. Alternatively, expression can be regulated by a promoter which is activated by the presence of grapevine leafroll virus. Suitable promoters for these purposes include those from genes expressed in response to grapevine leafroll virus infiltration.
The isolated DNA molecules of the present invention can be utilized to impart grapevine leafroll virus resistance for a wide variety of grapevine plants. The DNA molecules are particularly well suited to imparting resistance to Vitis scion or rootstock cultivars. Scion cultivars which can be protected include those commonly referred to as Table or Raisin Grapes, 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, Fiesta, Flame seedless, Flame Tokay, Gasconade, Gold, Himrod, Hunisa, Hussiene, Isabella, Italia, July Muscat, Khandahar, Katta, Kourgane, Kishmishi, Loose 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 seedless, land Thomuscat. They also include those used in wine production, such as Aleatico, Alicante Bouschet, Aligote, Alvarelhao, Aramon, Baco blanc (22A), Burger, Cabernet franc, Cabernet, Sauvignon, Calzin, Carignane, Charbono, Chardonnay, Chasselas dore, Chenin blanc, Clairette blanche, Early Burgundy, Emerald Riesling, Feher Szagos, Fernao Pires, Flora, French Colombard, Fresia, Furmint, Gamay, Gewurztraminer, Grand noir, Gray Riesling, Green Hungarian, Green Veltliner, Grenache, Grillo, Helena, Inzolia, Lalgrein, 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, Rubired, Ruby Cabernet, Saint-Emilion, Saint Macaire, Salvador, Sangiovese, Sauvignon blanc, Sauvignon gris, Sauvignon vert, Scarlet, Seibel 5279, Seibel 9110, Seibel 13053, Semillon, Servant, Shiraz, Souzao, Sultana Crimson, Sylvaner, Tannat, Teroldico, Tinta Madeira, Tinto cao, Touriga, Traminer, Trebbiano Toscano, Trousseau, Valdepen as, Viognier, Walschriesling, White Riesling, and Zinfandel. Rootstock cultivars which can be protected include Couderc 1202, Couderc 1613, Couderc 1616, Couderc 3309, Dog Ridge, Foex 33 EM, Freedom, Ganzin 1 (A×R #1), Harmony, Kober 5BB, LN33, Millardet & de Grasset 41 B, Millardet & de Grasset 420A, Millardet & de Grasset 101-14, Oppenheim 4 (SO4), Paulsen 775, Paulsen 1045, Paulsen 1103, Richter 99, Richter 110, Riparia Gloire, Ruggeri 225, Saint-George, Salt Creek, Teleki 5A, Vitis rupestris Constantia, Vitis california, and Vitis girdiana.
There exists an extensive similarity in the hsp70-related sequence regions of GLRaV-2 and other closteroviruses, such as tristeza virus and beet yellows virus. Consequently, the GLRaV-2 hsp70-related gene can also be used to produce transgenic plants or cultivars other than grape, such as citrus or sugar beet, which are resistant to closteroviruses other than grapevine leafroll, such as tristeza virus and beet yellows virus.
Suitable citrus cultivars include lemon, lime, orange, grapefruit, pineapple, tangerine, and the like, such as Joppa, Maltaise Ovale, Parson (Parson Brown), Pera, Pineapple, Queen, Shamouti, Valencia, Tenerife, Imperial Doblefina, Washington Sanguine, Moro, Sanguinello Moscato, Spanish Sanguinelli, Tarocco, Atwood, Australian, Bahia, Baiana, Cram, Dalmau, Eddy, Fisher, Frost Washington, Gillette, LengNavelina, Washington, Satsuma Mandarin, Dancy, Robinson, Ponkan, Duncan, Marsh, Pink Marsh, Ruby Red, Red Seedless, Smooth Seville, Orlando Tangelo, Eureka, Lisbon, Meyer Lemon, Rough Lemon, Sour Orange, Persian Lime, West Indian Lime, Bearss, Sweet Lime, Troyer Citrange, and Citrus Trifoliata. Each of these citrus cultivars is suitable for producing transgenic citrus plants resistant to tristeza virus.
The economically important species of sugar beet is Beta vulgaris L., which has four important cultivar types: sugar beet, table beet, fodder beet, and Swiss chard. Each of these beet cultivars is suitable for producing transgenic beet plants resistant to beet yellows virus, as described above.
Because GLRaV-2 has been known to infect tobacco plants (e.g., Nicotiana benthamiana), it is also desirable to produce transgenic tobacco plants which are resistant to grapevine leafroll viruses, such as GLRaV-2.
Plant tissue suitable for transformation include leaf tissue, root tissue, meristems, zygotic and somatic embryos, and anthers. It is particularly preferred to utilize embryos obtained from anther cultures.
The expression system of the present invention can be used to transform virtually any plant tissue under suitable conditions. Tissue cells transformed in accordance with the present invention can be grown in vitro in a suitable medium to impart grapevine leafroll virus resistance. Transformed cells can be regenerated into whole plants such that the protein or polypeptide imparts resistance to grapevine leafroll virus in the intact transgenic plants. In either case, the plant cells transformed with the recombinant DNA expression system of the present invention are grown and caused to express that DNA molecule to produce one of the above-described grapevine leafroll virus proteins or polypeptides and, thus, to impart grapevine leafroll virus resistance.
In producing transgenic plants, the DNA construct in a vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA. Crossway, Mol. Gen. Genetics, 202:179-85 (1985), which is hereby incorporated by reference. The genetic material may also be transferred into the plant cell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982), which is hereby incorporated by reference.
One technique of transforming plants with the DNA molecules in accordance with the present invention is by contacting the tissue of such plants with an inoculum of a bacteria transformed with a vector comprising a gene in accordance with the present invention which imparts grapevine leafroll resistance. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28° C.
Bacteria from the genus Agrobacterium can be utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacterium tumefaciens (e.g., strains C58, LBA4404, or EHA105) is particularly useful due to its well-known ability to transform plants.
Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science, 237:1176-83 (1987), which is hereby incorporated by reference.
After transformation, the transformed plant cells must be regenerated.
Plant regeneration from cultured protoplasts is described in Evans eat al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III (1986), which are hereby incorporated by reference.
It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugarcane, sugar beets, cotton, fruit trees, and legumes.
Means for regeneration vary from species to species of plants, but generally suspension of transformed protoplasts or a petri plate containing explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
Once transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure so that the DNA construct is present in the resulting plants. Alternatively, transgenic seeds are recovered from the transgenic plants. These seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants.
