# Patent

## Title
Closterovirus vectors and methods

## Abstract

The present disclosure relates to the development and use of Closterovirus-based vectors for the delivery of nucleotides to plants. Specifically, the present disclosure provides viral vectors based on Grapevine leafroll-associated virus-2 for the delivery and expression of genes in plants, particularly grape plants. Methods of making and using these vectors, as well as the plants transformed by these vectors, are also contemplated.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This is the U.S. National Stage of International Application No. PCT/US2009/032380, filed Jan. 29, 2009, which was published in English under PCT Article 21(2), which in turn claims the benefit of U.S. Provisional Application No. 61/063,305, filed Jan. 31, 2008, and No. 61/083,504, filed Jul. 24, 2008, both of which provisional applications are incorporated herein by reference in their entirety.


FIELD
The present disclosure relates to the field of closteroviruses and their use as gene delivery vehicles for plants.
BACKGROUND
Grapevines (Vitis) are a major global fruit crop with enormous economic and cultural significance, particularly Vitis vinifera, which is used for wine cultivation. A relatively small number of V. vinifera cultivars are used commercially to maintain consistency in the fruit as the plants are heterozygous and do not breed true. Thus, because so much of the commercial grape crop is dependent on these cultivars, which have limited diversity, disease resistance is of major concern. Classical breeding techniques to increase disease resistance generally erode fruit quality, making grapevines a prime candidate for genetic manipulation for improving disease resistance. However, the production of transgenic grapevines has proven difficult, as woody perennials, such as grapevines, are known to be recalcitrant to transformation, and the selection process required by Agrobacterium-mediated transformation is significantly more stringent due to out competition by the untransformed cells, leading to highly variable success rates (Mullins et al., Meth. Mol. Bio. 344:273-285, 1990); Bouquet et al., Methods Mol. Biol. 344:273-285, 2006). Thus, a need exists for reliable, efficient method for the delivery of genes to grapevines for disease treatments and the modification of grapevines for desired characteristics.
During the last two decades, viral vectors for the transient expression of the proteins in plants and animals became indispensable tools of molecular biology and biomedicine (Pogue et al., Annu. Rev. Phytopathol. 40: 45-74, 2002; Gleba et al., Curr Opin Biotechnol 18: 134-141, 1007). With the advent of RNA interference (RNAi) or RNA silencing, viral vectors were also developed for virus-induced gene silencing or VIGS (Godge et al., Plant Cell Rep 27(2):209-219, 2008; e-pub ahead of print, 2007). Taken together, an ability to rapidly overexpress or silence genes of interest made viral vectors important tools in functional genomics.
A number of plant viruses have been engineered into viral vectors, each with limitations and plant specificity. Most are suitable only for use in dicotyledonous herbaceous plants. By and large, icosahedral viruses are ill suited for accommodating foreign genes mostly due to the limited size of their capsids. In general, the elongated viruses have exhibited a better ability to tolerate recombinant genes and to express them to very high levels. Currently, the most commonly used vectors are those based on the rod-shaped Tobacco mosaic virus (TMV, genus Tobamovirus) (Pogue et al., Annu. Rev. Phytopathol. 40: 45-74, 2002; Gleba et al., Curr Opin Biotechnol 18: 134-141, 1007). These vectors are characterized by high expression levels but relatively low genetic stability, especially when it comes to large foreign inserts.
Another series of vectors is based on rod-shaped Tobacco rattle virus (TRV, genus Tobravirus) (Godge et al., Plant Cell Rep 27(2):209-219, 2008; e-pub ahead of print, 2007). Vectors derived from filamentous viruses are commonly based on Potato virus X (PVX, genus Potexvirus) (Chapman et al., Plant J 2: 549-557, 1992) and Tobacco etch virus (TEV, genus Potyvirus) (Dolja et al., Proc. Natl. Acad. Sci. USA 89: 10208-10212, 1992). TMV, TRV, and PVX vectors contain an expression cassette with a subgenomic RNA promoter, while TEV vectors use an alternative principle of protein expression based on a polyprotein processing. This latter feature provides the potyviral vectors with much higher genetic stability than that found in promoter-containing vectors (Dolja et al., Virology 252: 269-274, 1998).
Gene expression vectors based on Beet yellows virus (BYV, genus Closterovirus) have been developed (Hagiwara et al., J. Virol. 73: 7988-7993, 1999; Peremyslov et al., Proc. Natl. Acad. Sci. USA 96, 14771-14776, 1999). Although the levels of protein expression achievable for closteroviral vectors may be lower than those for TMV or TRV, these vectors have proved to be very stable genetically and capable of accommodating several expression cassettes based either on additional heterologous subgenomic RNA promoters or polyprotein processing. Such versatility of closteroviral vectors is most likely due to the large size of closteroviral genomes and presence of genes that dramatically increase genome replication and gene expression ability and possibly provide for increased fidelity of RNA copying (Dolja et al., Virus Res. 117: 38-51, 2006). Strong suppressors of RNAi (Reed et al., Virology 306: 203-209, 2003; Chiba et al., Virology 346: 7-14, 2006) and the leader proteinases of closteroviruses (Peng et al., J. Virol. 75(24), 12153-12160, 2001) are among the genes that ensure high genetic and evolutionary performance of closteroviruses and precondition their genomes for accommodating additional genes, viral or foreign.
One of the most critical characteristics of the viral vector is its host range that severely limits its potential utility for the desired crop plants. All of the vectors described above are able to infect only dicotyledonous herbaceous plants. In other words, the need to generate a viral vector for monocots or for woody crops such as grapevine dictates the need of using viruses that naturally infect such plants as a platform for vector development.
To date, very few viral vectors potentially suitable for woody plants have been developed, and data showing expression has typically been limited to a narrow range of model plants. One of these vectors is based on Apple latent spherical virus RNA 2 (ALSV, family Sequiviridae) (Li et al., Arch. Virol. 149: 1541-1558, 2004). Although the authors claim that ALSV vector was able to express the green fluorescent protein (GFP) by polyprotein processing upon mechanical inoculation to apple seedlings, no convincing experimental proof of such ability was presented in the paper. Similarly, no data is available to support recent claims of a ‘universal’ vector based on Tomato yellow leaf curl geminivirus, allegedly capable of systemically infecting a vast variety of plants from dicots to monocots to trees and vines (Peretz et al., Plant Physiol. 145(4):1251-1263, 2007). Another vector was developed using Grapevine virus A (GVA, a Vitivirus). Its ability to express a foreign protein was demonstrated in tobacco (Haviv et al., J. Virol. Meth. 132: 227-231, 2006) and remains unproven for grapevine. Another vector is based on Citrus tristeza virus (CTV), a closterovirus closely related to BYV (Folimonov et al., Virology 368(1):205-216, 2007). However, CTV is useful only in Citrus species, and its propagation involves cumbersome process of cycling in protoplasts prior to slash-inoculation of citrus trees with isolated virions. Accordingly, there exists a strong need for viral vectors suitable for transforming woody plants, particularly grapevines.
SUMMARY
The present disclosure relates to replication-competent plant gene transfer vectors comprising a nucleic acid encoding viral genes from Grapevine leafroll-associated virus-2 (LR-2) selected from the group consisting of methyltransferase, RNA helicase, RNA-dependent RNA polymerase, and p24; leader proteases L1 and L2; and a heterologous polynucleotide operably linked to a promoter, wherein the heterologous polynucleotide is expressed in a plant cell; and wherein the vector is capable of infectious replication in the plant cell.
The present disclosure further encompasses conditionally-replicating plant gene transfer vectors comprising a nucleic acid encoding viral genes from Grapevine leafroll-associated virus-2 (LR-2) selected from the group consisting of methyltransferase, RNA helicase, RNA-dependent RNA polymerase, and p24; leader proteases L1 and L2, wherein at least one leader protease is inactivated such that the vector cannot infectiously replicate independently; and a heterologous polynucleotide operably linked to a promoter, wherein the heterologous polynucleotide is expressed in a plant cell.
Optionally, vectors provided herein may also include one or more viral genes from Grapevine leafroll-associated virus-2 (LR-2) that are involved in virion assembly and/or transport within plants. Such genes include for instance p6, Hsp70h, p63, CPm, CP and p19. In particular embodiments, all of these genes are included in a vector, thereby facilitating systemic infection. Such a vector may be referred to as a full-length vector; examples of such are described herein.
The heterologous polynucleotide in the described vectors may encode one or more of a reporter molecule, a selectable marker, or a therapeutic gene, which may encode a desired protein, such as to improve the nutritional or aesthetic properties of the plant or a disease resistance gene, which may be antifungal, antibacterial or antiviral. The therapeutic gene may be for the treatment of Pierce's Disease, such as a polynucleotide which triggers viral induced gene silencing or encodes a lysozyme polypeptide.
The leader proteases may be L1 and/or L2 from LR-2. The leader proteases (e.g., SEQ ID NOs: 4 or 6) may be encoded by SEQ ID NO: 3 and/or SEQ ID NO: 5. One or both of the leader proteases may be inactivated by substitution, insertion, partial deletion or complete disruption of the coding sequence for the leader protease in the vector.
The vector may further comprise a T DNA sequence for the transformation of a plant cell. The vector may comprise a beet yellows virus or other related closterovirus promoter or a native LR-2 promoter. More than one vector may be introduced into the plant, such as a vector encoding a therapeutic gene and a vector encoding the p24 RNAi suppressor.
A plant cell or plant comprising a vector described herein is also contemplated, such as a grapevine cell or grapevine.
Also provided are methods for producing the described vectors, comprising culturing a cell comprising the vector and recovering vector from the cell or medium in which the cell is grown. The vector may optionally be in a plasmid, such as a plasmid suitable for bacterial amplification and/or a binary plasmid suitable for agroinoculation.
Another embodiment is a method for expressing a heterologous gene in a plant cell comprising introducing the vector of the present invention into the plant cell. In one embodiment, the plant cell is a grapevine cell. In another embodiment, the vector is introduced by agroinoculation.
Another embodiment is a method for expressing a heterologous gene in a plant cell comprising introducing a replication-competent vector into the plant cell such that the vector subsequently replicates and infects at least one additional plant cell. The method further comprises the systemic infection of a plant structure selected from the group consisting of tissue, leaf, steam, root, fruit, seed or entire plant.
Another embodiment is a method for inducing disease resistance comprising introducing the vector of the present invention into a plant cell. The vector may be introduced more than one time. The vector may comprise a heterologous polynucleotide encoding a gene that confers resistance to the disease. Such heterologous polynucleotide may be, for example, a polynucleotide which triggers viral induced gene silencing, a Run1 polynucleotide, or encodes a lysozyme polypeptide.
Another embodiment is a method for treating or preventing Pierce's Disease or powdery mildew in a grapevine comprising introducing into a grapevine cell a vector of the present invention. The vector may be introduced by agroinoculation and may be introduced more than one time.
Another embodiment is a method for modifying the aesthetic properties, such as taste and aroma of the juice, or enhancing the nutritional or other agricultural characteristic of a plant comprising introducing the vector of the present invention into a plant cell.
Another embodiment is a method for making a transgenic plant comprising introducing the vector of the present invention into a plant cell, culturing the plant cell under conditions that promote growth of a plant, wherein the heterologous gene is expressed in the transgenic plant. The transgenic plant may be a grapevine.
The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1C are genetic maps of GLRaV-2 genome indicating the functions of the viral genes (FIG. 1A), cassette for recombinant gene expression (FIG. 1B), and binary vector containing the full-length GLRaV-2 genome with ER-GFP insert (FIG. 1C). (FIG. 1A) Genes are designated in accord with the encoded proteins: (FIG. 1B) LR-2 CP promoter, a natural LR-2 promoter driving expression of CP gene; ER-GFP, endoplasmic reticulum-targeted green fluorescent protein; BYV CP promoter, an engineered heterologous promoter derived from Beet yellows virus where it drives expression of CP gene; Pac I and Fse I, engineered sites for corresponding restriction endonucleases. (FIG. 1C) From left, clockwise: 35S, 35S RNA polymerase II promoter derived from Cauliflower mosaic virus (arrow);  ̃17, 500 nts, the  ̃17, 500-nucleotide-long, full-length cDNA clone of LR-2 tagged by insertion of ER-GFP (rectangle, labeled  ̃17,500 nts); RZ, a custom-designed ribozyme that promotes the autocatalytic release of the 3′-terminus of LR-r RNA upon transcription of the inserted DNA in plant cell nucleus (rectangle with an arrow, labeled RZ); NOS terminator of RNA polymerase II; Right border, recognition sequence where plasmid DNA is cleaved by Agrobacterium for transfer to the plant cell (curved arrow); Ori, origin of plasmid replication (arrow); KanR, gene for kanamycin resistance; Left border, recognition sequence where plasmid DNA is cleaved by Agrobacterium for transfer to the plant cell (curved arrow).
FIG. 2 shows N. benthamiana cells infected with GLRaV-2/ER-GFP upon agroinoculation at two different scales using confocal laser scanning microscopy. Bright fluorescence marks endoplasmic reticulum of the virus-infected cell due to expression of ER-GFP marker by virus. Background fluorescence is due to autofluorescence of chloroplasts. Bars, 50 μm.
FIG. 3 shows images taken by epifluorescent microscopy of tissues from plants of N. benthamiana systemically infected with GLRaV-2/ER-GFP upon agroinoculation (bottom row) compared to control, uninfected plants (top row). Bright fluorescence highlights predominantly phloem cells infected by virus and expressing ER-GFP. Stems and petioles were manually cross sectioned prior to microscopic imaging. Background fluorescence is due to autofluorescence of chloroplasts.
FIG. 4 (FIG. 4A) Immunoblot analysis of the extracts from plants infected with the wild type virus and virus modified to express ER-GFP using CP-specific antibodies as shown below the image. Dilutions of the original leaf extracts are shown at the top. (FIG. 4B) RT-PCR analysis of the RNAs isolated from N. benthamiana plants infected with the wild type virus and virus modified to express ER-GFP. The products of RT-PCR were separated in 1% agarose gel and stained with ethidium bromide. M, DNA size markers; bands corresponding to 1- and 2-kb DNAs are marked by arrows.
FIG. 5 Top, a diagram showing the design of a binary vector that expresses p24, a 24-kDa LR-2 suppressor of RNA silencing cloned to a binary vector pCB302. TEV leader, cDNA sequence corresponding to the 5′-untranslated region of the Tobacco etch virus and used to enhance translation of the p24. Bottom, confocal images of the rare cell infected by miniBYV-GFP alone (left panel) and miniBYV-GFP with p24 co-expression (right panel). Images are from Chiba et al. (Virology 346: 7-14, 2006).
FIG. 6 Silencing of the GFP transgene by the viral vector LR-GFP in N. benthamiana line 16c. In the control, all cells are brightly fluorescent due to production of transgenic GFP. In the infected plants, bright fluorescent cells are those in which virus makes additional GFP. The areas of background fluorescence contain cells in which transgenic GFP was silenced due to virus-induced RNA interference.
FIG. 7 (FIG. 7A) Maximized expression of GFP using Agrobacterium introduced by sonication in a micropropagated Cabernet franc leaf. (FIG. 7B) A less susceptible leaf that, however, shows GFP expression in the vein. GFP is expressed directly from a binary vector engineered as shown in (FIG. 7C).
FIG. 8 Grapevine infection with miniLR-2-GFP launched by agroinfiltration showing images of the inoculated leaf and individual GFP-expressing fluorescent cells (top row) and a genetic map of the miniLR-GFP replicon. L1 and L2, leader proteinases; MET, methyltransferase domain; HEL, RNA helicase domain; POL, RNA-dependent RNA polymerase; GFP, green fluorescent protein; p24, a 24-kDa protein.
FIG. 9 Grapevine infection with the full-length LR-2-GFP GFP launched by agroinfiltration showing images of the inoculated leaf and individual GFP-expressing fluorescent cells.
FIG. 10 (FIG. 10A) Schematic of the GLRaV-2 virus, the full-length vector (LR_GFP) and minivector (mLR-GFP/GUS). (FIG. 10B) Domains of the leader proteases L1 and L2 with proteolytic processing, gene expression and infection results in N. benthamiana indicated.
FIG. 11 Processing of vectors as shown by HA-tagging (FIG. 11A) and radiolabeling (FIG. 11B).
FIG. 12 (FIG. 12A) Long distance transport and systemic infection of N. benthamiana leaves with vectors. (FIG. 12B) GFP accumulation in N. benthamiana leaves. (FIG. 12C and FIG. 12D) Absence of gene expression in upper leaves of N. benthamiana after inoculation.
FIG. 13 Immunoblot analysis of gradient from sucrose fractionation of virions isolated from infected N. benthamiana leaves.
FIG. 14 Alignment of the nucleotide sequences of the N. benthamiana-derived (Nb; top lanes; SEQ ID NO: 1, nucleotides 4064-2163) and V. vinifera-derived (Vv, bottom lanes; SEQ ID NO: 7) variants of the GLRaV-2 vector. The nucleotides different in two isolates are shown in bold and underlined.



