WO2017136895A1

WO2017136895A1 - Constructs and methods for conferring virus resistance

Info

Publication number: WO2017136895A1
Application number: PCT/AU2017/050114
Authority: WO
Inventors: James Dale; Peter Waterhouse; Benjamin Dugdale
Original assignee: Queensland University Of Technology
Priority date: 2016-02-10
Filing date: 2017-02-10
Publication date: 2017-08-17

Abstract

Disclosed are construct systems for inhibiting viral replication, and thereby establishing virus resistance, in plants. More particularly, the present invention discloses transgenic plants that are resistant to viruses and methods of producing them.

Description

TITLE OF THE INVENTION

"CONSTRUCTS AND METHODS FOR CONFERRING VIRUS RESISTANCE"

FIELD OF THE INVENTION

[0001] This application claims priority to Australian Provisional Application No.

2016900443 entitled "Constructs and Methods for Conferring Virus Resistance" filed 10 February 2016, the contents of which are incorporated herein by reference in their entirety.

[0002] This invention relates generally to construct systems for inhibiting viral replication, and thereby establishing virus resistance, in plants. The invention also relates to transgenic plants that are resistant to viruses and methods of producing them.

BACKGROUND OF THE INVENTION

[0003] Plant viruses are known to infect every economically viable horticultural and agricultural crop around the world (including both mono- and di-cotyledon), causing severe losses to crop yields and/or additional costs in control methods.

[0004] Two groups of single-stranded DNA (ssDNA) viruses are a particular threat to horticultural and agricultural crops: Geminiviruses and Nanoviruses. Members of the Geminiviridae have geminate virions and either a monopartite or bipartite circular ssDNA genome. Each molecule is about 2.7 kb in length. Of the Geminiviridae genera, the Begomoviruses and the Mastreviruses are the most important viruses that threaten horticultural and agricultural crops.

[0005] Begomoviruses are whitefly-transmitted and have either monopartite of bipartite genomes. Members of their genus include some of the most economically devastating viruses of modern agriculture such as tomato yellow leaf curl (consisting of a range of different viruses spread through most tropical and sub-tropical regions), African cassava mosaic (Africa), bean golden mosaic (South and Central America), mung bean yellow mosaic (India) and cotton leaf curl (South and South-East Asia) viruses. The impact of many of the Begomoviruses has increased dramatically over recent years as a result of the widespread introduction of the aggressive "B biotype" of the whitefly vector, Bemesia tabaci.

[0006] Mastreviruses have had a lesser impact on agriculture but are responsible for significant losses in some crops. These viruses are transmitted by the leafhoppers and have monopartite genomes. Members of this genus include maize streak (Africa), wheat dwarf (Europe) and tobacco yellow dwarf (Australia) viruses.

[0007] Nanoviruses have isometric virions and circular ssDNA genomes but these genomes are multi-component with at least six different integral genomic components each of which is approximately 1 kb. These viruses are transmitted by aphids except for one tentative nanovirus, coconut foliar decay virus, which is transmitted by a treehopper and has only been reported from Vanuatu. The economically most important nanovirus is banana bunchy top virus (BBTV), which nearly destroyed the Australian banana industry in the 1920s and causes major losses in the South Pacific, Asia and Africa. Subterranean clover stunt (Australia), faba bean necrotic yellows (Mediterranean) and coconut foliar decay (Vanuatu) viruses all cause significant yield loss.

[0008] The genome organization among and between Geminiviruses and Nanoviruses differs significantly, including differences in the number and size of genomic components and number and size of genes, the processing of transcripts, the orientation of genes and the like. There are, however, remarkable similarities. All of the Geminiviruses and Nanoviruses encode (i) a replication initiation (Rep) protein which has nicking and joining activity and directs rolling circle replication of the viral genome; (ii) a virion coat protein; (iii) a protein that is involved in binding host cell retinoblastoma-like proteins resulting in the cell moving to S phase; (iv) a cell-to-cell movement protein; and (v) a nuclear shuttle protein. Further, the viruses have functionally similar intergenic regions which contain (i) a stem/loop structure, the nonanucleotide loop sequence of which is highly conserved between all Geminiviruses and Nanoviruses and is the site of nicking and ligation by the Rep protein; and (ii) iterons within this region that recognizes the Rep protein.

[0009] Although there has been considerable progress in the development of resistance to RNA plant viruses, this success has not necessarily translated to ssDNA plant viruses.

Accordingly, there is a need for improved methods for developing resistance of plants to ssDNA viruses, such as Geminiviruses and Nanoviruses.

[0010] To establish infection, plant viruses must interact with the host plant for replication, gene expression, and cell-to-cell and long-distance movement. During this process, the virus must penetrate the cell walls in order to spread throughout the plant. This is the primary role of movement proteins that can interact with plasmodesmata (PD), the plasma membrane-lined channels that interconnect plant cells, to facilitate the cell-to-cell transport of the infectious form of the virus (see, Lucas and Gilbertson, 1994).

SUMMARY OF THE INVENTION

[0011] The present invention is predicated in part on the determination that resistance of a plant to virus infection can be better achieved by combining Rep-mediated activation and expression of a lethal gene that inhibits the viability of a plant cell when infected by a virus, together with siRNA targeting of a gene of the virus that is associated with virus spread and/or replication, so as to impede viability of the virus, including its ability to infect a plant host. The present inventors unexpectedly found that consistently high-levels of virus resistance were achieved using such an approach.

[0012] Accordingly, in one aspect, the present invention provides an expression system for conferring virus resistance to a plant, the expression system comprising a first expression system component (e.g. , comprising at least one expression cassette or construct) and a second expression system component (e.g. , comprising at least one expression cassette or construct); wherein an expression cassette of the first expression system component comprises a toxicant nucleic acid sequence encoding a toxicity protein operably connected to at least one transcriptional control sequence (e.g., a promoter, transcription terminator, c/s-acting sequence, etc.) ; and wherein a virus viability impairment (VVI) nucleic acid sequence is expressible from the second expression system component, and the expression of the VVI nucleic acid sequence in a plant cell produces a double stranded RNA molecule that induces silencing of a gene of the virus that is essential for virus replication and/or virus spread.

[0013] Suitably, one or both of the first expression system component and the second expression system component comprises an expression cassette that comprises an effector nucleic acid sequence of the invention {i.e., a toxicant or VVI nucleic acid sequence) operably connected to at least one transcriptional control sequence (e.g., a promoter, transcription terminator, c/^'s-acting sequence, etc. ). The effector nucleic acid sequence can be in the form of a contiguous sequence (a lso referred to herei n as a "contig uous nucleic acid entity" or "contiguous gene") or a pl ura lity of non-contiguous seq uences (also referred to herei n as a "non-contiguous nucleic acid entity", "noncontiguous gene" or "split gene") that ca n cond itionally form a contig uous seq uence.

[0014] In some embod iments, the toxica nt nucleic acid sequence is in the form of a contiguous sequence. Alternatively, the toxicant nucleic acid sequence may be present in the form of a plural ity of non-contiguous sequences that can conditionally form a contiguous sequence.

[0015] In some embod iments, one or both of the toxicant nucleic acid sequence and the VVI nucleic acid seq uence is conditiona lly expressible. In some embod iments, one or both of the toxica nt nucleic acid sequence and the VVI nucleic acid sequence is constitutively expressible. By way of a n illustrative example, the toxicant nucleic acid seq uence a nd the VVI nucleic acid sequence ca n both be conditional ly expressible.

[0016] In some embod iments, the expression cassette of the fi rst expression system component comprises an inactive replicon that comprises replicase c/s-acting elements, which facilitate, in the presence of a replicase, ci rcula rization and release from the inactive replicon of a corresponding replicon, and autonomous episomal replication (e.g., rolling ci rcle replication) of the replicon, wherein the replicon comprises a n expression cassette from which the toxica nt nucleic acid sequence is expressi ble.

[0017] In some embod iments, the expression cassette of the second expression system component comprises an inactive replicon that comprises replicase c/s-acting elements, which facilitate, in the presence of a replicase, ci rcula rization and release from the inactive replicon of a corresponding replicon, and autonomous episomal replication {e.g., rolling ci rcle replication) of the replicon, wherein the replicon comprises a n expression cassette from which the vi rus impa irment nucleic acid seq uence is expressible.