Another approach to transforming plant cells with a gene which imparts resistance to pathogens is particle bombardment (also known as biolistic transformation) of the host cell. This can be accomplished in one of several ways. The first involves propelling inert or biologically active particles at cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., and in Emerschad et al., “Somatic Embryogenesis and Plant Development from Immature Zygotic Embryos of Seedless Grapes (Vitis vinifera),” Plant Cell Reports, 14:6-12 (1995) (“Emerschad (1995)”), which are hereby incorporated by reference. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant, cells.
Once a grape plant tissue, citrus plant tissue, beet plant tissue, or tobacco plant tissue is transformed in accordance with the present invention, the transformed tissue is regenerated to form a transgenic plant. Generally, regeneration is accomplished by culturing transformed tissue on medium containing the appropriate growth regulators and nutrients to allow for the initiation of shoot meristems. Appropriate antibiotics are added to the regeneration medium to inhibit the growth of Agrobacterium and to select for the development of transformed cells. Following shoot initiation, shoots are allowed to develop tissue culture and are screened for marker gene activity.
The DNA molecules of the present invention can be made capable of transcription to a messenger RNA, which, although encoding for a grapevine leafroll virus (type 2) protein or polypeptide, does not translate to the protein. This is known as RNA-mediated resistance. When a Vitis scion or rootstock cultivar, or a citrus, beet, or tobacco cultivar, is transformed with such a DNA molecule, the DNA molecule can be transcribed under conditions effective to maintain the messenger RNA in the plant cell at low level density readings. Density readings of between 15 and 50 using a Hewlet ScanJet and Image Analysis Program are preferred.
A portion of one or more DNA molecules of the present invention as well as other DNA molecules can be used in a transgenic grape plant, citrus plant, beet plant, or tobacco plant in accordance with U.S. patent application Ser. No. 09/025,635, which is hereby incorporated herein by reference.
The grapevine leafroll virus (type 2) protein or polypeptide of the present invention can also be used to raise antibodies or binding portions thereof or probes. The antibodies can be monoclonal or polyclonal.
Monoclonal antibody production may be effected by techniques which are well-known in the art. Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a mammal (e.g., mouse) which has been previously immunized with the antigen of interest either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an-immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, Nature, 256:495 (1975), which is hereby incorporated by reference.
Mammalian lymphocytes are immunized by in vivo immunization of the animal (e.g., a mouse) with the protein or polypeptide of the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Following the last antigen boost, the animals are sacrificed and spleen cells removed.
Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol (“PEG”) or other fusing agents. (See Milstein and Kohler, Eur. J. Immunol., 6:511 (1976), which is hereby incorporated by reference.) This immortal cell line, which is preferably murine, but may also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capability. Many such cell lines are known,to those skilled in the art, and others are regularly described.
Procedures for raising polyclonal antibodies are also well known. Typically, such antibodies can be raised by administering the protein or polypeptide of the present invention subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The antigens can be injected at a total volume of 100 μl per site at six different sites. Each injected material will contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Ultimately, the rabbits are euthenized with pentobarbital 150 mg/Kg IV. This and other procedures for raising polyclonal antibodies are disclosed in Harlow et. al., editors, Antibodies: A Laboratory Manual (1988), which is hereby incorporated by reference.
In addition to utilizing whole antibodies, binding portions of such antibodies can be used. Such binding portions include Fab fragments, F(ab′)2 fragments, and Fv fragments. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in Goding, Monoclonal Antibodies: Principles and Practice, New York: Academic Press, pp. 98-118 (1983), which is hereby incorporated by reference.
The present invention also relates to probes found either in nature or prepared synthetically by recombinant DNA procedures or other biological procedures. Suitable probes are molecules which bind to grapevine leafroll (type 2) viral antigens identified by the monoclonal antibodies of the present invention. Such probes can be, for example, proteins, peptides, lectins, or nucleic acid probes.
The antibodies or binding portions thereof or probes can be administered to grapevine leafroll virus infected scion cultivars or rootstock cultivars. Alternatively, at least the binding portions of these antibodies can be sequenced, and the encoding DNA synthesized. The encoding DNA molecule can be used to transform plants together with a promoter which causes expression of the encoded antibody when the plant is infected by grapevine leafroll virus. In either case, the antibody or binding portion thereof or probe will bind to the virus and help prevent the usual leafroll response.
Antibodies raised against the GLRaV-2 proteins or polypeptides of the present invention or binding portions of these antibodies can be utilized in a method for detection of grapevine leafroll virus in a sample of tissue, such as tissue (e.g., scion or rootstock) from a grape plant or tobacco plant. Antibodies or binding portions thereof suitable for use in the detection method include those raised against a helicase, a methyltransferase, a papain-like protease, an RNA-dependent RNA polymerase, a heat shock 70 protein, a heat shock 90 protein, a coat protein, a diverged coat protein, or other proteins or polypeptides in accordance with the present invention. Any reaction of the sample with the antibody is detected using an assay system which indicates the presence of grapevine leafroll virus in the sample. A variety of assay systems can be employed, such as enzyme-linked immunosorbent assays, radioimmunoassays, gel diffusion precipitin reaction assays, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays, or immunoelectrophoresis assays.
Alternatively, grapevine leafroll virus can be detected in such a sample using a nucleotide sequence of the DNA molecule, or a fragment thereof, encoding for a protein or polypeptide of the present invention. The nucleotide sequence is provided as a probe in a nucleic acid hybridization assay or a gene amplification detection procedure (e.g., using a polymerase chain reaction procedure). The nucleic acid probes of the present invention may be used in any nucleic acid hybridization assay system known in the art, including, but not limited to, Southern blots (Southern, E. M., “Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis,” J. Mol. Biol., 98:503-17 (1975), which is hereby incorporated by reference), Northern blots (Thomas, P. S., “Hybridization of Denatured RNA and Small DNA Fragments Transferred to Nitrocellulose,” Proc. Nat'l Acad. Sci. USA, 77:5201-05 (1980), which is hereby incorporated by reference), and Colony blots (Grunstein, M., et al., “Colony Hybridization: A Method for the Isolation of Cloned cDNAs that Contain a Specific Gene,” Proc. Nat'l Acad. Sci. USA, 72:3961-65 (1975), which is hereby incorporated by reference). Alternatively, the probes can be used in a gene amplification detection procedure (e.g., a polymerase chain reaction). Erlich, H. A., et. al., “Recent Advances in the Polymerase Chain Reaction,” Science 252:1643-51 (1991), which is hereby incorporated by reference. Any reaction with the probe is detected so that the presence of a grapevine leafroll virus in the sample is indicated. Such detection is facilitated by providing the probe of the present invention with a label. Suitable labels include a radioactive compound, a fluorescent compound, a chemiluminescent compound, an enzymatic compound, or other equivalent nucleic acid labels.