SEQUENCE LISTING
The nucleic and/or amino acid sequences listed herein and/or in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. §1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:
SEQ ID NO: 1 is the nucleic acid sequence of p35S-LR-2/ERGFP, a full-length, Grapevine leafroll virus-2-derived gene expression and silencing vector containing a recombinant gene encoding ER-targeted GFP reporter. The sequence includes binary vector pCB301 (1-3305), and the entire viral expression cassette (3306-21957). The following features that are shown in FIG. 1C are also reflected in the sequence: 35S promoter (3306-4063), viral sequence (4064-21643) including GFP-reporter expressing cassette (18648-19439), heterologous BYV CP promoter (18440-19723), ribozyme (21644-21698) and NOS terminator (21705-21957).
SEQ ID NO: 2 is the nucleic acid sequence of MiniLR-GFP/GUS. SEQ ID NOs: 3 and 4 are the nucleotide and amino acid sequences of protease L1.
SEQ ID NOs: 5 and 6 are the nucleotide and amino acid sequences of protease L2.
SEQ ID NO: 7 is the nucleotide sequence of full-length, Grapevine leafroll-associated virus-2-derived gene expression and silencing vector containing a recombinant gene encoding ER-targeted GFP reporter (LR2-Vitis). All Grapevine leafroll-associated virus-2 nucleotide sequence corresponds to a consensus sequence of the viral isolate naturally present in Pinot Noir grapevine. Nucleotides distinct from those present in the original, N. benthamiana-derived virus (SEQ ID NO: 1) are highlighted in FIG. 14. The nucleotide sequences outside the viral expression cassette are the same as in SEQ ID NO: 1.
SEQ ID NO: 8 is a representative V. vinifera chromosome genomic sequence encompassing a putative phloem-specific promoter, AtSUC2 orthologous promoter (GSVIVT00002302001_VvSUC27_AF021810_Genomic), suitable for phloem-specific expression of the LR-2 vectors.
SEQ ID NO: 9 is a representative V. vinifera chromosome genomic sequence encompassing a putative phloem-specific promoter, AtAHA3 orthologous promoter (VV78X258876_VITISV—014422_AM487422_CAN64375_Genomic), suitable for phloem-specific expression of the LR-2 vectors.
SEQ ID NO: 10 is a representative V. vinifera chromosome genomic sequence encompassing a putative phloem-specific promoter, AtAsus1 orthologous promoter (VV78X051063_CAN82840_VITISV—024563_GI147856448_Genomic), suitable for phloem-specific expression of the LR-2 vectors.
SEQ ID NO: 11 is the amino acid sequence of the hemagglutinin epitope (HA) tag.
DETAILED DESCRIPTION
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). In order to facilitate review of the various embodiments of the invention, explanations of specific terms are provided herein.
The term “grapevine”, “grape plant” or “grapevine plant” refers to any plant of the genus Vitis, for example V. vinifera, V. labrusca, V. riparia, V. rotundifolia, V. aestivalis, or of the genus Muscadinia, and species thereof. The grapevine may be a scion, rootstock, cultivars or a hybrid plant. The term “grape” is the berry or fruit of the grapevine, which may be eaten whole or the juice extracted therefrom for drinking and/or fermentation into wine. Other edible portions of a grapevine include the leaves and the seeds.
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Hence “comprising A or B” means including A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including any explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Overview of Several Embodiments
The present disclosure provides gene transfer vectors comprising at least the replication genes of grapevine leafroll-associated virus-2 (LR-2). LR-2 has been sequenced (Meng et al., Virus Genes 31:31-41, 2005) and functionally compared to other known closteroviruses (Dolja et al, Virus Res. 117(1):38-51, 2006). Both references are hereby incorporated by reference. The core viral genes of LR-2 include Met (methyltransferase), Hel (RNA helicase) and Pol (RNA-dependent RNA polymerase), while L1, L2, and p24, by comparison to homologous genes of BYV, play accessory roles in genome replication. Other genes may also be included in the present vector, such as p6, Hsp70h, p63, CPm, CP, and p19 (see, e.g., Peremyslov et al., J. Virol. 72:5870-5876, 1998, which is hereby incorporated by reference).
The present disclosure relates to vectors comprising the leader proteases, L1 and L2, and vectors with one or both leader proteases inactivated. One of skill in the art will instantly recognize that there are myriad ways in which a protease could be inactivated, and all such methods are contemplated herein. It has been shown previously that for a related closterovirus, BYV, the corresponding leader protease L-Pro is required for efficient RNA amplification and virus long-distance transport (Peng et al., J. Virol. 77, 2843-2849, 2003; Peng & Dolja, J. Virol. 74, 9766-9770, 2000). Interestingly, replacement of L-Pro by the proteases from other closteroviruses (Peng et al., J. Virol. 75(24), 12153-12160, 2001) or even from an animal arterivirus (Peng et al., Virology 294, 75-84, 2002) can rescue the RNA amplification, but not the transport function of the leader protease. The Grapevine leafroll-associated virus-2 (GLRaV-2) is a close BYV relative in the Closterovirus genus whose genetic organization is almost identical to that of BYV (Zhu et al., J. Gen. Virol. 79: 1289-1298, 1998). However, unlike BYV that possesses one leader protease, GLRaV-2 codes for two leader proteases, L1 and L2 (Meng et al., Virus Genes 31, 31-41, 2005; Peng et al., J. Virol. 75(24), 12153-12160, 2001) (FIG. 1A, top diagram). Herein is shown for the first time that L1 and L2 have complementary functions in establishment of the GLRaV-2 infection in the initially inoculated cells and systemic transport. Strikingly, overall contribution of L1 and L2 into virus infection is much more critical in a natural virus host, grapevine, compared to an experimental herbaceous host, N. benthamiana. Thus, using the properties of L1 and L2, vectors for the introduction of heterologous genes into grapevines may be constructed as replication-competent (comprising both L1 and L2) or conditionally-replicating (with one or both leader proteases inactivated).
Replication-competent vectors are those vectors that are capable of producing infectious virions without needing additional factors supplied in trans. Such vectors, after inoculation or transduction into a plant cell may produce infectious virions and infect other cells. Such vectors may be useful when systemic infection of a plant is desired, as the vector may spread beyond the originally transduced cell and increase overall transduction. For example, the vector may spread within a tissue, between tissues, throughout the plant or even between plants. For instance, the vector may infect an entire leaf, more than one leaf, one or more leaves and the stem or fruit of the plant, as well as the roots.
Conditionally-replicating vectors lack necessary viral factors for the production of infectious virions independently. The present disclosure encompasses vectors that have one or both leader proteases inactivated such that the vector is not capable of producing infectious virions and cannot infect additional cells after inoculation or transduction. Such vectors may be useful when spread of the vector is not desired, or when limited spread is beneficial. Such vectors may have increased safety as the spread of the vector as well as expression of the heterologous gene does not go beyond the original transduction.
In specific embodiments, the plant transformation vectors provided herein include one or more viral genes from Grapevine leafroll-associated virus-2 (LR-2) that are involved in virion assembly and/or transport within plants. Such genes include for instance p6, Hsp70h, p63, CPm, CP and p19. In particular embodiments, all of these genes are included in the vector, thereby facilitating systemic infection. Such a vector may be referred to as a full-length vector.
The entire LR-2 genome (or essentially all of the genome, or the equivalent thereof) may be included in the vector. The vector may comprise additional heterologous sequences, such as sequences that facilitate propagation or transformation. Such sequences may be control elements for agroinfection, such as a binary vector, which are well known in the art. The vector may have the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, either as provided herein or with different heterologous sequences replacing the reporter genes provided as examples (GFP and GUS). The leader proteases of the vector may be L1 (SEQ ID NO: 4) and/or L2 (SEQ ID NO: 6) from LR-2 and may be encoded by SEQ ID NO: 3 and 5, respectively. Further, a suppressor of RNA silencing may also be introduced with the vector. Such suppressors may be LR-2 p24 or p21 from Beet yellows virus (Chiba et al., Virology 346: 7-14, 2006).
The vector includes a heterologous polynucleotide operably linked to a promoter. The promoter may be a native LR-2 promoter, e.g. LR-2 CP promoter, or it may be a heterologous promoter, such as a promoter from other viruses that belong to genus Closterovirus, such as Beet yellows virus, e.g. BYV CP promoter, Beet yellow stunt virus, Carnation yellow fleck virus, Citrus tristeza virus, Mint virus 1, etc. Although CP promoter of these viruses normally provides highest expression levels, several additional promoters from these viruses might be useful. Many suitable promoters are known in the art. The heterologous polynucleotide may encode a reporter molecule, a selectable marker and/or a therapeutic gene. Examples of reporter molecules include fluorescent proteins, such as green fluorescent protein, luciferase, beta-galactosidase, beta-glucuronidase, and other reporter molecules known in the art. Examples of selectable markers also include antibiotic resistant markers, epitope tags useful for affinity purification and the like. Examples of therapeutic genes include those that confer resistance to a pathology or disease.
The present disclosure encompasses methods for inducing disease resistance in a plant or plant cell by introducing the plant gene expression vector of the present disclosure into the cell. Such diseases of grape plants include, but are not limited to fungal agents, bacterial agents, viral agents, parasites and environmental stress. Examples of fungal diseases include Guignardia bidwellii (black rot), Plasmopara viticola (downy mildew), Erysiphe necator (powdery mildew, formerly Uncinula necator), Botrytis cinerea (bunch rot), Sclerotinia sclerotiorum (Sclerotinia shoot rot), Eutypia armenicae (Eutypia dieback), Elsinoe ampelina (anthracnose), Alternaria alternata (alternia rot), Pseudopezicula tetraspora, Cercospora brachypus, Sphaceloma ampelinum, Ascochyta sp. Aspergillus aculeatus, Cladosporium spp., Fusarium spp., Helminthosporium spp., Monilia sp., Stemphylium botryosumv, Pleospora tarda, Torula sp., Greeneria uvicola, Botryosphaeria stevensii, Diplodia mutila, Penicillium spp., Botryotinia fuckeliana, Anthostomella pullulans, Clostridium spp., Libertella blepharis, Lasiodiplodia theobromae, Rosellinia necatrix, Dematophora necatrix, Roesleria subterranea, Mycosphaerella personata, Pseudocercospora vitis, Isariopsis clavispora, Briosia ampelophaga, Phymatotrichopsis omnivora, Dematophora necatrix, Cristulariella moricola, Grovesinia pyramidalis, Cephalosporium spp., Phellinus igniarius, Stereum hirsutum, Physopella ampelopsidis, and Phompopsis viticola (phomopsis leaf and cane spot disease).
Examples of bacterial diseases include Xylella fastidiosa (Pierce's disease), Agrobacterium tumefaciens (crown gall), Pseudomonas syringae, and Xylophilus ampelinus (bacterial blight).