[0018] In some embod iments, the inactive repl icon or proreplicon comprises a toxicant nucleic acid seq uence or a VVI nucleic acid sequence, which is in the form of a contig uous sequence and which is operably connected to at least one transcriptional control seq uence (e.g. , a promoter, tra nscription termi nator, c/s-acting seq uence, etc.). Accord ingly, in some of these embodiments, the contiguous seq uence is opera bly linked to a constitutive promoter for constitutively expressi ng the contiguous seq uence.

[0019] In some other embodiments, the proreplicon comprises a toxicant nucleic acid sequence, which is i n the form of non-contiguous sequences (e.g., a pair of discontinuous sequences), wherein an upstream member of the non-contig uous sequences corresponds to a 3' portion of the toxicant nucleic acid seq uence a nd a downstream member of the non-contiguous sequences corresponds to a 5' portion of the toxicant nucleic acid seq uence, wherein the 5' portion is operably connected to at least one transcri ptiona l control sequence (e.g. , a promoter, tra nscription terminator, c/s-acting sequence, etc.). Simi larly, i n some embod iments, the proreplicon comprises a VVI nucleic acid sequence, which is in the form of non-contiguous sequences (e.g. , a pai r of d iscontinuous seq uences), wherein an upstrea m member of the noncontiguous sequences corresponds to a 3' portion of the virus i mpairment nucleic acid sequence and a downstrea m member of the non-contig uous sequences corresponds to a 5' portion of the VVI nucleic acid sequence, wherein the 5' portion is operably connected to at least one tra nscri ptional control sequence (e.g., a promoter, transcription terminator, c/s-acting sequence, etc. ) . [0020] Accordingly, in some aspects the toxicant nucleic acid sequence and/or VVI nucleic acid sequence comprises a proreplicon that lacks a functional rep gene for autonomous episomal replication (e.g. , rolling circle replication) but comprises Rep recognition elements, which facilitate, in the presence of a Rep protein (e.g., a viral Rep protein), circularization and release from the proreplicon of a corresponding replicon, and autonomous episomal replication (e.g., rolling circle replication) of the replicon, wherein the replicon comprises an expression cassette from which a toxicant nucleic acid sequence or a VVI of the invention is expressible.

[0021] In some aspects, the expression of the toxicant nucleic acid sequence and/or the VVI nucleic acid sequence is optionally boosted in the presence of a Rep protein, which is suitably produced from a rep gene in an ancillary expression cassette. The Rep protein interacts with the Rep recognition elements of the proreplicon to facilitate circularization and release from the proreplicon of a corresponding replicon and autonomous episomal replication of the replicon, to thereby boost expression of the toxicant nucleic acid sequence and/or the VVI nucleic acid sequence. In other embodiments, the contiguous sequence is operably linked to a regulatory promotor for conditionally expressing the contiguous sequence. In representative examples of this type, expression of the rep gene and the toxicant nucleic acid sequence and/or the VVI nucleic acid sequence occurs under control of regulated promoters whose transcriptional activity is stimulated or induced under the same conditions to thereby concurrently stimulate or induce expression of the rep gene and the toxicant nucleic acid sequence and/or the VVI nucleic acid sequence.

[0022] In other representative examples, the proreplicon comprises a toxicant nucleic acid sequence and/or a VVI nucleic acid sequence, which is in the form of non-contiguous sequences (e.g., a pair of discontinuous sequences), wherein an upstream member of the noncontiguous sequences corresponds to a 3' portion of the toxicant nucleic acid sequence, and/or the VVI nucleic acid sequence, and a downstream member of the non-contiguous sequences corresponds to a 5' portion of the toxicant nucleic acid sequence and/or the VVI nucleic acid sequence, wherein the 5' portion is operably connected to at least one transcriptional control sequence (e.g. , a promoter, transcription terminator, c/s-acting sequence, ere). Interaction of the Rep recognition elements of the proreplicon with a Rep protein, which is suitably produced from a rep gene in an ancillary expression cassette, facilitates circularization and release from the proreplicon of a corresponding replicon, and autonomous episomal replication (e.g., rolling circle replication) of the replicon comprising the expression cassette. Circularization of the replicon results in rearrangement of the expression cassette such that the non-contiguous sequences become operably connected with one another to form a contiguous toxicant nucleic acid sequence and/or a VVI nucleic acid sequence [i.e., a contiguous nucleic acid entity). Autonomous episomal replication of the replicon results in amplification of the replicon with expression of the contiguous toxicant nucleic acid sequence and/or the VVI nucleic acid sequence. In some embodiments, one of the Rep recognition sequences ("downstream Rep recognition sequence") is present in the expression cassette at a position downstream of the 5' portion of the toxicant nucleic acid sequence and/or the VVI nucleic acid sequence so that when circularization of the replicon occurs the downstream Rep recognition sequence is present in the circularized replicon at a location intermediate an upstream 5' portion and a downstream 3' portion of the toxicant nucleic acid sequence and/or the VVI nucleic acid sequence.

[0023] In some embodiments of the expression system described above and elsewhere herein, the Rep protein interacts with the Rep recognition elements of the proreplicon to facilitate circularization and release from the proreplicon of a corresponding replicon and autonomous episomal replication of the replicon, to thereby boost expression of the toxicant nucleic acid sequence and/or the VVI nucleic acid sequence. The Rep protein may be a virus-originating Rep protein. In some embodiments of the present invention, the Rep protein that serves to enhance or stimulate expression of the toxicant nucleic acid sequence and/or the VVI nucleic acid sequence activates replication of the toxicant nucleic acid sequence and/or the VVI nucleic acid sequence.

[0024] In some embodiments, the interaction of the Rep recognition elements of the proreplicon with a Rep protein, which is suitably produced from a virus rep gene or a rep gene in an ancillary expression cassette, facilitates circularization and release from the proreplicon of a corresponding replicon, and autonomous episomal replication {e.g., rolling circle replication) of the replicon comprising the expression cassette, wherein circularization of the replicon results in rearrangement of the expression cassette such that the non-contiguous sequences become operably connected with one another to form a contiguous toxicant nucleic acid sequence (i.e., a contiguous nucleic acid entity).

[0025] In some preferred embodiments, the toxicant nucleic acid sequence is in the form of a construct comprising non-contiguous sequences (e.g., a pair of discontinuous sequences that encode protein or a functional RNA) separated by a non-coding sequence. In illustrative examples of this type, an individual non-contiguous sequence is also separated from an upstream or downstream Rep recognition element by a non-coding sequence {e.g., an intron). Suitably, in these examples, the 3' portion of the toxicant nucleic acid sequence is separated from an upstream Rep recognition element by a 3' portion of an intron and the 5' portion of the toxicant nucleic acid sequence is separated from a downstream Rep recognition element by a 5' portion of the intron, wherein circularization and release of the replicon in the presence of the Rep protein facilitates rearrangement of the construct to form a contiguous toxicant nucleic acid sequence, which comprises in operable linkage, from 5' to 3', the 5' portion of the toxicant nucleic acid sequence, the 5' portion of the intron, the downstream Rep recognition element, the 3' portion of the intron and the 3' portion of the toxicant nucleic acid sequence. Suitably, a promoter {e.g., regulated or constitutive) is operably connected upstream of the 5' portion of the toxicant nucleic acid sequence to form an expression cassette.