Depending upon the desired scope of detection, it is possible to utilize probes having nucleotide sequences that correspond with conserved or variable regions of the ORF or UTR. For example, to distinguish a grapevine leafroll virus from other related viruses (e.g., other closteroviruses), it is desirable to use probes which contain nucleotide sequences that correspond to sequences more highly conserved among all grapevine leafroll viruses. Also, to distinguish between different grapevine leafroll viruses (i.e., GLRaV-2 from GLRaV-1, GLRaV-3, GLRaV-4, GLRaV-5, and GLRaV-6), it is desirable to utilize probes containing nucleotide sequences that correspond to sequences less highly conserve among the different grapevine leafroll viruses.
Nucleic acid (DNA or RNA) probes of the present invention will hybridize to complementary GLRaV-2 nucleic acid under stringent conditions. Generally, stringent conditions are selected to be about 50° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The Tm is dependent upon the solution conditions and the base composition of the probe, and may be calculated using the following equation:
Nonspecific binding may also be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with, RNase. Wash conditions are typically performed at or below stringency. Generally, suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected.
The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.
Specificity of the selected clones was confirmed by Northern hybridization. Northern hybridization was performed after electrophoresis of the dsRNA of GLRaV-2 in 1% agarose non-denaturing condition gel. The agarose gel was denatured by soaking in 50 mM NaOH containing 0.4 M NaCl for 30 min, and then neutralized with 0.1 M Tris-HCl (PH7.5) containing 0.5 M NaCl for another 30 min. RNA was sandwich blotted overnight onto Genescreen™ plus membrane (Dupont NEN Research Product) in 10×SSC buffer and hybridized as described by the manufacturer's instructions (DuPont, NEN).
DNA inserts were sequenced in pBluescript SK+ by using T3 and T7 universal primers for the terminal region sequence and additional oligonucleotide primers designed according to the known sequence for the internal region sequence. Purification of plasmid DNA was performed by a modified mini alkaline-lysis/PEG precipitation procedure described by the manufacturer (Applied Biosystems, Inc.). Nucleotide sequencing was performed on both strands of cDNA by using ABI TaqDyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc.). Automatic sequencing was performed on an ABI373 Automated Sequencer (Applied Biosystems, Inc.) at Cornell University, Geneva, N.Y.
The nucleotide sequences of GLRaV-2 were assembled and analyzed with the programs of EditSeq and SeqMan, respectively, of DNASTAR package (Madison, Wis.). Amino acid sequences deduced from nucleotide sequences and its encoding open reading frames were conducted using the MapDraw program. Multiple alignments of amino acid sequences, identification of consensus amino acid sequences, and generation of phylogenetic trees were performed using the Clustal method in the MegAlign program. The nucleotide and amino acid sequences of other closteroviruses were obtained with the Entrez Program; and sequence comparisons with nonredundant databases were searched with the Blast Program from the National Center for Biotechnology Information.
Several vines of GLRaV-2 infected Vitis vinifera cv Pinot Noir that originated from a central New York vineyard served as the source for dsRNA isolation and cDNA cloning. dsRNA was extracted from phloem tissue of infected grapevines according to the method described by Hu et al., “Characterization of Closterovirus-Like Particles Associated with Grapevine Leafroll Disease,” J. Phytopathology 128:1-14 (1990), which is hereby incorporated by reference. Purification of the high molecular weight dsRNA (ca 15 kb) was carried out by electrophoretic separation of the total dsRNA on a 0.7% low melting point agarose gel and extraction by phenol/chloroform following the method described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989), which is hereby incorporated by reference. Concentration of dsRNA was estimated with UV fluorescent density of an ethidium bromide stained dsRNA band in comparison with a known concentration of DNA marker.
cDNA synthesis was performed following the method initially described by Jelkmann et al., “Cloning of Four Plant Viruses From Small Quantities of Double-Stranded RNA,” Phytopathology 79:1250-53 (1989) and modified by Ling et al., “The Coat Protein Gene of Grapevine Leafroll Associated Closterovirus-3: Cloning, Nucleotide Sequencing and Expression in Transgenic Plants,” Arch. Virology 142:1101-16 (1997), both of which are hereby incorporated by reference. About 100 ng of high molecular weight dsRNA purified from low melting agarose gel was denatured in 20 mM methylmercuric hydroxide and incubated at room temperature for 10 min with 350 ng of random primers. First strand cDNA was synthesized by using avian myeloblastosis virus (AMV) reverse transcriptase. Second strand cDNA was obtained by using RNase H and E.coli DNA polymerase I. Double-stranded cDNA was blunt ended with T4 DNA polymerase and ligated with EcoRI adapters. The cDNA, which had EcoRI adapters at the ends, was activated by kinase reaction and ligated into Lambda ZAPII/EcoRI prepared arms following the manufacturer's instruction (Stratagene). The recombinant DNA was then packaged in vitro to Gigapack® II packaging extract (Stratagene). The packaged phage particles were amplified and titered according to the manufacturer's instruction.
Two kinds of probes were used to identify GLRaV-2 specific clones from the library. One type was prepared from the synthesized cDNA that was amplified by, PCR after ligation to the specific EcoRI Uni-Amp™ adapters (Clontech); and the other type was DNA inserts or PCR products from already sequenced clones. Clones from the cDNA library were selected by colony-lifting hybridization onto the colony/plaque Screen membrane (NEN Research Product) with the probe described above. The probe was prepared by labeling with 32P [α-dATP] using Klenow fragment of E. coli DNA polymerase I. Prehybridization, hybridization, and washing steps were carried out at 65° C. according to the manufacturer's instruction (Dupont, NEN Research Product). Selected plaques were converted to recombinant pBluescript by in vivo excision method according to the manufacturer's instruction (Stratagene).