Examples of viral diseases include grapevine leafroll-associated viruses, grapevine fanleaf virus, grapevine virus A, grapevine virus B, tomato ringspot, Rupestris stem pitting associated virus, tobacco ringspot, Arabis mosaic virus, Artichoke Italian latent virus, Alfalfa mosaic virus, Bratislava mosaic virus, Broad bean wilt virus, grapevine strawberry latent ringspot virus, tobacco necrosis virus, tobacco mosaic virus, grapevine chrome mosaic virus, petunia asteroid mosaic virus, and Tomato black ring virus.
Examples of pests include Daktulosphaira vitifoliae, Lepidoptera, Otiorhynchus sulcatus, Tylenchulus semipenetrans, Xiphinema spp., Pratylenchus spp., Longidorus spp., Paratylenchus hamatus, Paratylenchus hamatus, Rotylenchulus spp., Criconemella xenoplax, Meloidogyne spp., Helicotylenchus spp., Paratrichodorus christiei, and Tylenchorhynchus spp.
Examples of environmental stress include drought, heat stress, salt stress, iron deficiency, zinc deficiency, ozone, and environmental toxins. Alternatively, the present vector may be used to confer resistance to herbicides, fungicides, pesticides and viricides.
Pierce's disease (PD), caused by an infection by the bacterium Xylella fastidiosa, is of particular importance to growers of grape cultivars. The bacteria multiply in the plant xylem, causing blockage of water movement and the characteristic leaf blight or necrosis, particularly during heat or water stress. Management presently focuses on prevention of infection by targeting the insect carrier which transmits the bacteria from plant to plant during feeding. A number of insect vectors are known, including a variety of sharpshooters, such as the glassy winged sharpshooter. Many commercially important cultivars are susceptible to PD, thereby making control of this disease urgently needed.
Treatments for such diseases can include the expression of therapeutic proteins, such as the genes of pathogenic or infectious organisms or repression of host genes to mitigate an undesired response. Examples of therapeutic proteins include proteins that target the pathogenic organism or agent directly, such as chitinases, which degrade the protective fungal walls (e.g., Adams, Microbiology 150: 2029-2035, 2004), replicases, which inhibit replication (e.g. WO 98/052964), lysozymes, which attack bacterial walls, or genes that have been found in disease resistant plants. Lysozymes have been shown to have antibacterial properties, and may be effective against Xyllela. U.S. Appl. No. 20020104126, which is hereby incorporated by reference, shows that bovine lysozyme, which is active at a very low pH, may be expressed in tobacco plants and retain activity in in vitro tests. However, the feasibility of expressing lysozyme in woody plants, namely grapevines, or its effectiveness against Xyllela or treating PD was untested. The grapevine powdery mildew resistance gene, Run1, has been shown to confer resistance to susceptible V. vinifera when transferred through a pseudo-backcross strategy and delivery using a bacterial artificial chromosome (Barker et al., Theor. Appl. Genet. 111:370-377, 2005). Therefore, Run1 and other genes associated with disease resistance are suitable for use in the methods and constructs/vectors described herein.
Alternatively, the present vector can be used to induce RNA silencing (virus-induced gene silencing or VIGS) to repress undesired gene expression, such as expression of genes from pathogenic organisms or undesired host genes. VIGS is a phenomenon well known in the art as a mechanism used to examine the functions of plant genes (Godge et al., Plant Cell Rep 27(2):209-219, 2008; e-pub ahead of print, 2007). To induce VIGS, the vector may encode a portion of a nucleotide sequence from the targeted gene. The nucleotide sequence may be from any portion of the transcript expressed from the gene, including a protein coding region or untranslated sequences.
Another embodiment of the present disclosure is the use of the inventive vector to introduce genes that are involved in aesthetic modification of the edible portions of the transduced or transgenic plant, such as taste or aroma of the juice. Such edible portions include the roots, stems, leaves, flowers, seeds and/or fruit of the plant, or any edible substance derived therefrom. In particular, grapes, or the juice derived therefrom, may be modified such that agents that interact with the taste receptors to block or enhance certain tastes, such as bitterness, sweetness, sourness and the like. Alternatively, the agents enhance the aromatic compounds found in the grapes or juice. Such agents may be proteins, such as monellin, thaumatins, gustducin, terpenoids and other such proteins known in the art. For aesthetic modification, the vector of the present disclosure may be engineered to encode an aesthetic modifying molecule, such as a protein, and the vector is then used to transform a plant cell, which then expresses the molecule.
The methods and vectors of the present disclosure may also be used to introduce genes that are involved with the metabolic pathways of grapes (metabolomics) for the improvement of nutritional characteristics. For example, resveratrol, a polyphenolic compound (3,4′,5-trihydroxystilbene) found in grapes, has been found to have certain health benefits, such as anti-cancer properties and association with a reduction in cardiovascular disease. Other compounds found in grapes with potential health benefits include phytonutrients such as quercetin, catechins, anthocyanins and proanthocyanidins. The sequencing of the pinot noir grape varietal as well as development of metabolic profiling techniques, such as nuclear magnetic resonance spectroscopic techniques, provide valuable information for developing genes suitable for use with the vectors and expression systems described herein. Additional genes of interest are involved in ripening, such the fib gene, as well as any aspect of plant growth, development, and agricultural production that may be desired.
The present disclosure encompasses methods for expressing a heterologous gene in a plant cell by introducing a described plant gene expression vector of into the cell. The plant gene transfer vector may be introduced into the plant cell by any of the methods known in the art, for example by vacuum infiltration, sonication, ballistically, calcium phosphate precipitation, electroporation, polyethylene glycol fusion, direct transformation (Lorz et al, Mol. Genet. 199:179-182, 1985) and other methods. The vector may be introduced as an infectious viral particle or as a noninfectious nucleotide. One method is agroinoculation, for example, incorporating the vector into a binary plasmid with suitable control elements for expressing the vector in an Agrobacterium species, such as A. tumefaciens, A. rhizogenes, and A. vitis. Such methods are known in the art, such as those described in Leiser et al., Proc. Natl. Acad. Sci. USA 89:9136-9140, 1992 and Bouquet et al, Methods in Mol. Bio. 344: 273-285, Agrobacterium Protocols, 2nd ed. vol. 2, Wang ed (2006), both of which are incorporated by reference. Agrobacterium may be introduced into entire micropropagated grapevine plants by means of vacuum infiltration or sonication. Agrobacterial strains engineered to express viral vector may be mixed with another strain engineered to express viral suppressor of RNAi in order to increase vector infectivity (Chiba et al., Virology 346: 7-14, 2006).
The vector may be introduced for stable expression or transient expression. Stable expression may be achieved by a variety of known methods, such as co-culturing embryonic grape tissue with Agrobacterium containing the vector using the methods of Bouquet et al. (Methods Mol. Biol. 344:273-285, 2006). Transient expression can also be achieved by known methods, such as the methods described in the Examples of the present application. Other methods include, but are not limited to, leaf infiltration, vacuum infiltration and bombardment of target tissues with DNA-coated particles.
The vector may also be applied to the grapevine or any part thereof through application of a solution containing the vector, such as by spraying. Such solutions contain the vector as DNA, DNA coated particles or contained with Agrobacterium, as well as salts and buffers. The solution may contain, for example, a phosphate buffer at 0.1 M, pH 7 or it may contain nicotine. Such formulations are well known in the art and may be used as suitable for the present methods. The solution may further contain an abrasive, such as carborundum, for example 500-mesh carborundum, kaolin or Celite® diatomaceous earth. The solution may also comprise a surfactant, such as Triton or Tween, which are well known in the art. The solution may be applied to the plant via swabbing, dripping, immersion, spraying or other means. See, e.g., Graft transmissible diseases of grapevines. Martelli ed. 1993, Rome, Italy, Food and Agriculture Organization of the United Nations publ.
The present disclosure further includes compositions comprising the vector(s) described herein. In addition to the vector and optionally other functional ingredients such as abrasives and/or surfactant, such compositions may include excipients suitable for introducing the vector into a plant cell, such as salts, e.g., magnesium chloride, and buffers, such as MES or phosphate, and are typically aqueous solutions. See, e.g., Graft transmissible diseases of grapevines. Martelli ed. 1993, Rome, Italy, Food and Agriculture Organization of the United Nations publ.
The vector may be applied, daily, monthly, seasonally, or annually. The vector may be applied during or prior to the onset of disease or the infestation of the disease carriers. For example, symptoms of disease may be read twice a year: late spring for leaf and cane deformations and necrosis and autumn for abnormal pigmentation and other deformities (Graft transmissible diseases of grapevines. Martelli ed. 1993, Rome, Italy, Food and Agriculture Organization of the United Nations publ). Upon identification of symptoms, the vector can by applied, such as by spraying with an abrasive-containing solution. Alternatively, the plants may be sprayed upon infestation with a known carrier of a disease, such as certain nematodes or grapevine leafhoppers, or upon certain weather conditions or seasons known to induce conditions favorable to a disease, such as wet weather and powdery mildew. Guidelines and indices for such conditions are well known in the art, such as the University of California Pest Management Guidelines, Statewide Integrated Pest Management Systems, available on the World Wide Web at.ipm.ucdavis.edu/PMG/r302101211.html (last accessed Jan. 27, 2008), which is hereby incorporated by reference. Further, the present vector may be applied with other known anti-disease agents, such as oils, fungicides and the like.
The present disclosure includes a method for transforming a plant comprising introducing the described plant gene transfer into the plant once or more than once. The vector may be introduced two, three, four, five, six, seven, eight, nine, ten or more than ten times to the plant. The plant may express the heterologous polynucleotide systemically or locally.
The plant cell may be grown into a transgenic plant, such as using the methods of Bouquet et al. (Methods Mol. Biol. 344:273-285, 2006). Alternatively, the plant cell may be part of a multicellular plant such that only a portion of the plant is transformed. For example, the root stock, the stem or the leaves may be transformed. The vector may be introduced prior to onset of disease to confer resistance, or it may be introduced after disease is observed to reduce or ameliorate the disease, to protect the remaining uninfected portions of the plant or to prevent the spread of the disease to other plants.
ADDITIONAL REFERENCES