[0026] In some preferred embodiments, the VVI nucleic acid sequence is in the form of a construct comprising non-contiguous sequences {e.g., a pair of discontinuous sequences that encode protein or a functional RNA) separated by a non-coding sequence. In illustrative examples of this type, an individual non-contiguous sequence is also separated from an upstream or downstream Rep recognition element by a non-coding sequence (e.g., an intron). Suitably, in these examples, the 3' portion of the VVI nucleic acid sequence is separated from an upstream Rep recognition element by a 3' portion of an intron and the 5' portion of the VVI nucleic acid sequence is separated from a downstream Rep recognition element by a 5' portion of the intron, wherein circularization and release of the replicon in the presence of the Rep protein facilitates

rearrangement of the construct to form a contiguous VVI nucleic acid sequence, which comprises in operable linkage, from 5' to 3', the 5' portion of the VVI nucleic acid sequence, the 5' portion of the intron, the downstream Rep recognition element, the 3' portion of the intron and the 3' portion of the VVI nucleic acid sequence. Suitably, a promoter (e.g., regulated or constitutive) is operably connected upstream of the 5' portion of the virus impairment nucleic acid sequence to form an expression cassette. [0027] In non-limiting examples, the first expression system component comprises a proreplicon (a "toxicant proreplicon") that includes an upstream first Rep recognition element and a downstream second Rep recognition element, which facilitate circularization, release and autonomous episomal replication {e.g., rolling circle replication) of a corresponding "toxicant replicon" in the presence of a Rep protein, and a construct that comprises, from 5' to 3', a 3' portion of the toxicant nucleic acid sequence, a 5' portion of the toxicant nucleic acid sequence, and the second Rep recognition element. A promoter is suitably operably linked to the 5' portion of the toxicant nucleic acid sequence and a transcription terminator is preferably but not essentially (optionally) operably linked to the 3' portion of the toxicant nucleic acid sequence. In these examples, a Rep protein interacts with the Rep recognition element(s) in the target proreplicon to facilitate circularization, release and autonomous episomal replication of the target replicon.

Circularization of the target replicon results in rearrangement of the construct such that the 3' and 5' portions of the toxicant nucleic acid sequence and the second Rep recognition element become operably connected to form a contiguous toxicant nucleic acid sequence comprising, from 5' to 3', the 5' portion of the toxicant nucleic acid sequence, the second Rep recognition element and the 3' portion of the toxicant nucleic acid sequence. Autonomous episomal replication of the target replicon results in amplification of the toxicant replicon with expression of the contiguous toxicant nucleic acid sequence.

[0028] In some embodiments, the VVI nucleic acid sequence encodes a double stranded RNA molecule comprising a duplex region formed by hybridization of complementary RNA sequences encoded respectively by the 5' and 3' portions, and a single stranded region that forms a loop connecting the complementary RNA sequences, which loop is encoded in whole or in part by the downstream Rep recognition sequence. In some embodiments of this type, the double stranded RNA molecule is selected from long dsRNA, siRNA, and shRNA.

[0029] In some embodiments, the VVI nucleic acid sequence comprises a non-coding sequence (e.g., an intron) that separates individual sequences {e.g., sequences that encode a protein or a functional RNA molecule) of the VVI nucleic acid sequence.

[0030] In non-limiting examples, the second expression system component may comprise a proreplicon (a "VVI proreplicon") that includes an upstream first Rep recognition element, a downstream second Rep recognition element, which facilitate circularization, release and autonomous episomal replication (e.g., rolling circle replication) of a corresponding "VVI replicon" in the presence of a Rep protein, and a construct that includes, from 5' to 3', a first Rep recognition element, a 3' portion of the VVI nucleic acid sequence, a 5' portion of the VVI nucleic acid sequence, and the second Rep recognition element. Suitably, a promoter is operably linked to the 5' portion of the VVI nucleic acid sequence and a transcription terminator is preferably but not essentially (optionally) operably linked to the 3' portion of the VVI nucleic acid sequence. In these examples, a Rep protein interacts with the Rep recognition element(s) in the VVI proreplicon to facilitate circularization, release and autonomous episomal replication of the VVI replicon.

Circularization of the VVI replicon results in rearrangement of the construct such that the 3' and 5' portions of the VVI nucleic acid sequence and the second Rep recognition element become operably connected to form a contiguous VVI nucleic acid sequence and comprising, from 5' to 3', the 5' portion of the VVI nucleic acid sequence, the second Rep recognition element and the 3' portion of the VVI nucleic acid sequence. Autonomous episomal replication of the VVI replicon results in amplification of the VVI replicon with expression of the contiguous VVI nucleic acid sequence. [0031] In specific embodiments, the first expression system component comprises a proreplicon for expressing the toxicant nucleic acid sequence and the second expression system component comprises a proreplicon for expressing a VVI nucleic acid sequence. In other embodiments, the first expression system component comprises a proreplicon for expressing the toxicant nucleic acid sequence and the second expression system component is in the form of a biphasic expression system component for expressing a VVI nucleic acid sequence.

[0032] In representative examples of the proreplicon embodiments broadly described above and elsewhere herein, the first and/or second expression system component further comprises an expression cassette from which a rep gene is expressible to produce a Rep protein in the plant cell. Suitably, the rep gene is selected from among Geminivirus (e.g., Mastrevirus,

Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Topocuvirus, Turncurtovirus), nanovirus (e.g., Nanovi^'rus, Babuvirus), circovirus {e.g., Circovirus), and bacterial rep genes.

[0033] In certain of the proreplicon embodiments broadly described above and elsewhere herein, the Rep recognition elements of a proreplicon are virus intergenic regions (IRs), illustrative examples of which include long intergenic regions (LIRs) and short intergenic regions (SIRs). In representative examples, the Rep recognition elements in a proreplicon are selected from among Geminivirus (e.g. , Mastrevirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Topocuvirus, Turncurtovirus) IRs, nanovirus (e.g. , Nanovirus, Babuvirus) IRs and circovirus (e.g., Circovirus) IRs. In some embodiments, the Rep recognition elements in a proreplicon are selected from Mastrevirus IRs. In other embodiments, the Rep recognition elements in a proreplicon are selected from Segomov/ri/s-associated DNA-β satellite IRs. In certain embodiments, the Rep recognition elements in a proreplicon are Mastrevirus LIRs and the first and/or second expression cassette further comprises a Mastrevirus SIR.

[0034] In some embodiments, the toxicity protein is a ribosome inhibiting protein or hypersensitive response-elicitor polypeptide. One illustrative example of a ribosome inhibiting protein that is particularly suitable for using with the present invention, is a barnase.

[0035] In some embodiments, the VVI nucleic acid silences a virus gene sequence selected from the group comprising or consisting of movement protein gene, silencing suppressor gene, coat protein gene, nuclear shuttle protein gene, transactivator gene, cell cycle (e.g., retinoblastoma-like binding protein gene), and replication initiation (associated) protein gene (e.g., rep). In preferred embodiments, a siRNA is expressible from the VVI nucleic acid that silences an essential gene of the virus that is required for viability, wherein the gene is selected from the group comprising or consisting of a movement protein gene, a silencing suppressor gene, a coat protein gene, a nuclear shuttle protein gene, a transactivator gene, a cell cycle, and a replication initiation (associated) protein gene.

[0036] In a related aspect, the present invention provides plant cells that contain an expression system as broadly described above and elsewhere herein. In some embodiments, the first and/or second expression system component is/are stably introduced in the genome of the plant cell. In some embodiments, the plant cells are monocotyledonous or dicotyledonous plant cells.

[0037] In another embodiment of the present invention, the construct system confers resistance to a Geminivirus or a Nanovirus to the plant host. In some embodiments, the Geminivirus is selected from among a Mastrevirus, Begomovirus, Curtovirus and Topocuvirus. Notably, in some embodiments, the expression system in accordance with the present invention confers resistance to multiple viruses.

[0038] In yet another aspect, the invention provides a method for producing a transgenic plant that is resistant to infection by a ssDNA virus, the method comprising transforming a plant cell with an expression construct system as described in detail above and elsewhere herein.

[0039] In a further aspect, the invention provides a plant (i.e., transgenic plant) that comprises s virus-resistant plant cell described in detail above and elsewhere herein.

[0040] In some embodiments of this aspect, the plant is a monocotyledonous plant or a dicotyledonous plant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is photograph of a Southern hybridization blot in which two transformed tobacco plant cell lines (#1-3 and #1-25) were digested using restriction endonucleases Hindlll and EcoRI, to check for the incorporation of the INPACT cassette.

[0042] Figure 2 is a photograph of PCR reactions on which agarose gel electrophoresis was performed over a three month period. Primers were used to amplify TYDV movement protein gene. 16 out of 20 (80%) of the wild-type tobacco plants were infected with TYDV. Conversely, only 3 out of 20 (15%) of cell lines representing #1-3, and 2 out of 20 (10%) of cell lines representing #1-25 tested positive for TYDV.

[0043] Figure 3 is a photograph of representative plants that tested negative ("TYDV - ve") and positive ("TYDV ve") for plants that were wild-type (A), or transformed with cell line #1-3 (B).