To obtain clones representing the extreme 3′-terminus of GLRaV-2, dsRNA was polyadenylated by yeast poly(A) polymerase. Using poly(A)-tailed dsRNA as template, cDNA was amplified by RT-PCR with oligo(dT)18 and a specific primer, CP-1/T7R, which is derived from the clone CP-1 and has a nucleotide sequence according to SEQ. ID. No. 20 as follows:
As shown in
Two kinds of probes were used for screening the cDNA library. The initial clones were identified by hybridization with Uni-Amp™ PCR-amplified cDNA as probes. The specificity of these clones (e.g., TC-1) ranging from 200 to 1,800 bp in size was confirmed by Northern hybridization to dsRNA of GLRaV-2 as shown in FIG. 1B. Additionally, over 40 different clones ranging form 800 to 7,500 bp in size were identified following hybridization with the probes generated from GLRaV-2 specific cDNA clones or from PCR products. Over 40 clones were then sequenced on the both strands (FIG. 2).
To determine that ORF6 was the coat protein gene of GLRaV-2, the complete ORF6 DNA molecule was subcloned from a PCR product and inserted into the fusion protein expression vector pMAL-C2 (New England Biolabs, Inc.). The specific primers used for the PCR reaction were CP-96F and CP-96R, in which an EcoRI or BamHI site was included to facilitate cloning. CP-96F was designed to include the start codon of the CP and comprises a nucleotide sequence according to SEQ. ID. NO. 21 as follows:
A total of 15,500 bp of the RNA genome of GLRaV-2 was sequenced and deposited in GenBank (accession number AF039204). About 85% of the total RNA genome was revealed from at least two different clones. The sequence in the coat protein gene region was determined and confirmed from several different overlapping clones. The genome organization of GLRaV-2, shown in
ORF1a and ORF1b: Analysis of the amino acid sequence of the N-terminal portion of GLRaV-2 ORF1a encoded product revealed two putative papain-like protease domains, which showed significant similarity to the papain-like leader protease of BYV (Agranovsky et al., “Beet Yellows Closterovirus: Complete Genome Structure and Identification of a Papain-like Thiol Protease,” Virology 198:311-24 (1994), which is hereby incorporated by reference). Thus, it allowed prediction of the catalytic cysteine and histidine residues for the putative GLRaV-2 protease. Upon alignment of the sequence of the papain-like protease of BYV with that of GLRaV-2, the cleavage site at residues Gly-Gly (amino acid 588-589) of BYV aligned with the corresponding alanine-glycine (Ala-Gly) and Gly-Gly dipeptide of GLRaV-2 (FIG. 3A). Cleavage at this site would result in a leader protein and a 234 kDa (2090 amino acid) C-terminal fragment consisting of MT and HEL domains. However, the region upstream of the papain-like protease domain in GLRaV-2 did not show similarity to the corresponding region of BYV. In addition, variability in the residues located at the scissible bond (Gly in the BYV and Ala in the GLRaV-2) was present. Similar variability of the cleavage site residue in the P-PRO domain has been described ink LChV (Jelkmann et al., “Complete Genome Structure and Phylogenetic Analysis of Little Cherry Virus, a Mealybug-Transmissible Closterovirus. J. General Virology 78:2067-71 (1997), which is hereby incorporated by reference).
Database searching with the deduced amino acid sequence of the ORF1a/1b encoded protein revealed a significant similarity to the MT, HEL and RdRP domains of the other closteroviruses. The region downstream of the P-PRO cleavage site showed a significant similarity (57.4% identity in a 266-residues alignment) to the putative methyltransferase domain of BYV and contained all the conserved motifs typical of positive-strand RNA viral type I MTs (FIG. 3B). The C-terminal portion of the ORF1a was identified as a helicase domain, the sequence of which showed a high similarity (57.1% identity in a 315-residues alignment) to the helicase domain of BYV and contained the seven conserved motifs characteristic of the Superfamily I helicase of positive-strand RNA viruses (
ORF1b encoded a 460 amino acid polypeptide with a molecular mass of 52,486 Da, counting from the frameshifting site. Database searching with the RdRP showed a significant similarity to the RdRP domains of positive strand RNA viruses. Comparison of the RdRP domains of GLRaV-2 and BYV showed the presence of the eight conserved motifs of RdRP (FIG. 3D).
As shown in
In closteroviruses, a +1 ribosomal frameshift mechanism has been suggested to be involved in the expression of ORF1b as a large fusion protein with ORF1a (Agranovsky et al., “Beet Yellows Closterovirus: Complete Genome Structure and Identification of a Papain-like Thiol Protease,” Virology 198:311-24 (1994); Karasev et al., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,” Virology 208:511-20 (1995); Klaassen et al., “Genome Structure and Phylogenetic Analysis of Lettuce Infectious Yellows Virus, a Whitefly-Transmitted, Bipartite Closterovirus,” Virology 208:99-110 (1995); Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221:199-207 (1996); Jelkmann et al., “Complete Genome Structure and Phylogenetic Analysis of Little Cherry Virus, a Mealybug-Transmissible Closterovirus,” J. General Virology 78:2067-71 (1997), all of which are hereby incorporated by reference). In the overlapping ORF1a/1b region of BYV, the slippery sequence of GGGUUUA and two hairpins structure (stem-loop and pseudoknot) ate believed to result in a +1 frameshift (Agranovsky et al., “Beet Yellows Closterovirus: Complete Genome Structure and Identification of a Papain-like Thiol Protease,” Virology 198:311-24 (1994), which is hereby incorporated by reference). None of these features are conserved in CTV and BYSV (Karasev et al., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,” Virology 208:511-20 (1995); Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221:199-207 (1996), both of which are hereby incorporated by reference), in which a ribosomal pausing at a terminator or at a rare codon was suggested to perform the same function. Comparisons of the nucleotide sequence of the C-terminal region of the helicase and the N-terminal region of RdRP of GLRaV-2 with the same region of other closteroviruses revealed a significant similarity to BYV, BYSV, and CTV. As shown in
ORF2 encodes a small protein consisting of 171 bp (57 amino acid) with a molecular mass of 6,297 Da. As predicted, the deduced amino acid sequence includes a stretch of nonpolar amino acids, which is presumed to form a transmembrane helix. A small hydrophobic analogous protein is also present in BYV, BYSV, CTV, LIYV, and LChV (Agranovsky et al. “Nucleotide Sequence of the 3′-Terminal Half of Beet Yellows, Closterovirus RNA Genome Unique Arrangement of Eight Virus Genes,” J. General Virology 72:15-24 (1991); Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221:199-207 (1996); Pappu et al., “Nucleotide Sequence and Organization of Eight 3′ Open Reading Frames of the Citrus Tristeza Closterovirus Genome,” Virology 199:35-46 (1994); Klaassen et al., “Partial Characterization of the Lettuce Infectious Yellows Virus Genomic RNAs, Identification of the Coat Protein Gene and Comparison of its Amino Acid Sequence With Those of Other Filamentous RNA Plant Viruses,” J. General Virology 75:152,5-33 (1994); Jelkmann et al., “Complete Genome Structure and Phylogenetic Analysis of Little Cherry Virus, a Mealybug-Transmissible Closterovirus,” J. General Virology 78:2067-71 (1997), all of which are hereby incorporated by reference).