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The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described.
EXAMPLES
Search for a local virus isolate. A survey of local vineyards was performed to determine if LR-2 is naturally present in Oregon, and vines were found that exhibited LR-2-like symptoms of early leaf reddening Immunoblot analysis was performed with the commercially-available LR-2 immunodetection kit (Bioreba AG, Reinach, Switzerland; Cat. No 120775) and custom antibodies generated using the LR-2 virions isolated from N. benthamiana as described below. RT-PCR analysis using the primers designed to amplify  ̃2 kb region of LR-2 genome containing viral capsid protein gene also demonstrated that the vines indeed contained the virus.
Molecular cloning and nucleotide sequencing. To facilitate accumulation and isolation of LR-2, material from infected vines was used to mechanically inoculate N. benthamiana. Purified virus particles were used to obtain viral RNA, amplify it by RT-PCR and clone the cDNA products into pBlueScript vector plasmid. The terminal sequences of the viral genome were cloned using RLM-RACE. A large number of the independent clones were sequenced to provide multiple coverage of the viral genome. The resulting consensus nucleotide sequence for the first time included the entire viral genome and contained exact 5′-terminal and 3′-terminal regions. Comparative analysis of this sequence revealed 99.9% identity with the previously published incomplete sequence of the New York LR-2 isolate over the regions of overlap (Zhu et al., J. Gen. Virol. 79: 1289-1298, 1998). A functional map of the  ̃16,500 nucleotides-long LR-2 genome is shown in FIG. 1A.
The initial inoculation of N. benthamiana with LR-2 was performed as described by Goszczynski et al. (Vitis 35:133-135, 1996). Subsequently, infected leaves of N. benthamiana were used for making inoculum for virus propagation on this experimental virus host. Routinely, 1 g of infected tissue was ground in 5 ml of 0.05 Na-phosphate buffer, pH 7.0, and used for manual inoculation of the new plants with the aid of carborundum.
RT-PCR amplification, cloning, and assembly of the full-length cDNA clone were performed as described in Peremyslov and Dolja (Curr. Protocols Microbiol., “Cloning of Large Positive-Strand RNA Viruses” Suppl. 7., Coico, ed., November, 2007), which is hereby incorporated by reference.
Insertion of the gene expression cassette. A strong sub-genomic RNA promoter that drives expression of the major capsid protein in LR-2 relative, Beet yellows virus (BYV), was cloned and inserted downstream from analogous LR-2 promoter along with unique restriction sites Pac I and Fse I into one of the partial LR-2 cDNA clones. A reporter gene encoding green fluorescent protein targeted to endoplasmic reticulum (ER-GFP) was inserted between the two promoters (FIG. 1B). This design was expected to result in a high-level production of ER-GFP from authentic LR-2 promoter and LR-2 capsid protein from a recombinant BYV promoter described in (Agranovsky et al., J. Gen. Virol. 72:15-23, 1991) and illustrated in SEQ ID NO: 1.
Generation of a full-length cDNA clone of LR-2. Using overlapping partial cDNA clones, several prototype full-length LR-2 clones containing expression cassette were assembled in pBlueScript® (Stratagene, La Jolla, Calif.) under control of phage SP6 RNA polymerase promoter. Corresponding capped RNA transcripts were obtained in vitro and used to transfect tobacco suspension culture protoplasts and inoculate N. benthamiana plants. Because none of these experiments resulted in virus multiplication and infection, the cloned viral genome was re-sequenced. Multiple detrimental mutations throughout the genome were detected suggesting that errors were introduced during RT-PCR amplification of genome fragments. To overcome this major obstacle, the entire LR-2 genome was reassembled from the cDNA fragments obtained using reverse transcription and Klenow DNA polymerase instead of PCR. Although this approach resulted in a dramatic reduction in the number of lethal mutations, none of the resulting clones were entirely free of them. These results indicated that LR-2 cDNA in functional, mutation-free form was likely toxic to E. coli used for cloning.
One of the approaches that may alleviate the toxicity of recombinant DNA is to use a low-copy number plasmid for the cloning. To test this possibility, pCB-302 (Xiang et al., Plant Mol. Biol. 40: 711-717, 1999), a mini-binary vector suitable for cloning in both E. coli and A. tumefaciens was used. This vector has been modified to accommodate all control elements required for subsequent expression of LR-2 genome in plants using agroinoculation. These elements included 35S RNA polymerase promoter, NOS terminator, and a ribozyme custom designed for a release of an authentic 3′-terminus of LR-2 RNA upon its transcription. The latter processing event is critical for the ability of viral RNA to replicate in the plant. Following these modifications, the full-length LR-2 cDNA was cloned into pCB-302 and designated plasmid pCB-LR-GFP (SEQ ID NO: 1).
The LR-2 cDNA was cloned between the right and left T-DNA borders of plasmid pCB-302. A map of the resulting plasmid is shown in FIG. 1C. Nucleotide sequencing of the entire cloned viral genome revealed no detrimental mutations.
Infectivity assays in N. benthamiana. pCB-LR-GFP was transformed into agrobacterium and the resulting strain was grown, induced, and used for agroinfiltration of the N. benthamiana plants. On average at 2-3 weeks post inoculation, some of the plants exhibited typical infection symptoms. Confocal laser scanning microscopy detected strong GFP expression in the epidermal cells of the infiltrated leaves. As expected, GFP fluorescence was confined to a characteristic network of endoplasmic reticulum (ER) and ER-derived viral replication complexes (FIG. 2). Examination of the upper leaves, stems, and petioles of the symptomatic plants revealed high levels of ER-GFP accumulation in the phloem tissues throughout the plant that is typical of LR-2 and other closteroviruses (FIG. 3). Ultra-structural analysis of the infected tissue confirmed accumulation of the vast amounts of filamentous LR-2 virions in the phloem cells, especially in the bundle sheath cells that surround xylem and sieve elements. Furthermore, immunoblot analysis confirmed efficient accumulation of LR-2 capsid protein (FIG. 4A). These experiments demonstrate that the cloned, recombinant LR-2 is highly infectious, is able to spread systemically, and express a recombinant protein in an experimental host N. benthamiana. 
Improvement of the agroinfection efficiency. Although LR-2 infection consistently occurred upon agroinoculation, the number of primarily-infected cells in the inoculated leaves was low and only fraction of the inoculated plants developed systemic infection. To enhance the ability of recombinant virus to establish infection, a method based on the expression of viral suppressors of RNAi at the time of inoculation was used (Chiba et al., Virology 346: 7-14, 2006). Furthermore, it was demonstrated that LR-2 encodes a very potent suppressor of RNAi, p24. Therefore, agrobacterium that was engineered to express p24 was added to inoculum. This technique resulted in a  ̃5,000-fold increase in the specific infectivity of the virus launched by agrobacterium (FIG. 5) (Chiba et al., Virology 346: 7-14, 2006). Now 100% infection of the agroinoculated N. benthamiana plants is routinely obtained.
Genetic stability of the LR-2 gene expression vector. To test if the LR-2 vector is able to retain an intact foreign insert encoding reporter protein, RNAs have been isolated from the top leaves of the four individual infected N. benthamiana plants. RT-PCR has been done using these RNAs and primers located downstream and upstream from the ER-GFP expression cassette. The resulting PCR products were of expected size indicating retention of the entire cassette in each of the tested plants (FIG. 4B) Importantly, no shorter products that could originate from partial or complete loss of the cassette were detected. These data confirmed that the engineered vector is genetically stable and can be used for efficient expression of the recombinant proteins.
Development of gene silencing vector for functional genomics and virus control. To test a potential of LR-2 vector for virus-induced gene silencing (VIGS), GFP-transgenic N. benthamiana line 16c and GFP-expressing LR-2 was used. Strong GFP silencing was observed in the systemically infected leaves of 16c plants following inoculation with LR-2-GFP (FIG. 6). This experiment demonstrated that LR-2-GFP acts similarly to known VIGS vectors by inducing RNAi of the transgene in response to overexpression of the cognate gene by virus, providing proof of concept for the utility of LR-2 vector for VIGS.
Micropropagation of grapevine and transfer to soil. A reliable supply of experimental grapevine plants year round was developed for testing the new vectors using a technique for micropropagation of grapevine under sterile conditions in plastic boxes using agar-based growth medium. Such plants can be used for infectivity assays directly. Furthermore, there are anecdotal reports indicating that micropropagated plants have increased susceptibility to viral infection. Alternatively, micropropagated plants can be transferred to soil and grow in the greenhouse to obtain woody plants that are more similar in their susceptibility to plants grown in the open soil. Conditions were optimized for such transfer and obtained constant supply of the micropropagated and greenhouse grapevine plants.
Briefly, 12 inch cuttings with at least two buds were bundled into groups of 20-30 and soaked in a 10% bleach solution for 10 minutes, rinsed three times in water, then immersed in water for 12-24 hours. The bundles were then dipped in rooting solution (Dip'N Grow, Dip'N Grow Inc., Clackamas, Oreg.) and planted in moist vermiculite warmed to 85° F. Rooting occurred in 3-4 weeks. The cuttings were then planted 4-6 inches deep in potting soil under a misting bed set for three seconds every eight solar units (daytime) and 3 seconds every 2 hours (nighttime). Once leaves appeared in approximately two weeks, misting was decreased to prevent mold and mildew growth.
To establish grapevines in tissue culture, new shoots were removed and all but one leaf was removed from the shoot tip. The shoots were incubated in 0.05% Ivory liquid soap (Proctor and Gamble, Cincinnati, Ohio) for 10 minutes, rinsed in water for one hour, then sonicated for ten minutes in a sonicating water bath. The shoots were surface sterilized in a 5% bleach solution for six minutes and moved to a laminar flow hood for sterile handling. The shoots were rinsed in sterile deionized water, trimmed at the base and planted in OH agar medium. The shoots were incubated in a growth chamber for two weeks; GS 1 liquid was added, incubated for another two weeks, and then transferred to GS 1 agar medium. Every two weeks, fresh medium was added, then after a month, the shoots were switched to the next stage medium (GS2, GS3). Roots developed in GS3 medium.