[0044] Figure 4 is a photograph of PCR reactions on which agarose gel electrophoresis was performed over a six week period. The photographs demonstrate that 80% of the wild-type ("Wt") were infected with TYDV, whereas infection rates in transgenic pBIN-MP.hp tobacco lines ("1-10") ranged from 20-100%.

[0045] Figure 5 is a photograph of PCR reactions on which agarose gel electrophoresis was performed over six weeks. The results demonstrate that 100% of wild-type ("Wt") tobacco plants were infected with TYDV, whereas only 40% of the elite INPACT line (#1-3) were infected. Notably, infection rates in elite INPACT cell lines (" 1-11") ranged from 0-100%. Three cell lines (2, 3 and 5) shoed complete absence of the virus.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below. [0047] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0048] As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0049] Further, the term "about", as used herein when referring to a measurable value such as an amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length and the like.

[0050] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0051] The term "amplicon" refers to a chimeric nucleic acid sequence in which the cDNA of a RNA virus is operably connected to regulatory sequences such that the primary transcript is the 'plus' strand of RNA virus.

[0052] The term "antisense" refers to a nucleotide sequence whose sequence of nucleotide residues is in reverse 5' to 3' orientation in relation to the sequence of deoxynucleotide residues in a sense strand of a nucleic acid (e.g., DNA or RNA) duplex. A "sense strand" of a DNA duplex refers to a strand in a DNA duplex which is transcribed by a cell in its natural state into a "sense mRNA." Thus an "antisense' sequence is a sequence having the same sequence as the non- coding strand in a DNA duplex. The term "antisense RNA" refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene by interfering with the processing, transport and/or translation of its primary transcript or mRNA. The complementarity of an antisense RNA may be with any part of the specific gene transcript, in other words, at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. In addition, as used herein, antisense RNA may contain regions of ribozyme sequences that increase the efficacy of antisense RNA to block gene expression. "Ribozyme" refers to a catalytic RNA and includes sequence-specific endoribonucleases. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of preventing the expression of the target protein.

[0053] "Autonomous" or "c/s" replication refers to replication of a replicon that contains all cis- and trans-acting sequences (such as the replication gene (rep)) required for replication.

[0054] "Cells", "plant cells", "transformed plant cells", "regenerate plant cells" and the like are terms that not only refer to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0055] The terms "c/s-acting element", "c/s-acting sequence" or "c/s-regulatory region" are used interchangeably herein to mean any sequence of nucleotides, which modulates transcriptional activity of an operably linked promoter and/or expression of an operably linked nucleotide sequence. Those skilled in the art will be aware that a c/s-sequence may be capable of activating, silencing, enhancing, repressing or otherwise altering the level of expression and/or cell-type-specificity and/or developmental specificity of any nucleotide sequence, including coding and non-coding sequences.

[0056] "Chromosomally-integrated", as used herein, refers to the integration of a heterologous nucleic acid sequence, typically in the form of a construct, into a host DNA by covalent bonds.

[0057] By "coding sequence" is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene or for the final mRNA product of a gene (e.g. the mRNA product of a gene following splicing). By contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene or for the final mRNA product of a gene.

[0058] As used herein the term "co-expression", "co-expressing" and the like mean that nucleotide sequences coding for two or more nucleic acid sequences are expressed in the same plant cell, suitably concurrently {i.e., the expression of a nucleotide sequence and that of another overlap with each other) or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all nucleotide sequences are expressed concurrently.

[0059] As used herein, "complementary" polynucleotides are those that are capable of hybridizing via base pairing according to the standard Watson-Crick complementarity rules.

Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A: U) in the case of RNA. For example, the sequence "A-G-T" binds to the

complementary sequence "T-C-A." It is understood that two polynucleotides may hybridize to each other even if they are not completely or fully complementary to each other, provided that each has at least one region that is substantially complementary to the other. The terms "complementary" or "complementarity", as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. Complementarity between two single stranded molecules (also referred to herein as "nucleobase polymers") may be "partial", in which only some of the nucleobases base pair, or it may be "complete" when total complementarity exists between the single stranded molecules either along the full length of the molecules or along a portion or region of the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. The term "complementary" includes within its scope nucleic acid sequences that are "fully complementary", "substantially complementary" or "partially complementary". As used herein, the term "fully complementary" indicates that 100% of the nucleobases in a particular nucleobase polymer are able to engage in base-pairing with another nucleobase polymer. The term "substantially complementary", as used herein, indicates that at least at about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the nucleobases in a particular nucleobase polymer are able to engage in base-pairing with another nucleobase polymer. As used herein, the term "partially complementary" indicates that at least at about 50%, 55% or 60% of the nucleobases in a particular nucleobase polymer are able to engage in base-pairing with another nucleobase polymer. The terms "substantially complementary" and "partially complementary" can also mean that two nucleic acid sequences can hybridize under high stringency or medium stringency conditions and such conditions are well known in the art.

[0060] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0061] The terms "conditional expression", "conditionally expressed" "conditionally expressing" and the like refer to the ability to activate or suppress expression of a gene of interest by the presence or absence of a stimulus or other signal (e.g., chemical, light, hormone, stress, or a pathogen). In specific embodiments, conditional expression of a nucleic acid sequence of interest is dependent on the presence of an inducer or the absence of an inhibitor.

[0062] As used herein, the term "concurrent stimulation", "concurrently stimulated" and the like means that the stimulation of a regulated promoter and that of another promoter overlap with each other.

[0063] "Constitutive expression", as used herein, refers to expression using a constitutive or regulated promoter. "Conditional" and "regulated expression" refer to expression controlled by a regulated promoter.

[0064] "Constitutive promoter" refers to an unregulated promoter that directs expression of an operably linked transcribable sequence in many or all tissues of a plant regardless of the surrounding environment and suitably at all times.

[0065] The term "construct" refers to a recombinant genetic molecule including one or more isolated nucleic acid sequences from different sources. Thus, constructs are chimeric molecules in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule and include any construct that contains (1) nucleic acid sequences, including regulatory and coding sequences that are not found together in nature (i.e., at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of functional RNA molecules or proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined. Representative constructs include a ny recombi nant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or l inea r or circular single stranded or double stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecules have been operably l inked . Constructs of the present invention will genera lly incl ude the necessary elements to di rect expression of a nucleic acid sequence of interest that is also conta ined i n the construct, such as, for example, a toxica nt nucleic acid sequence or a VVI nucleic acid sequence. Such elements may i nclude control elements such as a promoter that is operably linked to (so as to d irect transcription of) the nucleic acid seq uence of interest, and often includes a polyadenylation seq uence as wel l. Within certai n embod iments of the i nvention, the construct may be contai ned within a vector. In addition to the components of the construct, the vector may i nclude, for exa mple, one or more selectable ma rkers, one or more origins of replication, such as prokaryotic and euka ryotic orig ins, at least one multiple cloning site, and/or elements to facilitate sta ble integration of the construct into the genome of a plant cell . Two or more constructs can be contained within a single nucleic acid molecule, such as a single vector, or ca n be contai ning withi n two or more separate nucleic acid molecules, such as two or more separate vectors. An "expression construct" genera lly incl udes at least a control seq uence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in opera ble connection with the nucleotide sequences to be expressed are provided in expression constructs for expression i n a n plant or pa rt thereof i ncludi ng a plant cell. For the practice of the present invention, conventional compositions and methods for prepa ring and using constructs and plant cells a re well known to one ski lled in the art, see for example, Molecula r Cloning : A Laboratory Manual, 3rd ed ition Volumes 1, 2, and 3. J . F. Sa mbrook, D. W. Russell, and N . Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0066] As used herei n, the term "contiguous" in the context of a nucleic acid sequence means that the sequence is a si ngle sequence, uninterrupted by any interveni ng seq uence or sequences.