ORF3 encodes a 600 amino acid polypeptide with a molecular mass of 65,111 Da, which is homologous to the HSP70 cellular heat shock protein. HSP70 is highly conserved among closteroviruses and is probably involved in ATPase activity and, the protein to protein interaction for chaperone activity (Agranovsky et al. “The Beet Yellows Closterovirus p65 Homologue of HSP70 Chaperones has ATPase Activity Associated with its Conserved N-terminal Domain but Interact with Unfolded Protein Chains,” J. General Virology 78:535-42 (1997); Agranovsky et al., “Bacterial Expression and Some Properties of the p65, a Homologue of Cell Heat Shock Protein HSP70 Encoded in RNA Genome of Beet Yellows Closterovirus,” Doklady Akademii Nauk. 340:416-18 (1995); Karasev et al., “HSP70-Related 65-kDa Protein of Beet Yellows Closterovirus is a Microtubule-Binding Protein,” FEBS Letters 304:12-14 (1992), all of which are hereby incorporated by reference). As shown in
ORF4 encodes a 551 amino acid protein with a molecular mass of 63,349 Da. Database searching with the ORF4 protein product did not identify similar proteins except those of its counterparts in closteroviruses, BYV (P64), BYSV (P61), CTV (P61), LIYV (P59), and LChV (P61). This protein is believed to be a putative heat shock 90 protein. As shown in
ORF5 and ORF6 encode polypeptides with molecular mass of 24,803 Da and 21,661 Da, respectively. The start codon for both ORFs is in a favorable context for translation. ORF6 was identified as the coat protein gene of GLRaV-2 based on the sequence comparison with other closteroviruses. The calculated molecular mass of the protein product of ORF6 (21,662 Da) is in good agreement with the previously estimated 22˜26 kDa based on SDS-PAGE (Zimmermann et al., “Characterization and Serological Detection of Four Closterovirus-like Particles Associated with Leafroll Disease on Grapevine,” J. Phytopathology 130:205-18 (1990); Boscia et al., “Nomenclature of Grapevine Leafroll-Associated Putative Closteroviruses,” Vitis 34:171-75 (1995), both of which are hereby incorporated by reference).
Database searching with the deduced amino acid sequence of the ORF6 of GLRaV-2 showed a similarity with the coat proteins of closteroviruses, BYV, BYSV, CTV, LIYV, LChV, and GLRaV-3. At the nucleotide level, the highest percentage similarity was with the coat protein of BYSV (34.8%); at the amino acid level, the highest percentage similarity was with the coat proteins of BYV (32.7%) and BYSV (32.7%). As shown in
Identification of ORF6 as the coat protein gene was further confirmed by Western blot following expression of a fusion protein, consisting of a 22 kDa of ORF6 CP and a 42 kDa of maltose binding protein, produced by transformed E. coli as described in Example 5 supra. As shown in
ORF7 and ORF8 encode polypeptides of 162 amino acid with a molecular mass of 18,800 Da and of 206 amino acid with a molecular mass of 23,659 Da, respectively. Database searching with the ORF7 and ORF8 showed no significant similarity with any other proteins. Nevertheless, these genes were of similar in size and location as those observed in the sequence of other closteroviruses, BYV (P20, P21), BYSV (P18, P22), and LChV (P21, P27) (FIG. 7). However, conserved regions were not observed between the ORF7 or ORF8 and its counterparts in BYV, BYSV, and LChV.
The 3′ terminal untranslated region (3′-UTR) consists of 216 nucleotides. Nucleotide sequence analysis revealed a long oligo(A) tract close to the end of the GLRaV-2 genome which is similar to that observed in the genome of BYV and BYSV (Agranovsky et al. “Nucleotide Sequence of the 3′-Terminal Half of Beet Yellows Closterovirus RNA Genome Unique Arrangement of Eight Virus Genes,” J. General Virology 72:15-24 (1991); Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221:199-207 (1996), both of which are hereby incorporated by reference). The genome of BYV ends in CCC, BYSV, and CTV ends in CC with an additional G or A in the double-stranded replicative form of BYSV (Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221:199-207 (1996), which is hereby incorporated by reference) and CTV (Karasev et al., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,” Virology 208:511-20 (1995), which is hereby incorporated by reference), respectively . GLRaV-2 had CGC at the 3′ terminus of the genome. Recently, a conserved 60 nt cis-element was identified in the 3′-UTR of three monopartite closteroviruses, which included a prominent conserved stem and loop structure (Karasev et al., 1996). As shown in
The closteroviruses studied so far (e.g., BYV, BYSV, CTV, LIYV, LChV, and GLRaV-3) have apparent similarities in genome organization, which include replication associated genes that consist of MT, HEL, and RdRP conserved domains and a five-gene array unique for closteroviruses (Dolja et al. “Molecular Biology and Evolution of Closteroviruses: Sophisticated Build-up of Large RNA Genomes,” Annual Rev. Photopathology 32:261-85 (1994); Agranovsky “Principles of Molecular Organization, Expression, and Evolution of Closteroviruses: Over the Barriers,” Adv. in Virus Res. 47:119-218 (1996); Jelkmann et al., “Complete Genome Structure and Phylogenetic Analysis of Little Cherry Virus, a Mealybug-Transmissible Closterovirus,” J. General Virology 78:2067-71 (1997); Ling et al., “Nucleotide Sequence of the 3′ Terminal Two-Thirds of the Grapevine Leafroll Associated Virus-3 Genome Reveals a Typical Monopartite Closterovirus,” J. General Virology 79(5):1289-1301 (1998), all of which are hereby incorporated by reference).