Grape OH Medium










Ingredient
Concentration g/L















M & S salts
3.22
g



Thiamine
0.8
ml stock ( )



Inositol
0.100
g 0.5 mg/ml



Sucrose
7
g



NaH2PO4
0.170
g



Adenine sulfate
0.08
g



Adjust pH to 5.7



Agar
3
g










Autoclave for 20 minutes, cool in water bath at 50° C. for 30 minutes then add antibiotic: Cefotaxime 0.2 g. Filter sterilize into cooled medium and mix. Dispense to sterile test tubes.










Grape Stage 1 (GS1) Medium










Ingredient
Concentration g/L















M & S salts
3.22
g



Thiamine
0.8
ml stock (0.5 mg/ml)



Inositol
0.1
g



Sucrose
20
g



BAP
2
mls stock (0.5 mg/ml)










Autoclave for 20 minutes, cool in water bath at 50° C. for 30 minutes then add antibiotic: Cefotaxime 0.2 g. Filter sterilize into cooled medium and mix. Dispense to 24 sterile Magenta boxes.










Grape Stage 2 Medium










Ingredient
Concentration g/L















M & S salts
3.22
g



Thiamine
0.8
ml stock (0.5 mg/ml)



Inositol
.025
g



Sucrose
15
g



NaH2PO4
0.05
g



BAP
4
mls stock (0.5 mg/ml)



IAA
0.5
mls IAA stock (1 mg/ml)



Adjust pH to 5.3



Agar
2
g



Gelrite ® gellan gum
1.2
g










Autoclave for 20 minutes, cool in water bath at 50° C. for 30 minutes then add antibiotic: Cefotaxime 0.2 g. Filter sterilize into cooled medium and mix. Dispense to 24 sterile Magenta boxes.










Grape Stage 3 (GS3) Medium










Ingredient
Concentration g/L















M & S salts
3.22
g



Thiamine
0.8
ml stock (0.5 mg/ml)



Inositol
.025
g



Sucrose
12.5
g



NaH2PO4
0.05



IAA
1
ml stock (1 mg/ml)



Adjust pH to 5.3



Agar
1
g



Gelrite ® gellan gum
1
g










Autoclave for 20 minutes, cool in water bath at 50° C. for 30 minutes then add antibiotic: Cefotaxime 0.2 g. Filter sterilize into cooled medium and mix. Dispense to 24 sterile Magenta boxes or 12 Double-Decker Magenta boxes.
Numerous grape varieties are being micropropagated and examined for their susceptibility to agroinfiltration and foreign protein expression. Cabernet franc and Sirah varieties have demonstrated desirable properties. Further, sterile plants transferred to antibiotic-free medium have been preliminarily found to be more susceptible to agroinfiltration.
Agrobacterium preparation. To prepare the Agrobacterium, the agro construct was streaked onto LB Kan (50 μg/ml) agar plate and incubated at 28° C. for three days. Single colonies were selected and added to a 5 ml culture on LB Kan (50 μg/ml), MES (10 mM) and acetosyringone (20 μM), and shaken at 220 rpm and 28° C. Rifampicin (50 μg/ml) was added if Agro strain C58 GV2260 was used. The starting cultures were transferred to 500 ml LB Kan (50 μg/ml), MES (10 mM) and acetosyringone (20 μM) and shaken overnight (1-20 hours) at 28° C. The culture was centrifuged at 6000 rpm for 10 minutes at room temp and the cell pellet suspended in 20 ml induction buffer for a final concentration of 2.0 OD600 for full virus constructs or 0.7 OD600 for GFP marker only constructs by combining with 0.1-0.14 OD600 for suppressor p24 and adding additional induction buffer.
To prepare the suppressor p24 construct, the Agro stock was prepared as above, then suspended in 20 ml induction buffer at a final concentration 0.1 OD600 to be combined with full virus construct suspension or 0.14 OD600 to combined with GFP marker only suspension.
Induction Buffer

    
    
        10 mM MgCl2=2 ml of 0.5 MgCl2 
        10 mM MES pH 5.85=2 ml of 0.5 M MES pH 5.85 per 100 ml
        150 μM acetosyringone=100 μl of 150 mM acetosyringone
    
    