[0067] The term "contiguous nucleic acid entity" defines an entity (e.g. , a gene) comprised of a linear series or complete seq uence of nucleotides, suitably within a larger polynucleotide seq uence, which defines the nucleic acid entity (e.g. , a VVI nucleic acid sequence, a toxica nt nucleic acid sequence etc. ) . A "non-contiguous nucleic acid entity" is an entity that is comprised of a series of nucleotides withi n a polynucleotide sequence, which is non-linear in alignment, that is that the nucleotides a re spaced or g rouped in a non-conti nuous manner along the length of a polynucleotide sequence. A non-contig uous nucleic acid entity (also referred to herei n as a "spl it gene") can be a discontinuous nucleic acid entity wherein the nucleotides a re grouped i nto 2 li near seq uences (e.g., each comprising a d ifferent open read ing fra me (ORF)) arra nged along the length of the polynucleotide, which together define the enti re sequence of the nucleic acid entity (e.g., a VVI nucleic acid sequence, a toxica nt nucleic acid seq uence etc. ) .

Alternatively, the non-contiguous nucleic acid entity ca n be a disconti nuous scattered nucleic acid entity wherein the nucleotides, which contribute the entire sequence of the nucleic acid entity, are provided in 3 or more groups of linear nucleotide sequences (e.g. , each comprising a different ORF) arra nged along the length of the polynucleotide. Illustrative non-contiguous nucleic acid entities i nclude those in which a 5' portion of a contiguous nucleic acid entity is located on a nucleic acid molecule downstrea m of a 3' portion of the contiguous nucleic acid entity, such that transcription of the full length RNA encoded by the contiguous nucleic acid entity can not occur unless the non- contiguous nucleic acid entity is first rearranged, such as described herein and in U.S. Pat. No. 7,863,430. Reference to non-contiguous nucleic acid entities also includes reference to entities in which two or more non-contiguous sequences [e.g., ORFs) are located on two or more nucleic acid molecules, such as described in U.S. Patent No. 6,531,316. In some embodiments, a non- contiguous nucleic acid entity (e.g., a non-contiguous VVI nucleic acid entity, a non-contiguous toxicant nucleic acid entity etc.) is in the form of two or more portions and positioned so that a 3' portion is upstream of a 5' portion and the 5' portion is operably connected to a promoter, the 5' portion generally does not contain greater than 80%, 85%, 95%, or more of the entire contiguous nucleic acid entity, to prevent any unintended expression of a functional expression product. In some embodiments, a 3' portion contains at least or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of a contiguous nucleic acid entity, and a 5' portion contains at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the contiguous nucleic acid entity, wherein the 5' and 3' portions together make up a contiguous nucleic acid entity that codes for a desired expression product.

[0068] By "control sequence", "control element" and the like is meant nucleic acid sequences (e.g., DNA) necessary for expression of an operably linked coding and/or non-coding sequences in a particular plant cell. Control sequences include nucleotide sequences located upstream, within, or downstream of a nucleic acid sequence of interest (which may comprise coding and/or non-coding sequences), and which influence the transcription, RNA processing or stability, or translation of the associated nucleic acid sequence of interest, either directly or indirectly. The control sequences that are suitable for prokaryotic cells for example, include a promoter, and optionally a c/s-acting sequence such as an operator sequence and a ribosome binding site. Control sequences that are suitable for eukaryotic cells include transcriptional control sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers, introns, Rep recognition elements, intergenic regions, polyadenylation signal sequences, internal ribosome binding sites (IRES), nucleic acid sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment. Control sequences include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.

[0069] By "corresponds to" or "corresponding to" is meant a nucleic acid sequence that displays substantial sequence identity to a reference nucleic acid sequence (e.g., at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence identity to all or a portion of the reference nucleic acid sequence) or an amino acid sequence that displays substantial sequence similarity or identity to a reference amino acid sequence (e.g., at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence similarity or identity to all or a portion of the reference amino acid sequence).

[0070] The term "double stranded RNA" or "dsRNA", as used herein, refers to a ribonucleic acid containing at least a region of nucleotides that are in a double stranded conformation. The double stranded RNA may be a single nucleotide polymer with one or more region(s) of self-complementarity such that nucleotides in one segment of the polymer base pair with nucleotides in another segment of the polymer. Alternatively, the double stranded RNA may include two nucleotide polymers that have one or more region(s) of complementarity to each other. The double stranded RNA will typically comprise a duplex region comprising two anti-parallel nucleic acid strands that are partially, substantially or fully complementary, as defined herein. As used herein, a "strand" refers to a contiguous sequence of nucleotides and reference herein to "two strands" includes the strands being, or each forming a part of, separate nucleotide polymers or molecules, or the strands being covalently interconnected, e.g., by a linker, to form but one nucleotide polymer or molecule. At least one strand can include a region which is sufficiently complementary to a target sequence. Such strand is termed the "antisense strand". A second strand comprised in the double stranded RNA, which comprises a region complementary to the antisense strand, is termed the "sense strand". However, a double stranded RNA can also be formed from a single RNA molecule which is at least partly self-complementary, forming a duplex region, e.g., a hairpin or panhandle. In such case, the term "strand" refers to one of the regions of the RNA molecule that is complementary to another region of the same RNA molecule. The term "antisense strand" refers to the strand of a double stranded RNA which includes a region that is complementary (typically substantially or fully complementary) to a sequence of nucleotides ("target sequence") located within the RNA transcript of target gene. This strand is also known as a "guide" sequence, and is used in a functioning RISC complex to guide the complex to the correct RNA {e.g., mRNA) for cleavage. As used herein, the term "region of complementarity" refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully

complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5' and/or 3' terminus. The term "sense strand", as used herein, refers to the strand of a double stranded RNA that includes a region that is substantially complementary to a region of the antisense strand. This strand is also known as an "anti-guide" sequence because it contains the same sequence of nucleotides as the target sequence and therefore binds specifically to the guide sequence.

[0071] As used herein, the terms "encode", "encoding" and the like refer to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide. For example, a nucleic acid sequence is said to "encode" a polypeptide if it can be transcribed and/or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and/or translated to produce the polypeptide. Such a nucleic acid sequence may include a coding sequence or both a coding sequence and a non-coding sequence. Thus, the terms "encode", "encoding" and the like include a RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of a RNA molecule, a protein resulting from transcription of a DNA molecule to form a RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide a RNA product, processing of the RNA product to provide a processed RNA product (e.g. , mRNA) and the subsequent translation of the processed RNA product.

[0072] The term "endogenous" refers to any polynucleotide or polypeptide which is present and/or naturally expressed within a host organism or cell thereof. For example, an "endogenous" nucleic acid refers to a nucleic acid molecule or nucleotide sequence that is naturally found in the cell into which an expression system component of the invention is introduced. [0073] As used herein, the term "episome" or "replicon" refers to a DNA or RNA virus or a vector that undergoes episomal replication in plant cells. It contains c/s-acting viral sequences, such as the Rep recognition element (also commonly referred to as a "replication origin"), necessary for replication. It may or may not contain trans-acting sequences necessary for replication, such as the viral replication genes (for example, the AC1 and AL1 genes in ACMV and TGMV Geminiviruses, respectively). It may or may not contain a nucleic acid sequence of interest for expression in the plant cell.

[0074] "Episomal replication" and "replicon replication" are used interchangeably herein to refer to replication of replicons, suitably DNA or RNA viruses or virus-derived replicons, that are not stably introduced in a host (e.g., chromosomally-integrated). Episomal replication generally requires the presence of viral replication protein(s) essential for replication, is independent of chromosomal replication, and results in the production of multiple copies of virus or replicons per host genome copy.

[0075] The term "expression" refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to the production of protein. Thus, as will be clear from the context, expression of a coding sequence results from transcription and translation of the coding sequence. Conversely, expression of a non-coding sequence results from the transcription of the non-coding sequence.

[0076] As used herein, the term "expression cassette" refers to a polynucleotide sequence capable of effecting expression of a gene of interest (e.g., a toxicant nucleic acid sequence) in a plant cell. Expression cassettes include at least one control sequence (e.g., a promoter, enhancer, transcription terminator and the like) operably linked with the gene of interest, which can be in the form of a contiguous or non-contiguous nucleic acid entity as defined herein. "Overexpression" refers to the level of expression in transgenic organisms that exceeds levels of expression in normal or untransformed organisms. The expression cassette may be naturally present in a plant cell or may be part of a construct.