The above data clearly shows that GLRaV-2 is a closterovirus. In the genome of GLRaV-2, two putative papain-like proteases were identified and an autoproteolytic cleavage process was predicted. The replication associated proteins consisting of MT, HEL, and RdRP conserved motifs were also identified, which were phylogenetically closely related to the replication associated proteins of other closteroviruses. A unique gene array including a small hydrophobic transmembrane protein, HSP70 homolog, HSP90 homolog, diverged CP and CP was also preserved in GLRaV-2. In addition, the calculated molecular mass (21,661 Da) of the coat protein (ORF6) of GLRaV-2 is in good agreement with that of the other closteroviruses (22 to 28 kDa) (Martelli and Bar-Joseph, “Closteroviruses: Classification and Nomenclature of Viruses,” Fifth Report of the International Committee on Taxonomy of Viruses, Francki et al., eds., Springer-Verlag Wein, New York, p. 345-47 (1991); Candresse and Martelli, “Genus Closterovirus,” in Virus Taxonomy, Report of the International Committee on Taxonomy of Viruses, Murphy et al., eds., Springer-Verlag., NY, p. 461-63 (1995), both of which are hereby incorporated by reference). Two ORFs downstream of the CP are of similar, in size and location, to those observed in the genome of BYV. Furthermore, lack of a poly(A) tail at the 3′ end of GLRaV-2 is also in good agreement with other closteroviruses. Like all other closteroviruses, the expression of ORF1b is suspected to occur via a +1 ribosomal frameshift and the 3′ proximal ORFs are probably expressed via formation of a nested set of subgenomic RNAs. Since the slippery sequence, stem-loop and pseudoknot structure involved in the frameshift of BYV were absent in GLRaV-2, the +1 frameshift of GLRaV-2 might be the same as proposed for CTV (Karasev et al., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,” Virology 208:511-20 (1995), which is hereby incorporated by reference) and BYSV (Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221:199-207 (1996), which is hereby incorporated by reference).
Overall, GLRaV-2 is more closely related to monopartite closteroviruses BYV, BYSV, and CTV than to GLRaV-3 (
Closteroviruses are a diverse group with complex and heterogeneous genome organizations. So far, GLRaV-2 is the only closterovirus that matches with the genome organization of BYV, the type member of the genus Closterovirus. In addition, the genomic RNA of GLRaV-2 is about the same size as that of BYV; however, the transmission vector of GLRaV-2 is unknown. The genome organization of GLRaV-2 is more closely related to the aphid transmissible closteroviruses (BYV and CTV) than to whitefly (LIYV) or mealybug transmissible closteroviruses (LChV and GLRaV-3). Thus, it is possible that GLRaV-2 is transmitted by aphids. Aphid transmission experiments with GLRaV-2 should provide information that might help develop methods for further control of GLRaV-2.
A total of 15,500 nucleotides or over 95% of the estimated GLRaV-2 genome has been cloned and sequenced. GLRaV-2 and GLRaV-3 (Ling et al., “Nucleotide Sequence of the 3′ Terminal Two-Thirds of the Grapevine Leafroll Associated Virus-3 Genome Reveals a Typical Monopartite Closterovirus,” J. General Virology 79(5):1289-1301 (1998), which is hereby incorporated by reference) are the first grapevine leafroll associated closteroviruses that have been almost completely sequenced. The above data clearly justify the inclusion of GLRaV-2 into the genus Closterovirus. In addition, the information regarding the genome of GLRaV-2 would provide a better understanding of this and related GLRaVs, and add fundamental knowledge to the group of closteroviruses.
GLRaV-2 infected Vitis vinifera, cv Pinot Noir grapevines originated from a vineyard in central New York was used as the virus isolate, from which the cp gene of GLRaV-2 was identified. Based on the sequence information, two oligonucleotide primers have been designed. The sense primer CP-96F (SEQ. ID. No. 21) starts from the ATG initiation codon of the coat protein gene and the complementary primer CP-96R (SEQ. ID. No. 22) starts from 56 nucleotides downstream of the stop codon of the CP gene. A NcoI restriction site (11 bp in SEQ. ID. No. 21 and 13 bp in SEQ. ID. No. 22) is introduced in the beginning of both primers to facilitate the cloning. The coat protein gene of GLRaV-2 was amplified from dsRNA extracted from GLRaV-2 infected grapevine using reverse transcriptase polymerase chain reaction (RT-PCR). The PCR-amplified CP product was purified from low melting temperature agarose gel, digested with NcoI and cloned into the same enzyme digested plant expression vector pEPT8 (shown at FIG. 11). After screening, the orientation of recombinant construct was checked by using the internal restriction site of the CP gene and directly sequencing the CP gene. The recombinant construct with translatable (sense) full length coat protein gene, pEPT8CP-GLRaV2, was going through for the further cloning. The plant expression cassette, which consisted of a double cauliflower mosaic virus (CaMV) 35S-enhancer, a CaMV 35S-promoter, an alfalfa mosaic virus (ALMV) RNA4 5′ leader sequence, a coat protein gene of GLRaV-2 (CP-GLRaV-2), and a CaMV 35S 3′ untranslated region as a terminator, was cut using the EcoRI restriction enzyme, isolated from low melting point temperature agarose gel, and cloned into the same restriction enzyme treated binary vector pGA482GG or pGA482G (a derivative of pGA482 (An et al., “Binary Vectors,” in Plant Molecular Biology Manual, pp. A3:1-19, Gelvin and Schilperoot, eds., Kinwer Academic Publishers, Dordrecht, Netherlands (1988), which is hereby incorporated by reference). The resulting recombinants constructs are pGA482GG/EPT8CP-GLRaV2 (shown at FIG. 11A), which contain both neomycin phosphotransferase (npt II) and β-glucuronidase (GUS) at the internal region of the T-DNA, and pGA482G/EPT8CP-GLRaV2 (shown at
NPT II-ELISA: Double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) was used to detect the npt II enzyme with an NPT II-ELISA kit (5′ prime to 3′ prime, Inc., Boulder, Colo.).