Agrobacterium Infiltration of Micropropagated Grapevine Leaves or Entire Plants. Healthy leaves from micropropagated plants were wounded with 31 g needle by poking the large veins and leaf surface. Single leaves were placed in tubes with 5-10 ml of induction suspension, or the full plant was loosely in large beaker with 200 ml induction suspension. The grapevine/agro suspensions were sonicated for 10 minutes in a Branson 3510 sonicating water bath, the soaked in induction suspension for 2-3 hrs after sonication. The grapevine leaves or plants were blotted on sterile paper towels, then leaves were placed on water agar plates (7 g agar/1 L water) and whole plants potted in 4 inch pots containing potting mix and watered liberally. Plastic cups were placed tightly over the plants to reduce transpiration in the growth chamber. Ambient air was gradually introduced to potted plants by tilting the angle of the cups over a two week period until eventually removing plastic cups.
Leaves were monitored for GFP expression by microscope after day 4 for GFP marker only constructs and after day 12-14 for full viral/GFP constructs.
Similar techniques may be used with other portions of the plant, such as the rootstock.
Expression of GFP in grapevine using agrobacterium. In parallel with the efforts to generate infectious clone of LR-2, experiments aimed at developing technologies for agrobacterium-mediated transient expression of recombinant genes in grapevine were conducted. These experiments were designed to facilitate future infectivity tests in grapevine. Using agrobacterium engineered to express free GFP reporter (FIG. 7C) and a technique of vacuum infiltration of bacterial suspension into leaves or entire plants, accumulation of GFP in micropropagated plants was demonstrated. To this end, detached agroinfiltrated leaves were kept in Petri dishes over water agar in a plant growth chamber at 22° C. for 4 days and screened for GFP expression using epifluorescent stereoscope Leica MZ 16F equipped with GFP2 filter and digital camera (as shown in FIGS. 7A and B) or confocal laser microscope. Confocal laser scanning microscopy was done using Zeiss LSM 510 META (Zeiss, Germany) microscope fitted with the 488 nm excitation and 508 nm emission filters. The software package provided by manufacturer was used for image processing.
An alternative technique of agroinoculation using immersion of the plants into ultrasonic bath with bacterial suspension was also tested as described above. This approach proved to be as successful as infiltration with additional benefit of simplicity (FIG. 7A). Moreover, sonication resulted in a frequent expression of GFP in the vascular leaf tissue (FIG. 7B). Because LR-2 is preferentially associated with the phloem, this technique is expected to facilitate infection.
Additional testing utilizing Agrobacterium vitis and A. rhizogenes isolates is being conducted to determine if these bacteria may provide better efficiency of agroinoculation in grapes compared to traditional A. tumefaciens. Further, optimization of the protocols for agroinfiltration and sonication, testing of the utility of agroinfection combined with grafting (Omega-grafted cuttings treated with bacterial suspension) and a peel-heal agroinoculation technique whereby bacterial suspension is applied to the phloem exposed by peeling the bark is ongoing to improve transformation.
Infectivity assays in grapevine. Two different LR-2 clones were used for agroinoculation: i) miniLR-GFP/GUS (this mini-genome includes only the genes required for replication plus a reporter GFP/GUS gene and lacks genes required for virion assembly and transport in plants; SEQ ID NO: 2). The GFP/GUS reporter represents a fusion of GFP that can be detected by epifluorescent microscopy and GUS (β-glucuronidase) that possesses enzymatic activity providing sensitive in situ and in vitro assays. The map of miniLR-GFP/GUS is shown in FIG. 8; the corresponding nucleotide sequence is provided as SEQ ID NO: 2; and ii) full-length LR-2-GFP (SEQ ID NO: 1). When the micropropagated plants agroinoculated with miniLR2-GFP/GUS mixed with p24-expressing plasmid were screened, large numbers of infected cells that expressed ER-GFP reporter in the ER were observed (FIG. 8) demonstrating an ability of the mini-vector to replicate and express reporter protein in the grapevine leaf cells. Therefore, each GFP-positive cell detected by epifluorescent or confocal laser scanning microscopy represents a successful event of launching the viral vector and obtaining expression of the reporter by the viral vector in this case presented by the mini-replicon.
However, similar experiments with the full-length vector resulted in infection of the limited number of cells (FIG. 9). In fact, in previous work with BYV, it was found the mini-genome had much higher infectivity upon agroinfection (Chiba et al., Virology 346: 7-14, 2006).
Further development of the full-length LR-2 vector. The original full-length LR-2 clone was generated using LR-2 genomic RNA isolated from the virus that was propagated in N. benthamiana. To improve grapevine infectivity and decrease possible mutation due to virus adaptation to an experimental host, the full-length clone was reassembled from cDNA fragments derived directly from LR-2 infected vine of Pinot Noir obtained from a local Oregonian vineyard.
Total RNA was purified from leaves using a Plant RNeasy kit (QIAGEN) and used as a template for random-primed cDNA construction. 2 to 4 kb contiguous fragments were then PCR amplified from this cDNA. Oligonucleotides for the PCRs were designed based on the published sequence of the GRLaV-2 and overlapped unique cloning sites present in the cDNA of this virus. The amplified PCR fragments were then cloned into a binary plasmid carrying the original variant of the LR-2 cDNA to replace existing parts with the ones derived from a virus present in grapevine. For each of the fragments at least 4 clones were sequenced to deduce consensus sequences that correspond to predominant and fully biologically active variant of LR2 genome present in grapevine. The complete genomic cDNA comprised of the consensus pieces was reassembled de novo. The new binary plasmid carrying the cDNA for the grapevine-derived virus derived from the Pinot Noir and never passed through N. benthamiana was designated LR2-Vitis. 
The entire nucleotide sequence of this grapevine-derived LR2-Vitis expression cassette is shown in SEQ ID NO: 7). This sequence contains 74 nucleotide differences from an original, N. benthamiana-derived viral cassette (FIG. 14). It seems likely that these differences reflect adaptations of the virus to the systemic infection of either natural (grapevine) or experimental (N. benthamiana) host plant. Therefore, it is expected that the grapevine-derived LR2-Vitis vector will possess an increased ability of propagation in the grapevine.
Utilization of the phloem-specific promoters of V. vinifera. The LR-2 and LR2-Vitis vectors are launched using the CaMV 35S RNA polymerase II (POL II) promoter that drives the transcription of viral RNA upon agroinoculation. Although 35S promoter is routinely used for such purposes, it could be suboptimal for grapevine infection using LR-2 vectors. Indeed, LR-2 is naturally infects grapevine and is limited to the phloem tissue, whereas 35S promoter is not specific to either grapevine or phloem. Therefore, we used bioinformatics to identify the candidate grapevine phloem-specific promoters that can be used for replacement of the 35S promoter in order to improve grapevine infection by LR2-Vitis. 
Three highly-expressed, phloem-specific A. thaliana genes were identified, and the corresponding protein sequences and BLASTP search were used to identify apparent V. vinifera orthologs. The V. vinifera genomic sequences upstream from corresponding protein-coding sequences (SEQ ID NOs: 8-10) are proposed to be useful as phloem-specific promoters in place of the exemplified 35S promoter in a LR2-Vitis cassette. The specific nature and properties of the V. vinifera promoters are outlined below.
1. The promoter of the A. thaliana SUCROSE TRANSPORTER 2 (SUC2 sucrose-H+ symporter) gene (At1g22710) was first characterized by Truernit and Sauer (Planta 196:564-570, 1995). In Arabidopsis, this promoter regulates expression of the phloem companion cell—specific AtSUC2 sucrose—H+ symporter gene in the entire veinal network of fully developed leaves (Imlau et al., Plant Cell 11:309-322, 1999). Imlau et al. was also found that a 939 nt 3′ fragment of this promoter is sufficient to drive phloem-specific, high-level expression of a reporter gene
2. Arabidopsis gene At5g57350 codes for a plasma membrane H(+)-ATPase 3 (proton pump) and is expressed in phloem throughout the plant (DeWit et al., Plant J. 1, 121-128, 1991). The 2,467-kb promoter fragment of this ORF was sufficient to drive gene expression in phloem companion cells present in leaves, stems, and roots (HongYu et al., Chinese Science Bulletin, 52: 1949-1956, 2007).
3. Arabidopsis Sucrose synthase 1 gene (At5g20830). A 2 kb promoter region of this ORF fused to a reporter gene directs its expression in the phloem of mature leaves (Bieniawska et al., The Plant Journal 49, 810-828, 2007). The mRNA 5′-end begins with ATCTTA (Martin et al., Plant J 4: 367-377. 1993) which is very close to the 5′-terminal sequence of LR-2 making this promoter a very attractive candidate for improvement of infectivity of LR2-Vitis in grapevine.
The nucleotide sequences of the grapevine promoters located upstream from the grapevine ORFs encoding apparent orthologs of the Arabidopsis genes At1g22710, At5g57350, and At5g57350 are shown in SEQ ID NOs: 8-10 (respectively), along with database identifiers providing their respective positions in the V. vitis genome.
Characterization of the Effects of Leader Proteases on Replication.
Generation of GLRaV-2 Replicons Tagged by Insertion of the Fluorescent, Enzymatic, and Epitope Reporters
Clones for GLRaV-2 were generated to determine functional profiles of L1 and L2. The entire, 16,486 nt-long GLRaV-2 genome was sequenced (GenBank accession number FJ436234; gene ID is: gi:213958313; incorporated herein by reference as of Jan. 26, 2009) and compared to the other isolates of this virus to reveal the closest relationship (99.6% nt identity) to the isolate 94/970 (Meng et al., Virus Genes 31, 31-41, 2005). The initial full-length clone was assembled using a binary vector and primarily conventional cDNA cloning to avoid introduction of the PCR-generated mutations, and sequenced to confirm its correspondence to the consensus nucleotide sequence of the viral genome. To facilitate launching of viral infection by agroinoculation, 35S RNA polymerase promoter of Cauliflower mosaic virus (CaMV) and a ribozyme sequence were inserted upstream and downstream of the GLRaV-2 sequence, respectively.
The resulting full-length GLRaV-2 clone was further modified to accommodate a reporter gene expression cassette immediately upstream of the CP open reading frame. This cassette contained GFP open reading frame followed by the BYV CP sub-genomic RNA promoter. As a result, the latter promoter directed expression of the GLRaV-2 CP, while the authentic GLRaV-2 CP promoter expressed the GFP reporter. This tagged full-length GLRaV-2 replicon was designated LR-GFP (FIG. 10A, middle diagram).
Deletion of the genes that are not required for the viral RNA amplification in the individual cells facilitates experimentation with the remaining genes that code for the leader protease, RNA replicase and RNAi suppressor. In the case of GLRaV-2, such minireplicon was generated by deletion of the gene block spanning genome region from p6 to p19 open reading frames and retention of the reporter gene. The reporter expression cassette was further modified to express a fusion of GFP with b-glucuronidase to result in the tagged GLRaV-2 minireplicon designated mLR-GFP/GUS (FIG. 10A, bottom diagram).