[0077] The term "expression system" refers to any nucleic acid based approach or system for expressing one or more nucleic acids of interest. Where expression of two or more nucleic acid sequences of interest is desired, the expression system will generally comprise a component ("expression system components") for expression of each nucleic acid sequence of interest. Such components may comprise one or more expression cassettes for expressing an individual nucleic acid sequence of interest. Where more than one expression cassette is used to express a nucleic acid sequence of interest, the expression cassettes may be on the same construct or vector or on different constructs or vectors. The expression cassettes may be endogenous or heterologous with respect to the plant cell in which they reside or are proposed to reside, provided that at least one them (e.g., used to express the VVI nucleic acid sequence) of the expression system is heterologous with respect to the plant cell. In specific embodiments, at least one component of the expression system is in the form of a binary expression system. As used herein, the term "binary expression system" describes an expression system component comprised of two constructs, at least one of which is chromosomally integrated. In specific embodiments, the binary expression system component is a binary viral expression system component comprising a first construct and a second construct in which the first construct comprises an inactive replicon or a proreplicon from which a nucleic acid sequence of interest is expressible in a plant cell and the second construct comprises a regulated promoter operably-linked to a transactivating gene. The inactive replicon or proreplicon and a chimeric transactivating gene, functioning together, will effect replicon replication and expression of the nucleic acid sequence of interest in a plant cell in a regulated manner. Both constructs may be stably introduced into the plant cell (e.g.,

chromosomally-integrated) and may be inherited independently. Stimulating the regulated promoter driving the transactivating gene releases the replicon from the chromosome and its subsequent episomal replication. The release can be physical excision of the replicon from the chromosome involving site-specific recombination, a replicative release from a master

chromosomal copy of a proreplicon in the presence of the replication protein, or transcriptional release from a master chromosomal copy of an amplicon.

[0078] As used herein, the terms "fragment" or "portion" when used in reference to a nucleic acid molecule or nucleotide sequence will be understood to mean a nucleic acid molecule or nucleotide sequence of reduced length relative to a reference nucleic acid molecule or nucleotide sequence and comprising, consisting essentially of and/or consisting of a nucleotide sequence of contiguous nucleotides identical or homologous {e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.

[0079] The term "functional nucleic acid" as used herein refers to a nucleic acid having specific biological functions in vivo or in cells, such as enzymatic functions, catalytic functions, or biologically inhibiting or enhancing functions (e.g., inhibition or enhancement of transcription or translation). Specific examples include siRNA, shRNA, miRNA (including pri-miRNA and pre- miRNA), nucleic acid aptamers (including RNA aptamers and DNA aptamers), ribozymes (including deoxyribozymes), riboswitches, Ul adaptors, molecular beacons, and transcriptional factor-binding regions.

[0080] As used herein, the term "gene" refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, siRNA, shRNA, miRNA, and the like. Genes may or may not be capable of being used to produce a functional protein. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements including promoters, enhancers, termination sequences and 5' and 3' untranslated regions). A gene may be "isolated" by which is meant a nucleic acid molecule that is substantially or essentially free from components normally found in association with the nucleic acid molecule in its natural state. Such components include other cellular material, culture medium from recombinant production, and/or various chemicals used in chemically synthesizing the nucleic acid molecule. Reference to a "gene" also includes within its scope reference to genes having a contiguous sequence, thus defining contiguous nucleic acid entities, as defined herein, or a non-contiguous sequence thus defining a non-contiguous nucleic acid entity as defined herein. In certain embodiments, the term "gene" includes within its scope the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non- coding nucleotide sequences involved in the regulation of expression. In this regard, the gene may further comprise control sequences such as promoters, enhancers, termination and/or

polyadenylation signals that are naturally associated with a given gene, or heterologous control sequences. The gene sequences may be cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for introduction into a host.

[0081] "Genome" as used herein includes the nuclear and/or plastid genome, and therefore includes introduction of the nucleic acid into, for example, the chloroplast genome.

[0082] The terms "growing" or "regeneration" as used herein mean growing a whole, differentiated plant from a plant cell, a group of plant cells, a plant part (including seeds), or a plant piece (e.g., from a protoplast, callus, or tissue part).

[0083] The term "heterologous" as used herein with reference to nucleic acids refers to a nucleic acid molecule or nucleotide sequence that either originates from another species or is from the same species or organism but is modified from either its original form or the form primarily expressed in the cell. Thus, a nucleotide sequence derived from an organism or species different from that of the cell into which the nucleotide sequence is introduced, is heterologous with respect to that cell and the cell's descendants. Such nucleotide sequences are also referred to herein as "foreign" nucleotide sequences. In addition, a heterologous nucleotide sequence includes a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g. present in a different copy number, and/or under the control of different regulatory sequences than that found in the native state of the nucleic acid molecule. The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, a nucleic acid may be recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a nucleic acid encoding a protein from one source and a nucleic acid encoding a peptide sequence from another source. Similarly, a "heterologous" protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0084] As used herein the terms "homolog", "homolog" or "homologous" refer to the level of similarity between two or more nucleic acid sequences in terms of percent of sequence identity. Generally, homologs, homologous sequences or sequences with homology refer to nucleic acid sequences that exhibit at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to one another. Alternatively, or in addition, homologs, homologous sequences or sequences with homology refer to nucleic acid sequences that hybridize under high stringency conditions to one another. High stringency conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 salt for hybridization at 42° C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42° C. High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, ImM EDTA, 40 mM NaHP04 (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C. By contrast, the terms "non-homologous", "non-homologous sequences" or "sequences that lack homology" and the like refer to nucleic acid sequences that exhibit no more than 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% sequence identity to one another. Alternatively, or in addition, non-homologous", "non-homologous sequences" or "sequences that lack homology" and the like refer to nucleic acid sequences that do not hybridize under high stringency conditions to one another but suitably hybridize under medium or low stringency conditions to one another. Reference herein to low stringency conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C, and at least about 1 M to at least about 2 M salt for washing at 42° C. Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2xSSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP04 (pH 7.2), 5% SDS for washing at room temperature. Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C, and at least about 0.5 M to at least about 0.9 M salt for washing at 42° C. Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP04 (pH 7.2), 5% SDS for washing at 42° C. In specific embodiments, the term "non-homologous", with reference to a double stranded RNA molecule comprising a duplex region formed by hybridization of complementary RNA sequences refers to the non-homology displayed by those complementary RNA sequences (particularly the complementary RNA sequence defining the "antisense strand" of the duplex) to a RNA expression product (e.g., mRNA) of a target nucleic acid, suitably over a comparison window as defined for example below.

[0085] The term "host" refers to any plant, or cell thereof, into which a construct of the invention can be introduced, particularly, hosts in which RNA silencing occurs. Illustrative examples of plant hosts include angiosperms, gymnosperms, monocotyledons (monocots) and dicotyledons (dicots). Thus, the term "host cell" suitably encompasses cells of such plants as well as cell lines derived from such plants.

[0086] The terms "in c/s" and "in trans" refer to the presence of nucleic acid elements, such as the Rep recognition element and the rep gene, on the same nucleic acid

molecule/construct or on different nucleic acid molecules/constructs, respectively. Consistent with these definitions, the terms "c/s-acting sequence" and " /s-acting element" refer to DNA or RNA sequence, whose function requires them to be on the same molecule. An example of a c/s-acting sequence on a replicon is a Rep recognition element.

[0087] As used herein, "inactive replicon" refers to a replication-defective replicon that contains c/s-acting viral sequences, such as the replication origin, necessary for replication but is defective in replication because it lacks either a functional viral gene necessary for replication and/or the ability to be released from the chromosome due to its DNA arrangement involving site- specific recombination sequences (e.g., Rep recognition elements). Consequently, an inactive replicon can replicate episomally only when it is provided with the essential replication protein in trans, as in the case of single stranded DNA virus (e.g., Geminivirus) proreplicon, or when its nonfunctional replication gene is rendered functional by site-specific recombination with or without release of the active replicon nucleic acid from the chromosome. "Activation of replicon replication" refers to the process in which an inactive replicon is rendered active for episomal replication.

[0088] "Inducible promoter", as used herein, refers to those regulated promoters that can be turned on in one or more cell types by an external stimulus, such as a chemical, light, hormone, stress, or a pathogen. [0089] By "intergenic region" or "IR" is meant a non-coding region in the genome of a virus, in particular a single stranded DNA virus. For the purposes of the present invention, reference to an intergenic region or IR includes ssDNA virus intergenic regions and fragments or variants thereof that retain the features necessary for binding of Rep and initiation of rolling circle replication. Accordingly, intergenic regions for use in the present invention include Geminivirus IRs (such as Mastrevirus long intergenic regions (LIRs), Begomovirus and Topocuvirus common regions (CRs), and Curtovirus IRs), Nanovirus IRs, IRs from Begomovirus beta-satellites (DNA-β satellites) or alphasatellites, as well as variants and fragments thereof that retain the necessary elements for binding of Rep and initiation of rolling circle replication.