Indirect ELISA: Polyclonal antibodies to GLRaV-2, which were prepared from the coat protein expressed in E. coli, were used. Plates were coated with homogenized samples in extraction buffer (1:10, w/v) (phosphate buffered saline containing 0.05% Tween 20 and 2% polyvinyl pyrrolidone) and incubated overnight at 4° C. After washing with phosphate buffered saline containing 0.05% Tween 20 (PBST), the plates were blocked with blocking buffer (phosphate buffered saline containing 2% BSA) and incubated at room temperature for 1 hr. The anti-GLRaV-2 IgG was added at 2 μg/ml after washing with PBST. After incubation at 30 C for 4 hr, the plates were washed with PBST, and the goat anti-rabbit IgG conjugate of alkaline phosphotase (Sigma) was added at 1:10,000 dilution. The absorbance was measured at 405 nm with a MicroELISA AutoReader. In addition, Western blot was also performed according to the method described by Hu et al., “Characterization of Closterovirus-like Particle Associated Grapevine Leafroll Disease,” J. Phytophathology 128:1-14, (1990), which is incorporated herein by reference.
PCR analysis: Genomic DNA was extracted from leaves of putative transgenic and non-transgenic plants according to the method described by Cheung et al., “A Simple and Rapid DNA Microextraction Method for Plants, Animal, and Insect Suitable for RAPD and other PCR analysis,” PCR Methods and Applications 3:69 (1996), which is incorporated herein by reference. The extracted total DNA served as the template for PCR reaction. The primers CP-96F and CP-96R (SEQ. ID. Nos. 21 and 22, respectively,) for the CP gene of GLRaV-2, as well as npt II 5′- and 3′-primers were used for PCR analysis. PCR reaction was performed at the 94° C.×3 min for one cycle, followed by 30 cycles of 94° C.×1 min, 50° C.×1 min, and 72° C.×2:30 min with an additional extension at 72° C. for 10 min. The PCR product was analyzed on agarose gel.
After transformation, a total of 42 kanamycin resistant Nicotiana benthamiana lines (R0) were obtained, of which the leaf samples were tested by NPT II enzyme activity. Among them, 37 lines were NPT II positive by ELISA, which took about 88.0% of total transformants. However, some of NPT II negative plants were obtained among these selected kanamycin resistant plants. All of the transgenic plants were self-pollinated in a greenhouse, and the seeds from these transgenic lines were germinated for further analysis.
The production of GLRaV-2 CP in transgenic plants was detected by indirect ELISA prior to inoculation, and the results showed that GLRaV-2 CP gene expression was not detectable in all transgenic plants tested. This result was further confirmed with Western blot. Using the antibody to GLRaV-2, the production of the CP was not detected in the transgenic and nontransgenic control plants. However, a protein of expected size (˜22 kDa) was detected in GLRaV-2 infected positive control plants. This result was consistent with the ELISA result. The presence of the CP gene of GLRaV-2 in transgenic plants was detected from total genomic DNA extracted from plants tissue by PCR analysis (FIG. 12). The DNA product of expected size (653 bp) was amplified from twenty tested transgenic lines, but not in non-transgenic plants. The result indicated that the CP gene of GLRaV-2 was present at these transgenic lines, which was also confirmed by Northern blot analysis.
Inoculation of transgenic plants: GLRaV-2 isolate 94/970, which was originally identified and transmitted from grapevine to Nicotiana benthamiana in South Africa (Goszczynski et al., “Detection of Two Strains of Grapevine Leafroll-Associated Virus 2,” Vitis 35:133-35 (1996), which is incorporated herein by reference), was used as inoculum. The CP gene of isolate 94/970 was sequenced; and it is identical to the CP gene used in construction. Nicotiana benthamiana is an experimental host of GLRaV-2. The infection on it produces chlorotic and occasional necrotic lesions followed by systemic vein clearing. The vein clearing results in vein necrosis. Eventually the infected plants died, starting from the top to the bottom.
At five to seven leaf stage, two youngest apical leaves were challenged with GLRaV-2 isolate 94/970. Inoculum was prepared by grinding 1.0 g GLRaV-2 infected Nicotiana benthamiana leaf tissue in 5 ml of phosphate buffer (0.01 M K2HPO4, PH7.0). The tested plants were dusted with carborundum and rubbed with the prepared inocululm. Non-transformed plants were simultaneously inoculated as above. The plants were observed for symptom development every other day for 60 days after inoculation. Resistant R1 transgenic plants were carried on to R2 generation for further evaluation.
Transgenic progenies from 20 R0 lines were initially screened for the resistance to GLRaV-2 followed by inoculation with GLRaV-2 isolate 94/970. The seedlings of the transgenic plants (NPT II positive), and nontransformed control plants were inoculated with GLRaV-2. After inoculation, the reaction of tested plants were divided into three types: highly susceptible (i.e. typical symptoms were observed two to four weeks postinoculation); tolerant (i.e. no symptom was developed in the early stage and typical symptoms was shown four to eight weeks postinoculation); and resistant (i.e. the plants remained asymptomatic eight weeks postinoculation). Based on the plant reaction, the resistant plants were obtained from fourteen different lines (listed in Table 1 below). In each of these fourteen lines, there was no virus detected within these plants by ELISA at 6 weeks postinoculation. In contrast, GLRaV-2 was detected in symptomatic plants by indirect ELISA. In the other six lines, although there were a few plants with some kind of delay in symptom development, all the inoculated transgenic plants died at three to eight weeks postinoculation. Based o(n the initial screening results, five representative lines consisting of three resistant lines (1, 4, and 19) and two susceptible lines (12 and 13) were selected for the further analysis.
Table 2 below shows the symptom development in transgenic plants relative to non-transgenic control plants in the five selected lines in separate experiments. Non-transgenic control plants were all infected two to four weeks after inoculation, which showed typical GLRaV-2 symptoms on Nicotiana benthamiana, including chlorotic and local lesions followed by systemic vein clearing and vein necrosis on the leaves. Three of the tested lines (1, 4, and 19) showed some resistance that was manifested by either an absence or a delay in symptom development. Two other lines, 12 and 13, developed symptoms at nearly the same time as the non-transformed control plants. From top to bottom, the leaves of infected plants gradually became yellow, wilted, and dried, and, eventually, the whole plants died. No matter when infection occurred, the eventual result was the same. Six weeks after inoculation, all non-transgenic plants and the susceptible plants were dead. Some tolerant plants started to die. In contrast, the asymptomatic plants were flowering normally and pollinating as the non-inoculated healthy control plants (FIG. 13).