To permit immunochemical detection of L2HA using commercial HA-specific monoclonal antibody, both LR-GFP and mLR-GFP/GUS were modified by an insertion of the triple hemagglutinin epitope (HA) tag into the N-terminal domain of L2 (FIGS. 10B and 11A). Infectivity of the full-length and minireplicon variants was tested using leaf agroinfiltration of N. benthamiana, a systemic experimental host of GLRaV-2 (Goszczynski et al., Vitis 35, 133-133, 1996). For mLR-GFP/GUS, such agroinfiltration resulted in minireplicon RNA accumulation and efficient expression of the fluorescent and enzymatically-active GFP/GUS reporter in the initially inoculated cells (FIG. 10B). Importantly, the level of GUS activity in a HA-tagged variant was  ̃85% of that in the original mLR-GFP/GUS. Because this modest reduction was only marginally statistically significant (p value  ̃0.001), it was concluded that the insertion of HA tag into L2 did not significantly affect viral genome amplification. Attempts to insert an HA tag into L1 resulted in non-infectious replicons and were abandoned.
Both the original and HA-tagged variants of the full-length LR-GFP were systemically infectious in N. benthamiana; typical symptoms of the viral infection and GFP fluorescence were detected in the upper non-inoculated leaves by 3 weeks post agroinfiltration of the bottom leaves (FIG. 10A). Therefore, a series of the infectious tagged GLRaV-2 replicons were generated that can be launched to N. benthamiana and used to address L1 and L2 functions in the viral infection cycle.
Mutation Analysis of the L1 and L2 Functions in Protein Processing and RNA Accumulation in the Initially Inoculated Cells of N. benthamiana 
To address L1 and L2 functions, seven point mutations and deletions were introduced into corresponding coding region (FIG. 10B). In particular, to determine the requirements for the self-processing at the respective C-termini of L1 and L2, the predicted catalytic cysteine residues of the each protease (Cys493 and Cys767) were replaced by alanine residues to result in M1 and M2 variants, respectively (FIG. 10B). The processing competence of each variant was investigated using in vitro translation of the capped mRNAs encompassing the 5′-terminal untranslated region, the entire L1-L2 open reading frame and a short downstream region that encodes a part of the methyltransferase domain (FIG. 10B). The resulting translation products were analyzed using either immunoblotting and HA-specific antibody (FIG. 11A), or 35S-methionine labeling (FIG. 11B). As expected, a tagged non-mutant variant produced single HA-positive band corresponding to the fully-processed, HA-tagged L2 (FIG. 10B and FIG. 11A, lane L2HA) and, in addition, isotope-labeled, fully processed L1 (FIG. 10B and FIG. 11B, lane L2HA).
In contrast, translation of the M1 variant resulted in accumulation of a single major product corresponding to a L1-L2 fusion (FIG. 1B; FIGS. 2A and 2B, lanes M1). Analogously, mutational replacement of the predicted catalytic cysteine in L2 resulted in a lack of L2 self-processing, but did not affect the autocatalytic release of L1 (FIG. 10B; FIGS. 11A and 11B, lanes M2). Because mutation of the predicted active site residues did inactivate autoproteolysis by each leader protease, it was concluded that L1 and L2 are indeed the catalytically active, papain-like proteases.
To determine if the processing by L1 and L2 is required for viral RNA amplification, M1 and M2 variants of mLR-GFP/GUS were used to agroinfiltrate N. benthamiana leaves and to determine the resulting GUS activity. As shown previously for BYV minireplicon, GUS activity provides a reliable surrogate marker for measuring accumulation of the viral RNAs in the infected cells (Peng & Dolja, J. Virol. 74, 9766-9770, 2000). Using this marker, it was found that, unexpectedly, inactivation of the L1 cleavage resulted in more efficient GUS expression; almost 2-fold increase in GUS activity was detected in three independent experiments (FIG. 10B). In contrast, inactivation of L2 cleavage virtually abolished minireplicon infectivity: the corresponding GUS expression level was less than 0.5% of that of the parental mLR-GFP/GUS (FIG. 10B). This result is in agreement with the strict requirement for the cleavage by L-Pro for BYV minireplicon infectivity (Peremyslov et al., J. Virol. 72, 5870-5876, 1998); indeed fusion of either L-Pro or L2 with the replicase is likely to interfere with the synthesis of viral RNAs.
To determine the individual roles of L1 and L2 in RNA accumulation, the mutants were generated in which the coding regions of L1, L2, or both, were deleted. Interestingly, the L1 null mutant DL1 was capable of replication, although a corresponding level of GUS activity was  ̃5-fold lower than that in the parental mLR-GFP/GUS variant (FIG. 10B). Unexpectedly, deletion of L2 in the DL2 variant resulted in a slight increase in GUS expression suggesting that L2 is not essential for minireplicon accumulation in the isolated N. benthamiana cells (FIG. 10B). However, simultaneous deletion of L1 and L2 yielded the minireplicon DL1/2 that expressed only  ̃1% of the GUS activity observed in a parental mLR-GFP/GUS variant (FIG. 10B). Taken together, these results indicated that although the role of L1 in viral RNA amplification is more prominent than that of L2, the latter protease can rescue RNA accumulation of the L1-deficient mutant, and therefore L1 and L2 have partially overlapping functions in this process.
Both L1 and L2 possess the C-terminal papain-like protease domains (Pro1 and 2, respectively) and the N-terminal domains (NTD1 and NTD2, respectively; FIG. 10B). To determine the relative contributions of NTD1 and Pro1 in the L1 function, DNTD1 and DPro1 variants were generated in which these domains were deleted (FIG. 10B). The former of these minireplicon variants exhibited  ̃3-fold reduction in accumulation of GUS, while the latter produced even more GUS than the parental variant (FIG. 10B). These data indicated that the non-proteolytic rather than the protease domain of L1 provided a major contribution to viral RNA accumulation in N. benthamiana cells. It should be emphasized that the observed requirement for NTD1 for optimal RNA accumulation can reflect either a role of a protein domain, or of a corresponding coding region at the RNA level, or both.
Roles of L1 and L2 in the Virion Infectivity and Systemic Spread of GLRaV-2 in N. benthamiana 
To define the potential functions of L1 and L2 in the viral cell-to-cell movement and long-distance transport, the DL1 and DL2 deletions were introduced into the background of the full-length LR-GFP variant. Following agroinfiltration, virions were isolated from the inoculated leaves and the virion suspensions of the equal concentrations were used to manually inoculate N. benthamiana leaves and to characterize the resulting infection foci using GFP fluorescence at 8 days post inoculation. For the parental LR-GFP variant, inoculation yielded 9.9±5.6 infection foci per leaf with the mean diameter of 4.3±1.4 cells. Very similar results (8.2±4.8 foci per leaf; mean diameter of 4.1±1.3 cells) were obtained for the LR-GFPDL2 variant indicating that L2 is dispensable for both the infectivity and cell-to-cell movement of the GLRaV-2 in N. benthamiana. Strikingly, deletion of L1 resulted in a dramatic, 25-fold reduction in the specific infectivity of the LR-GFPDL1 variant (0.4 cells per leaf). Furthermore, the very few detected GFP-positive foci were unicellular suggesting that either L1 or the corresponding coding region is essential for the virion ability to establish infection in the initially inoculated cells and to move to the neighboring cells.
To determine if L1 and L2 are involved in the systemic transport of GLRaV-2, six replication-competent variants were tested in a context of the full-length LR-GFP launched to N. benthamiana plants using agroinfiltration. The inoculated plants were screened for the symptom, GFP, and CP expression at 3, 4, and 5 weeks post inoculation. Interestingly, most or all of the plants inoculated with M1 and DL2 variants became systemically infected indicating that neither L2 not the cleavage between L1 and L2 is required for the long-distance transport of the virus in N. benthamiana (FIG. 10B and FIG. 12A). Similar competence for the systemic spread was found in the case of DPro1 mutant. However, deletion of the L1 or its N-terminal domain resulted in complete loss of the replicon ability to establish systemic infection (FIGS. 10B and 12A).
Observation of the systemically infected leaves revealed apparent differences in the GFP accumulation between the experimental variants (FIG. 10A). To further assess these differences, GLRaV-2 CP accumulation in the non-inoculated upper leaves was evaluated. Conspicuously, it was found that only the DPro1 mutant accumulated to the levels comparable to those of the parental variant (FIG. 12B). The remaining two mutant variants, M1, and especially DL2, each accumulated to the significantly lower levels than that of the parental LR-GFP variant both at 3 and 4 weeks post inoculation (FIG. 3B). Collectively, these results demonstrated that the L2 per se, and the cleavage between L1 and L2 are required for optimal systemic spread of GLRaV-2 in N. benthamiana. In addition, L1 and its N-terminal non-proteolytic domain or the corresponding coding regions are essential for the ability of GLRaV-2 to establish systemic infection since neither GFP nor viral CP were detectable in the upper leaves of the plants inoculated with the DNTD1 or DL1 variants even at five weeks post inoculation (FIGS. 12C and 12D).
In BYV, both p20 and L-Pro are involved into viral systemic spread (Peng et al., J. Virol. 77, 2843-2849, 2003; Prokhnevsky et al., J. Virol. 76, 11003-11011, 2002). Of these, p20 is an integral component of the virion tail (Peremyslov et al., Proc. Natl. Acad. Sci. U S A 101, 5030-5035, 2004), while it is not known if L-Pro is present in the virions due to unavailability of the L-Pro-specific antibody. Because functional, HA-tagged variant of L2, were generated, it was used to determine if this protease is associated with the virions. The GLRaV-2 virions were isolated from systemically infected leaves and fractionated using sucrose density gradient. The peak of virions was detected in fractions 12-14 using CP-specific antibody (FIG. 13). However, the immunoblot analysis of the same gradient fractions using HA-specific antibodies showed the peak of L2 in fractions 15-17, suggesting that L2 present in the virion suspension is not physically associated with the virions (FIG. 13). This conclusion was further supported by the immunogold-specific electron microscopy used to detect HA epitopes present in L2. Indeed, only very weak gold labeling was found in the fractions 12-14 that contained bulk of the virions. Furthermore, a few gold microspheres detected in these fractions were not directly associated with the virions (FIG. 13, upper inset). The L2 peak fractions 15-17 contained much larger numbers of gold microspheres, but virtually no virions (FIG. 13, bottom inset) suggesting that L2 is not directly associated with GLRaV-2 virions.
L1 and L2 are Critical for Minireplicon Infection of the V. vinifera 
It is generally accepted that N. benthamiana is, perhaps, the most promiscuous host for a great variety of plant viruses. To determine if the seemingly non-essential and largely redundant roles played by L1 and L2 in GLRaV-2 infection in this experimental host do faithfully reflect their roles in a grapevine infection, four minireplicon variants were agroinfiltrated to V. vinifera (Grenache) leaves (Table 1). At 8 days post inoculation with the parental mLR-GFP/GUS variant,  ̃300 unicellular, GFP-fluorescent infection foci per leaf were observed. Strikingly, infiltration using DL1 and DL2 variants resulted in a  ̃100-fold and  ̃7-fold reduction in the foci numbers, respectively, indicating that each of the leader proteases is required for the ability of minireplicon to establish infection in grapevine cells (Table 1). However, similar to what was observed in N. benthamiana, infectivity of the M1 variant was not significantly different from that of the parental variant.
Remarkably, measurements of GUS activity in the infiltrated leaves correlated well with the data on the numbers of the infected cells (Table 1) suggesting that the principal function of the leader proteases is to aid the establishment of viral infection rather than to increase accumulation of viral RNA in the infected cells. Because the effects of L1 and L2 deletion in V. vinifera were much more dramatic compared to those in N. benthamiana, it was concluded that each protease provides a significant and specific contribution into GLRaV-2 infection in its natural host plant.