[0090] "Introducing" in the context of a plant cell, plant part and/or plant organ means contacting a nucleic acid molecule with the plant, plant part, and/or plant cell in such a manner that the nucleic acid molecule gains access to the interior of the plant cell, plant part and/or plant organ. Where more than one nucleic acid molecule is to be introduced these nucleic acid molecules can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, these polynucleotides can be introduced into plant cells in a single transformation event, in separate transformation events, or, e.g., as part of a breeding protocol. Thus, the term "transformation" as used herein refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell may be stable or transient. "Transient transformation" in the context of a polynucleotide means that a polynucleotide is introduced into the cell and does not integrate into the genome or heritable extrachromosomal element of the cell. By "stably introducing" or "stably introduced" in the context of a polynucleotide introduced into a cell, it is intended that the introduced polynucleotide is stably incorporated or integrated into the genome or stable extra-chromosomal element of the cell, and thus the cell is stably transformed with the polynucleotide. "Stable transformation" or "stably transformed" as used herein means that a nucleic acid molecule is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. Stable transformation as used herein can also refer to a nucleic acid molecule that is maintained extrachromosomally, for example, as a minichromosome.

[0091] The term "intron" refers to a nucleotide sequence within or adjacent to a coding sequence that is removed by RNA splicing, and necessarily contains sequences required for splicing, such as a 3' splice site and a 5' splice site. Reference to introns includes reference to intact introns and split introns, such as an intron split into two regions: a 3' region comprising a 3' splice site, and a 5' region comprising a 5' splice site.

[0092] The term "inverted repeat" refers to a nucleic acid sequence comprising a sense and an antisense element positioned so that they are able to form a RNA duplex when the repeat is transcribed. The inverted repeat may optionally include a linker or a heterologous sequence between the two elements of the repeat, which defines a loop structure. The inverted repeat need not be perfect; non-complementary bases are tolerated provided there is a sufficient degree of complementarity between the repeats for the sense and antisense elements to anneal to one other and form the duplex. [0093] The term "microRNA" or "miRNA" refers to small, noncoding RNA molecules that have been found in a diverse array of eukaryotes, including plants. miRNA precursors share a characteristic secondary structure, forming short 'hairpin' RNAs. The term "miRNA" includes processed sequences as well as corresponding long primary transcripts (pri-miRNAs) and processed precursors (pre-miRNAs). Genetic and biochemical studies have indicated that miRNAs are processed to their mature forms by Dicer, a RNAse III family nuclease, and function through RNA- mediated interference (RNAi) and related pathways to regulate the expression of target genes (Hannon (2002) Nature 418, 244-251 ; Pasquinelli, et al. (2002) Annu. Rev. Cell. Dev. Biol. 18, 495-513). miRNAs may be configured to permit experimental manipulation of gene expression in cells as synthetic silencing triggers 'short hairpin RNAs' (shRNAs) (Paddison et al. (2002) Cancer

Cell 2, 17-23). Silencing by shRNAs involves the RNAi machinery and correlates with the production of small interfering RNAs (siRNAs), which are a signature of RNAi.

[0094] The term "non-coding" refers to sequences of nucleic acid molecules that do not encode part or all of an expressed protein. Non-coding sequences include but are not limited to introns, enhancers, promoter regions, 3' untranslated regions, and 5' untranslated regions. Thus, the term "5'-non-coding region" shall be taken in its broadest context to include all nucleotide sequences which are derived from the upstream region of a gene. Such regions may include an intron, e.g. , an intron. As used herein, the term "3' non-coding region" refers to nucleic acid sequences located downstream of a coding sequence and include polyadenylation recognition sequences (normally limited to eukaryotes) and other sequences encoding regulatory elements capable of affecting mRNA processing or gene expression. The polyadenylation signal (normally limited to eukaryotes) is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[0095] As used herein, the term "nucleic acid sequence" or "nucleotide sequence" refers to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, a DNA fragment, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, any of which can be single stranded or double stranded. The terms "nucleotide sequence" "nucleic acid", "nucleic acid molecule", "oligonucleotide" and "polynucleotide" are also used interchangeably herein to refer to a heteropolymer of nucleotides, and include RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids.. Nucleic acid sequences provided herein are presented herein in the 5' to 3' direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR 1.821 - 1.825 and the World Intellectual Property Organization (WIPO) Standard ST.25.

[0096] The term "operably connected" or "operably linked" as used herein refers to a j uxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a control sequence (e.g., a promoter) "operably linked" to a nucleotide sequence of interest (e.g., a coding and/or non-coding sequence) refers to positioning and/or orientation of the control sequence relative to the nucleotide sequence of interest to permit expression of that sequence under conditions compatible with the control sequence. The control sequences need not be contiguous with the nucleotide sequence of interest, so long as they function to direct its expression. Thus, for example, intervening non-coding sequences (e.g., untranslated, yet transcribed, sequences) can be present between a promoter and a coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence. Likewise, "operably connecting" a cis-acting sequence to a promoter encompasses positioning and/or orientation of the cis-acting sequence relative to the promoter so that the cis-acting sequence regulates (e.g., inhibits, abrogates, stimulates or enhances) promoter activity. Alternatively, "operably connecting" non-contiguous nucleic acid sequences of a noncontiguous nucleic acid entity encompasses rearrangement (e.g., positioning and/or orientation) of the non-contiguous nucleic acid sequences relative to each other so that (1) the reassembled nucleic acid sequences form the sequence of a contiguous nucleic acid entity (e.g., a contiguous toxicant or VVI nucleic acid entity) and optionally (2) if the non-contiguous nucleic acid sequences each comprise a coding sequence, each coding sequence is Ίη-frame' with another to produce a complete open reading frame corresponding to the coding sequence of the contiguous nucleic acid entity.

[0097] As used herein, "plant" and "differentiated plant" refer to a whole plant or plant part containing differentiated plant cell types, tissues and/or organ systems. Plantlets and seeds are also included within the meaning of the foregoing terms. Plants included in the invention are any plants amenable to transformation techniques, including angiosperms, gymnosperms, monocotyledons (monocots) and dicotyledons (dicots). Non-limiting examples of monocot plants of the present invention include sugar cane, corn, barley, rye, oats, wheat, rice, flax, millet, sorghum, grasses, banana, onion, asparagus, lily, coconut, and the like.

[0098] As used herein, "plant cell" refers to a structural and physiological unit of the plant, which comprises a cell wall and also may refer to a protoplast, gamete-producing cell, or cell which regenerates into whole plants. A plant cell of the present invention can be in the form of an isolated single cell or can be a cultured cell or can be a part of a higher-organized unit such as, for example, a plant tissue or a plant organ.

[0099] As used herein, the term "plant part" includes but is not limited to embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, plant cells including plant cells that are intact in plants and/or parts of plants, plant protoplasts, plant tissues, plant cell tissue cultures, plant calli, plant clumps, and the like.

[0100] The term "plant organ" refers to plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant.

[0101] "Polypeptide", "peptide", "protein" and "proteinaceous molecule" are used interchangeably herein to refer to molecules comprising or consisting of a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. This term also includes within its scope two or more complementing or interactive polypeptides comprising different parts or portions (e.g., polypeptide domains, polypeptide chains etc.) of a polypeptide of the present invention, wherein the individual complementing polypeptides together reconstitute the activity of the different parts or portions to form a functional polypeptide.

[0102] The term "promoter" refers to a nucleotide sequence, usually upstream (5') to a transcribable sequence, which controls the expression of the transcribable sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoter that is a short nucleic acid sequence comprised of a TATA-box and other sequences that serve to specify the site of transcription initiation, to which control elements {e.g., c/s-acting elements) are added for control of expression. "Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus control elements (e.g., c/s-acting elements) that are capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a nucleic acid sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific nucleic acid-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic nucleic acid segments. A promoter may also contain nucleic acid sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions. Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters." In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription. A "minimal or core promoter" thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and/or an initiator.