ELISA was performed at 6 weeks postinoculation to test the GLRaV-2 replication in the plants. Presumably, the increased level of CP reflected virus replication. The result showed that the absorbance value in symptomatic plants reached (OD) 0.7 to 3.2, compared to (OD) 0.10-0.13 prior to inoculation. In contrast, GLRaV-2 was not detected in asymptomatic plants, of which the absorbance value was the same or nearly the same as that of healthy nontransformed control plants. The data confirmed that virus replicated in symptomatic plants, but not in asymptomatic plants. The replication of GLRaV-2 was suppressed in asymptomatic plants. This result implicated that another mechanism other than the CP-mediated resistance was probably involved.
Three 2 progenies derived from transgenic resistant plants of lines 1, 4, and 19 were generated and utilized to examine the stable transmission and whether resistance was maintained in R2 generation. These results are shown in Table 3 below. NPT II analysis revealed that R2 progeny were still segregating. The CP expression in R2 progeny was still undetectable. After inoculation, all the nontransgenic plants were infected and showed GLRaV-2 symptoms on the leaves after 24 days postinoculation. In contrast, the inoculated transgenic R2 progeny showed different levels of resistance from those highly susceptible to highly resistant. The tolerant and resistant plants were manifested by a delay in symptom development and absence of symptoms, respectively. At 6 weeks postinoculation, GLRaV-2 was detected in the tolerant symptomatic infected plants by indirect ELISA; but not in asymptomatic plants. This result indicated that virus replication was suppressed in these resistant plants, which was confirmed by Western blot. These resistant plants remained asymptomatic eight weeks postinoculation, and they were flowering normally and pollinating.
Northern blot analysis: Total RNA was extracted from leaves prior to inoculation following the method described by Napoli et al., Plant Cell 2:279-89 (1990), which is hereby incorporated by reference. The concentration of the extracted RNA was measured by spectrophotometer at OD 260. About 10 g of total RNA was used for each sample. The probe used was the 3′ one third of GLRaV-2 CP gene, which was randomly labeled with 32P (α-dATP) using Klenow fragment of DNA polymerase I.
Using a DNA corresponding to the 3′ one third CP gene sequence as probe, a single band was detected in the RNA extracted from susceptible plants from R1 progeny of lines 5, 12, and 13 by Northern hybridization. There was little or no signal detected in the transgenic plants from R1 progeny of line 1, 4, and 19. This RNA is not present in nontransformed control plants. The size of the hybridization signal was estimated to an approximately 0.9 kb nucleic acid, which was about the same as estimated (FIG. 14). In lines of 1, 4, and 19, the steady state level of RNA expression was also low in R2 progeny. This data showed that susceptible plants from lines 12 and 13 had high mRNA level and all transgenic plants from lines 1, 4, and 19 had low mRNA level.
Plant materials: The rootstock cultivars Couderc 3309 (3309C) (V. riparia×V. rupestris), Vitis riparia ‘Gloire de Montpellier’ (Gloire), Teleki 5C (5C) (V. berlandieri×V. riparia), Millardet et De Grasset 101-14 (101-14 MGT) (V. riparia×V. rupestris), and Richter 110 (110R) (V. rupestris×V. berlandieri) were utilized. Initial embryogenic calli of Gloire were provided by Mozsar and Süle (Plant Protection Institute, Hungarian Academy of Science, Budapest). All other plant materials came from a vineyard at the New York State Agricultural Experiment Station, Geneva, N.Y. Buds were removed from the clusters and surface sterilized in 70% ethanol for 1-2 min. The buds (from the greenhouse and the field) were transferred to 1% sodium hypochlorite for 15 min, then rinsed three times in sterile, double-distilled water. Anthers were excised aseptically from flower buds with the aid of a stereo microscope. The pollen was crushed on a microscope slide under a coverslip with a drop of acetocarmine to observe the cytological stage. This was done to determine which stage was most favorable for callus induction.
Somatic embryogenesis and regeneration: Anthers were plated under aseptic conditions at a density of 40 to 50 per 9 cm diameter Petri dish containing MSE. Plates were cultured at 28° C. in the dark. Callus was initiated, and, after 60 days, embryos were induced and were transferred to hormone-free HMG medium for differentiation. Torpedo stage embryos were then transferred from HMG to MGC medium to promote embryo germination. Cultures were maintained in the dark at 26-28° C. and transferred to fresh medium at 34 week intervals. Elongated embryos were transferred to rooting medium in baby food jars (5-8 embryos per jar). The embryos were grown in a tissue culture room at 25° C. with a daily 16 h photoperiod (76:mol. s) to induce shoot and root formation. After plants developed roots, they were transplanted to soil in the greenhouse.
Transformation: The protocols used for transformation were modified from those described by Scorza et. al., “Transformation of Grape (Vitis vinifera L.) Zygotic-derived Somatic Embryos and Regeneration of Transgenic Plants,” Plant Cell Rpt. 14:589-92 (1995), which is hereby incorporated by reference. Overnight cultures of Agrobacterium strain C58Z707 or LBA4404 were grown in LB medium at 28° C. in a shaking incubator. Bacteria were centrifuged for 5 min at 3000-5000 rpm and resuspended in MS liquid medium (OD 1.0 at A600 nm ). Calli with embryos were immersed in the bacterial suspension for 15-30 min, blotted dry, and transferred to HMG medium with or without acetosyringone (100 μM). Embryogenic calli were co-cultivated with the bacteria for 48 h in the dark at 28° C. Then, the plant material was washed in MS liquid plus cefotaxime (300 mg/ml) and carbenicillin (200 mg/ml) 2-3 times. To select transgenic embryos, the material was transferred to HMG medium containing either 20 or 40 mg/L kanamycin, 300 mg/L cefotaxime, and 200 mg/L carbenicillin. Alternatively, after co-cultivation, embryogenic calli were transferred to initiation MSE medium containing 25 mg/l kanamycin plus the same antibiotics listed above. All plant materials were incubated in continuous dark at 28° C. After growth on selection medium for 3 months, embryos were transferred to HMG or MGC without kanamycin to promote elongation of embryos. They were then transferred to rooting medium Without antibiotics. Nontransformed calli were grown on the same media with and without kanamycin to verify the efficiency of the kanamycin selection process.
Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.
This application is Continuation of a U.S. application Ser. No. 09/080,983, filed May 19, 1998 now U.S. Pat. No. 6,197,948, which claims the benefit of U.S. Provisional Patent Application Ser. No. 60/047,194, filed May 20, 1997.
This work was supported by the U.S. Department of Agriculture Cooperative Grant No. 58-2349-9-01. The U.S. Government may have certain rights in the invention.
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| Child | 09613486 | US |