TABLE 1







Infectivity and GUS expression by mLR-GFP/GUS


minireplicon variants in V. vinifera












Mean number of the





infection foci
Mean GUS activity


Experi-

(% of that in
(% of the level in


ment
Variant
parental variant)
parental variant)













1
mLR-GFP/GUS
100.00
100.00


1
ΔL1
1.03
4.16


1
ΔL2
14.96
10.03


1
M1
104.44
103.11


2
mLR-GFP/GUS
100.00
100.00


2
ΔL1
1.58
2.93


2
ΔL2
10.44
10.87


2
M1
133.43
118.08









Discussion

Without being bounded by any particular theory, the following discussion is provided. The instant disclosure allowed delineation of three major functions of L1 and L2 in the GLRaV-2 infection cycle: i) polyprotein processing; ii) virus accumulation in the initially infected cells; and iii) systemic transport of the infection.
In particular, it was found that both L1 and L2 are the active proteases with the conserved catalytic cysteines (FIGS. 10B and 11). The cleavage upstream from the methyltransferase domain of the viral RNA replicase polyprotein is essential for GLRaV-2 viability (FIG. 10B). Surprisingly, although L1 does cleave at its own C-terminus both in vitro (FIG. 11) and in vivo (FIG. 13), neither this cleavage nor the L1 protease domain per se are essential for systemic infection in N. benthamiana as evident from the phenotypes of M1 and DPro1 variants (FIGS. 10B and 12). However, slower virus accumulation in the non-inoculated leaves in these mutants (FIGS. 12A and 12B) suggests that the L1-mediated cleavage is required for the optimal development of systemic infection.
The deletion analysis indicated that L1 and L2 play partially overlapping roles in the viral RNA accumulation in the initially inoculated cells. When viral minireplicon was launched by agroinfiltration, complete deletion of L1 resulted in a  ̃5-fold reduction of RNA accumulation and expression. Similar effect was observed upon deletion of the non-proteolytic N-terminal domain of L1 indicating its principal role in L1 function (FIG. 10B). Although the deletion of L2 did not affect RNA accumulation, combined deletion of L1 and L2 resulted in a virtually nonviable minireplicon indicating that L2 provided a significant contribution into viral infectivity in the absence of L1.
Interestingly, when isolated virions containing full-length genome were used for plant inoculation, the infectivity and cell-to-cell movement of the DL2 variant were indistinguishable from those of the parental variant, while the virions of DL1 variant have lost their infectivity. The deletion of L1 but not L2 coding region could affect virion structure, stability, and infectivity. Therefore, it is possible that in addition to L1 function in RNA accumulation revealed by minireplicon agroinoculation, the corresponding coding region also functions at the RNA level to facilitate formation of the tailed virions capable of the local and systemic transport.
In accord with the latter assumption, DL1 and DNTD1 mutants were unable to establish a systemic infection upon agroinfiltration using full-length replicons (FIG. 12C). In contrast, deletion of the protease domain in DPro1 variant did not affect systemic infectivity indicating that virion tail formation was likely unaffected. The deletion of L2 resulted in a systemically infectious DL2 variant, which, however, exhibited much slower accumulation in the upper leaves (FIGS. 11A and 11B). This result indicated that L2 is required for the efficient systemic spread of GLRaV-2 in N. benthamiana. 
Perhaps the most significant results of this study were obtained when the minireplicon variants were agroinoculated to the leaves of the GLRaV-2's natural host, grapevine. In a sharp contrast to a permissive experimental host N. benthamiana where L2 was superfluous for minireplicon infectivity, DL2 variant exhibited a  ̃10-fold reduction in RNA accumulation upon agroinfiltration into V. vinifera leaves (Table 1). The specific infectivity of the DL2 variant measured as a mean number of the GFP-fluorescent infected cells per leaf was also reduced  ̃10-fold. This correlation in the accumulation of the minireplicon RNA and the numbers of infected cells clearly points to the critical role of L2 in the virus invasiveness, i.e. the ability to establish infection in the inoculated cells. A role in GLRaV-2 invasiveness in grapevine is even more dramatic in the case of L1. Indeed, L1 deletion resulted in  ̃100-fold reduction in the RNA accumulation and specific infectivity of DL1 variant (Table 1). It was concluded that both L1 and L2 are essential for the optimal GLRaV-2 infection of the grapevine.
What is a specific functional significance of duplication and diversification of the leader proteases in GLRaV-2? It seems that the answer, at least in part, lies in the host-specific effects of L1 and L2 whose functional cooperation is required for the infection of grapevine but not N. benthamiana. In other words, a tandem of viral proteases could have evolved to boost the function of a single protease in order to subvert a perennial woody host potentially recalcitrant to virus infection. This hypothesis is compatible with the fact that in addition to GLRaV-2, protease duplication is found in CTV (Karasev et al., Virology 208, 511-520, 1995), Raspberry mottle virus (Tzanetakis et al., Virus Res. 127, 26-33, 2007), and Strawberry chlorotic fleck virus (Tzanetakis & Martin, Virus Res. 124, 88-94, 2007), each of which infects woody and/or perennial hosts, but not in BYV, Mint virus 1 (Tzanetakis et al., Virus Res. 127, 26-33, 2007), or Carnation yellow fleck virus that infect herbaceous annual hosts.
What is the possible mechanism by which L1 and L2 facilitate GLRaV-2 infection? The fact that each of these leader proteases acts in a host-specific manner to boost viral infectivity suggests that L1 and L2 could be involved in suppression of antiviral defense response. One possibility is that L1 and L2 are involved in suppression of RNA interference (RNAi) either independently from or in cooperation with the RNAi suppressor p24 (FIG. 1A) (Chiba et al., Virology 346: 7-14, 2006). However, efforts to identify effects of GLRaV-2 L1 and L2 or BYV L-Pro on the RNAi response in several model systems invariably failed. Moreover, it was found that the ectopic co-expression of L-Pro with the reporter reduced accumulation of the latter, suggesting possible involvement of the leader proteases in gene regulation at the RNA or protein level.
By analogy to papain-like proteases of coronaviruses (Lindner, Virology 362, 245-256, 2007), it can be hypothesized that closteroviral proteases act as deubiquitination enzymes (DUBs) whereby affecting regulation of the plant defense. The only fact that it is in a disagreement with DUB hypothesis is that inactivation of the L1 proteolytic activity in M1 variant has little effect on the viral infection. This apparent discrepancy can be explained if the L1-mediated binding rather than cleavage of the host defense-related proteins is sufficient to exert L1 function.
The generation of the full-size and minireplicons of GLRaV-2 tagged via insertion of the reporter genes or epitopes highlights a potential of this virus as a gene expression vector for the grapevine. In general, closterovirus-derived vectors provide strong advantages of relatively large genetic capacity and stability (Dolja et al., Virus Res. 117: 38-51, 2006; Folimonov et al., Virology 368(1):205-216, 2007). Utility of closteroviral vectors is further enhanced by a dramatic increase in the vector infectivity by co-expression of the homologous RNAi suppressors of which p24 of GLRaV-2 appears to be the strongest (Chiba et al., Virology 346: 7-14, 2006). Full realization of the GLRaV-2 vector potential requires development of the efficient inoculation technique for the grapevine.
Materials and Methods
Generation of the GFP-Tagged, Full-Length cDNA Clone of GLRaV-2
The GLRaV-2 isolate obtained from a local Oregonian vineyard was propagated on N. benthamiana plants as described earlier (Goszczynski et al., Vitis 35, 133-133, 1996). Virions were isolated (Napuli et al., Virology 274(1), 232-239, 2000) and the viral RNA was obtained using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. A strategy for nucleotide sequencing of the viral genome and the generation of the intermediate and full-length viral cDNA clones was as described for BYV (Peremyslov & Dolja, Curr. Protocols Microbiol. (Suppl. 7), 16F.1.1-16F.1.26, 2007). The resulting sequence of GLRaV-2 RNA was deposited to GenBank (Accession No. FJ436234; incorporated herein by reference as of Jan. 26, 2009). The sequences of the numerous primers used in cloning procedures are available upon request.
In brief, a full-length cDNA clone of GLRaV-2 was assembled using pCB301 mini-binary vector (Xiang et al., Plant Mol. Biol. 40: 711-717. 1999), while the cDNA cloning was done using reverse transcription and either conventional synthesis of a double-stranded (ds) cDNA or PCR amplification. The NOS terminator was added to the pCB301 by using unique sites Sac I and Kpn I and a polylinker containing restriction sites Sac I, Barn HI, AatI I, Bbvc I, Rsr II, Bst EII, and SmaI was inserted between Sad site and NOS terminator to produce pCB301-NOS-PL. To add a CaMV 35S RNA polymerase promoter fused to the 5′-fragment of the viral cDNA (nts 1-2,034), a PCR-mediated DNA splicing technique was used. Separate PCRs were done to amplify the 35S promoter and the 5′end of GLRaV2 cDNA and to generate products with overlapping ends. These products were combined and used as templates for another round of PCR using primers complementary to the 5′- and 3′-ends of the full-length product. The latter product was cloned into pCB301-NOS-PL using Sac I (added to the 5′-end of 35S promoter) and BamH I (nt 2034) to produce p35S5′LR.
To add a ribozyme to the 3′-end of the viral cDNA, a megaprimer with a virus-specific part complementary to the 3′-end of the viral cDNA followed by a ribozyme sequence designed as described (Prokhnevsky et al., J. Virol. 76, 11003-11011, 2002) and a Sma I site was used in combination with a regular primer to amplify the 3′-terminal region of the GLRaV-2 cDNA (nts 14,842-16,486). Resulting PCR product was cloned into p35S5′LR using restriction sites BstE II (nt 14,842) and Sma I (added at the 3′-terminus of the megaprimer) to produce a p35S5′3′LR-Rib.
For cloning the internal region of viral cDNA (nts 2,029-10,827), three partially overlapping fragments of ds cDNA were obtained using conventional cDNA cloning and Gibco-BRL protocol for SuperScript II reverse transcriptase. These fragments were inserted into p35S5′3′LR-Rib using restriction sites Bam HI (nt 2,029), Aat II (nt 3,394), Bbv CI (nt 6,281), and Rsr II (nt 10,821) to generate p35S-5′BR3′LR-Rib. The remaining part of the viral cDNA (nts 10,821-14,848) was PCR-amplified and cloned into an intermediate vector pGEM-3Zf(+) (Promega). A nucleotide sequence encoding an endoplasmic reticulum-targeted GFP (Haseloff et al., Proc Natl Acad Sci USA 94(6), 2122-2127, 1997) followed by a BYV CP promoter was inserted upstream from the 5′-end of GLRaV-2 CP ORF. The resulting cDNA fragment was cloned into p35S5′-BR-3′LR-Rib using Rsr II (nt 10,821) and Bst EII (nt 14,842) sites to generate the full-length GLRaV-2 cDNA clone p35S-LR-GFP or LR-GFP for the brevity.
Generation of the Modified and Mutant GLRaV-2 Variants
The minireplicon variant mLR-GFP/GUS was engineered by modifying the LR-GFP cDNA via deletion of the cDNA fragments from the start codon of the p6 ORF (FIG. 1A) to nt 14,185 and from the Fse I site at the 3′-end of the GFP ORF to nt 15,285 (nt numbers correspond to the original GLRaV-2 cDNA). As a result, GLRaV-2 ORFs encoding p6, Hsp70h, p63, CPm, CP and p19 were deleted (FIG. 1A). The GFP ORF was then replaced with a hybrid GFP/GUS ORF described earlier (Peng et al., Virology 294, 75-84, 2002) using Pac I at the 5′-terminus of the GFP ORF and Fse I at the 3′-terminus of the GUS ORF.
Two plasmids, pGEM-35SLR-Pro and pGEM-SP6LR-Pro, containing the whole L1 and L2 coding region and a fragment of the methyltranferase coding region (nts 1-3,071) were generated by cloning the corresponding PCR-amplified fragments (FIG. 1B) into pGEM-3Zf(+). Both pGEM-35SLR-Pro and pGEM-SP6LR-Pro were used to generate pGEM-35SLR-L2HA and pGEM-SP635SLR-L2HA by inserting three copies of the hemagglutinin epitope (HA) tag (YPYDVPDYA; SEQ ID NO: 11) coding sequence downstream from codon 663 within L2 coding region. Each of these plasmids was used to introduce the following mutations into the L1 or L2.
Mutation 1 (M1) was generated by replacing the catalytic Cys493 residue of L1 with Ala using site-directed mutagenesis. Analogously, mutation 2 (M2) was obtained via substitution of Ala for Cys767 of L2. In DL2 mutation, the entire L2-coding region was deleted and Lys848 residue downstream from L2 scissile bond was replaced with Gly to regenerate an authentic L1 cleavage site. Mutation DL1 was made by deleting the entire L1 coding region except for the 5′-terminal start codon. In mutation DNTD1, the entire N-terminal, non-proteolytic region of L1 was deleted, again except for the start codon. In mutation DPro1, the C-terminal proteinase domain of L1 was deleted while the N-terminal region of L1 was fused to the N-terminal region of L2. In the last mutation DL1/2, both L1 and L2 were deleted except for the start codon that was fused with the first Lys codon of the GLRaV-2 replicase, resulting in the formation of a replicase that differed from the proteolytically processed, wild-type replicase only by the presence of the N-terminal Met. The diagrams of all mutations are shown in FIG. 1B.
The pGEM-SP6LR-L2HA variants were used to analyze the proteolytic activity of the mutated proteases in vitro. The DNA fragments from the mutant derivatives of pGEM-35SLR-L2HA were cloned into mLR-GFP/GUS using Sbf I (located in the vector part of the plasmid) and Stu I (nt 3,063) sites. The DNA fragments from mutant derivatives of p35S-miniV94-GFPGUS were also cloned into the full-length cDNA clone LR-GFP using Sfi I (located in the vector part of the plasmid) and Bbv CI (nt 6,282).
Mutation Analysis of the Proteolytic Activity of L1 and L2
The pGEM-SP6LR-L2HA variants were linearized using Sma I and the corresponding in vitro RNA transcripts were generated using mMessage Machine kit (Ambion). To assay the proteolytic activity of the leader proteases, the resulting capped RNA transcripts were translated using the wheat germ extracts (Promega) and [35S]-Met (Amersham/Pharmacia Biotech) or a non-labeled amino acid mixture. After 1 hr of incubation at 25° C., the products were separated by PAGE, electroblotted onto a PROTRAN nitrocellulose membrane and used for autoradiography or for immunoblotting using anti-HA rat monoclonal antibody (Roche) as first antibody and goat anti rat-peroxidase as secondary antibody.
Mutation Analysis of the L1 and L2 Roles in RNA Accumulation
Agrobacterium tumefaciens strain C58 GV2260 was transformed by each of the mLR-GFP/GUS variants by electroporation. Corresponding cultures were grown overnight at 28° C. with shaking, spun down and resuspended in a buffer containing 10 mM MES-KOH (pH 5.85), 10 mM MgCl2, and 150 mM acetosyringone. Bacterial suspensions of each variant were mixed with corresponding cultures transformed to express an RNAi suppressor P1/HC-Pro from Turnip mosaic virus to enhance minireplicon infectivity (Chiba et al., Virology 346: 7-14, 2006). The final bacterial concentrations were 1.0 OD600 for minireplicon-expressing variants and 0.1 OD600 for the P1/HC-Pro-expressing variant. The induced bacterial cultures were infiltrated into lower surface of the N. benthamiana leaves using a syringe without a needle or vacuum infiltrated into the grapevine leaves. The GFP-fluorescent leaf cells were visualized using epifluorescent stereomicroscope Leica MZ 16F (Deerfield, Ill.) at 8 days post infiltration. Samples for GUS assays were prepared and GUS activity was measured using Hoefer TKO100 DNA fluorimeter (Hoefer Scientific Instruments) as previously described (Dolja et al., Proc. Natl. Acad. Sci. USA 89: 10208-10212, 1992).
Analysis of the Local and Systemic Virus Transport
To assay the cell-to-cell movement of the GFP-tagged virus variants, virions were isolated from the agroinfiltrated leaves of N. benthamiana at 2 weeks post inoculation (Napuli et al., Virology 274(1), 232-239, 2000), resuspended in a buffer containing 20 mM sodium phosphate (pH 7.4) and 1 mM Na2-EDTA and inoculated manually to leaves of N. benthamiana. The fluorescent infection foci were analyzed using the epifluorescent stereomicroscope at 8 days post inoculation.
To investigate the systemic spread in N. benthamiana, plasmids carrying the corresponding variants in a context of the LR-GFP were mobilized into A. tumefaciens, the resulting bacterial suspensions were mixed with those engineered to express P1/HC-Pro as described above, and infiltrated into leaves of young N. benthamiana (6-8 leaf stage) plants. After 3, 4, or 5 weeks, the upper leaves of these plants were screened for the symptom development, whereas epifluorescence microscopy and a spot camera MicroPublisher3.3 RTV (QImaging) were used to document accumulation of the virus-expressed GFP Immunoblotting and custom-made GLRaV-2-specific antiserum in 1:5,000 dilution were used to document accumulation of CP.
Virion Analyses
To determine if HA-tagged L2 was associated with the virions, the sucrose gradient fractionation followed by immunoblotting was used. Virions isolated as described above were resuspended in a buffer containing 20 mM Na-phosphate (pH 7.4) and 1 mM Na2-EDTA, loaded to the top of 10-40% sucrose gradients prepared in the same buffer, and centrifuged at 25,000 RPM for 4 hours in a Beckman SW40 rotor at 4° C. Gradients were separated into 25 fractions and the immunoblot analysis was done using anti-HA rat monoclonal antibody (Roche) and GLRaV-2-specific antibody to detect L2HA and CP, respectively. The immunogold-specific electron microscopy to detect L2HA was done essentially as described (Medina et al., Virology 260(1), 173-181, 1999).
It will be apparent that the precise details of the methods and compositions described may be varied or modified without departing from the spirit of the described invention. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

## Claims

1. A replication-competent plant gene transfer vector comprising a nucleic acid encoding:
a) viral genes from Grapevine leafroll-associated virus-2 (LR-2) comprising a methyltransferase, RNA helicase, and RNA-dependent RNA polymerase;
b) an RNAi suppressor selected from the group consisting of LR-2 p24 and Beet yellows virus p21;
c) LR-2 leader proteases L1 and L2; and
d) a heterologous polynucleotide operably linked to a promoter, wherein the heterologous polynucleotide is expressed in a plant cell, and wherein the vector is capable of infectious replication in the plant cell, wherein the L1 protease has the amino acid sequence shown in SEQ ID NO: 4, and wherein the vector is adapted for virus-induced gene silencing.

2. The vector of claim 1, further comprising viral genes from Grapevine leafroll-associated virus-2 (LR-2) that are involved in virion assembly, transport within plants, or both.

3. The vector of claim 2, wherein the viral genes are selected from the group consisting of p6, Hsp70h, p63, CPm, CP and p19 from Grapevine leafroll-associated virus-2 (LR-2).

4. The vector of claim 1, wherein the heterologous polynucleotide encodes at least one polypeptide selected from the group consisting of a reporter molecule, a selectable marker, and a therapeutic gene.

5. The vector of claim 4, wherein the therapeutic gene is antifungal, antibacterial, antiviral or a taste modifier.

6. The vector of claim 4, wherein the therapeutic gene is for the treatment of Pierce's Disease or powdery mildew.

7. The vector of claim 6, wherein the therapeutic gene is a polynucleotide which triggers viral induced gene silencing, a Run1 polynucleotide, or encodes a lysozyme polypeptide.

8. The vector of claim 1, wherein the L2 protease has the amino acid sequence shown in SEQ ID NO: 6.

9. A plant cell comprising the vector of claim 1.

10. A plant comprising the vector of claim 1.

11. A method for virus-induced gene silencing in a plant cell, comprising introducing into the plant cell the vector of claim 1.

12. The method of claim 11, wherein introducing the vector into the plant cell comprises agroinoculation.

13. The method of claim 11, wherein the vector is a replication-competent vector, wherein the vector is introduced into a plant cell and subsequently replicates and infects at least one additional plant cell.

14. The method of claim 13, wherein the vector is a replication-competent vector, wherein the vector systemically infects a plant structure selected from the group consisting of tissue, leaf, stem, root, fruit, seed or entire plant.

15. The method of claim 11, wherein introducing the vector into the plant cell comprises grafting a plant part comprising the vector to a plant part that does not comprise the vector.

16. The method of claim 12, further comprising grafting a plant part comprising the plant cell comprising the vector to a plant part that does not comprise the vector.

17. The vector of claim 1, wherein the RNAi suppressor is p24.