[0103] "Promoter activity" refers to the ability of a promoter to drive expression of a nucleic acid sequence operably linked to the promoter. Promoter activity of a sequence can be assessed by operably linking the sequence to a reporter gene, and determining expression of the reporter.

[0104] As used herein "proreplicon' refers to an inactive replicon that is comprised of c/s-acting viral sequences required for replication, and flanking sequences that enable the release of the replicon from it. It is generally integrated into a vector or host chromosome and may contain a nucleic acid sequence of interest (e.g. , toxicant or VVI nucleic acid sequence). A proreplicon generally lacks a gene encoding a replication protein (Rep) essential for replication. Therefore, it is unable to undergo episomal replication in the absence of the Rep. Its replication requires both release from the integration and the presence of the essential replication gene {rep) in trans. The release from integration can be triggered in different ways. For example, the proreplicon can be present as a partial or complete tandem duplication, such that a full-length replicon sequence is flanked by virus sequences, typically intergenic region sequences, and such that the duplicated viral sequence includes the viral replication origin. Thus, in this case, the proreplicon serves as a master copy from which replicons can be excised by replicational release in the presence of Rep. Alternatively, the proreplicon can be excised by site-specific recombination between sequences flanking it in the presence of an appropriate site-specific recombinase. In the case of RNA virus proreplicons, the amplicon sequences flanking the inactive replicon, which include regulatory sequences, allow generation of the replicon as RNA transcripts that can replicate in trans in the presence of Rep. These regulatory sequences can be for constitutive or regulated expression. [0105] The term "rearrangement" refers to the rearrangement of non-contiguous nucleic acid sequences such that they become operably connected with one another to form a contiguous nucleic acid entity (e.g., a contiguous toxicant or VVI nucleic acid sequence). This term encompasses one or more changes in the order of spaced subsequences of a VVI or toxicant nucleic acid sequence, and can include the insertion of a new subsequence or replacement of a subsequence with a new subsequence. This includes combinations of re-ordering, substitution, and insertion of subsequences. Thus, for example, rearrangement of a split rep gene comprising two or more spaced rep subsequences, to form an intact rep gene, will result in transcription of rep mRNA and subsequent translation of a Rep protein.

[0106] The term "regulated promoter" refers to promoters that direct gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and include both tissue- specific and inducible promoters. It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in host cells are constantly being discovered. Since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity. Illustrative regulated promoters include but are not limited to safener- inducible promoters, promoters derived from the tetracycline-inducible system, promoters derived from salicylate-inducible systems, promoters derived from alcohol-inducible systems, promoters derived from glucocorticoid-inducible systems, promoters derived from pathogen-inducible systems, promoters derived from carbohydrate inducible systems, promoters derived from hormone inducible systems, promoters derived from antibiotic inducible systems, promoters derived from metal inducible systems, promoters derived from heat shock inducible systems, and promoters derived from ecdysone-inducible systems.

[0107] "Regulatory elements", "regulatory sequences" and the like refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence, either directly or indirectly. Regulatory elements include enhancers, promoters, translation leader sequences, introns, Rep recognition element, intergenic regions and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.

[0108] A "rep gene", as used herein, refers to a gene that encodes a replication initiation (Rep) protein or replicase. The term "rep gene" also encompasses genes that encode wild-type Rep proteins as well as modified or variant Rep proteins, including Rep protein fragments. Such modified or variant Rep proteins are biologically active and retain the ability to initiate rolling circle replication (i.e. are functional). Reference to a rep gene includes reference to viral and bacterial rep genes, as well as rep genes that are endogenous to the host plant and those that are heterologous to the host plant (such as introduced into the host plant by recombinant techniques). Further, reference to a rep gene includes reference to split rep genes. In addition to the ORF for the Rep protein, the rep gene may contain other overlapping or non-overlapping ORF(s) as are found, for example, in viral sequences in nature. While not essential for replication, these additional ORFs may enhance replication and/or viral DNA accumulation. Non-limiting examples of such additional ORFs are AC3 and AL3 in ACMV and TGMV Geminiviruses, respectively.

[0109] A Yep coding sequence" as used herein refers to a sequence of nucleic acids from which a single transcript encoding a Rep protein can be produced.

[0110] A "Rep protein" of the present invention refers to a replicase or replication initiation protein or polypeptide that is encoded by a rep gene. Reference to a Rep protein includes reference to wild-type Rep proteins as well as modified or variant Rep proteins, including Rep protein fragments. Such modified or variant Rep proteins are biologically active and retain the ability to initiate rolling circle replication {i.e. are functional). Reference to a Rep protein includes reference to Rep proteins that are encoded by viral and bacterial rep genes, as well as rep genes that are endogenous to the host plant and those that are heterologous to the host organism (such as introduced into the host organism by recombinant techniques). For example, reference to a Rep protein includes, but is not limited to, reference to a Rep protein produced by expression of an endogenous rep gene encoded in a virus genome (i.e. a virus-originating Rep protein) and reference to a Rep protein produced by expression of a heterologous rep gene encoded in a construct introduced into a host cell.

[0111] The term "Rep recognition element" refers to a nucleic acid element that contains features necessary to facilitate binding of Rep and initiate rolling circle replication. Rep recognition elements therefore can contain iterons, which are the small repeat sequences required for virus Rep recognition and binding, and an inverted repeat and the consensus nonanucleotide, which together form a stem loop structure. The consensus nonanucleotide contains the initiation site of rolling circle replication. Reference to Rep recognition elements includes reference to Rep recognition elements that contain all of the necessary features for Rep binding and initiation of rolling circle replication, as well as Rep recognition elements that contain necessary features for Rep binding and initiation of rolling circle replication but that further require the presence of one or more additional nucleic element in cis for rolling circle replication to occur. Thus, reference to a Rep recognition element includes, for example, Geminivirus and Nanovirus intergenic regions (IRs), including Mastrevirus long intergenic regions (LIRs; which typically require a Mastrevirus short intergenic region for rolling circle replication), Begomovirus and Topocuvirus common regions (CRs), and Curtovirus IRs, IRs from Begomovirus betasatellites (DNA-β satellites) or

alphasatellites, and origins of replication from bacterial rolling circle replication plasmids, as well as variants and fragments thereof that retain the necessary elements for binding of Rep and initiation of rolling circle replication.

[0112] As used herein, the terms "RNA interference" and "RNAi" refer to sequence- specific, post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) in animals and plants, initiated by double stranded RNA that is homologous in sequence to the silenced gene. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs) triggered by dsRNA fragments cleaved from longer dsRNA which direct the degradative mechanism to other RNA sequences having closely homologous sequences. As practiced as a technology, RNAi can be initiated by human intervention to reduce or even silence the expression of target genes using either exogenously synthesized dsRNA or dsRNA transcribed in the cell (e.g., synthesized as a sequence that forms a short hairpin structure). The terms "RNA interference" and "RNAi" are used interchangeably herein to refer to "RNA silencing" (also referred to herein as "RNA-mediated gene silencing") as the result of RNAi inhibition or "silencing" at the RNA level of the expression of a corresponding gene or nucleic acid sequence of interest. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0113] As used herein, the terms "small interfering RNA" and "short interfering RNA" ("siRNA") refer to a short RNA molecule, generally a double stranded RNA molecule about 10-50 nucleotides in length (the term "nucleotides" including nucleotide analogs), preferably between about 15-25 nucleotides in length. In most cases, the siRNA is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. Such siRNA can have overhanging ends {e.g. , 3'-overhangs of 1, 2, or 3 nucleotides (or nucleotide analogs). Such siRNA can mediate RNA interference.

[0114] As used in connection with the present invention, the term "shRNA", and in some embodiments the terms "double stranded RNA molecule", dsRNA and the like, refer to a RNA molecule having a stem-loop structure. The stem-loop structure includes two mutually

complementary sequences, where the respective orientations and the degree of complementarity allow base pairing between the two sequences. The mutually complementary sequences are linked by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.

[0115] The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Useful methods for determining sequence identity are also disclosed in Guide to Huge Computers (Martin J. Bishop, ed., Academic Press, San Diego (1994)), and Carillo et al. (Applied Math 48: 1073(1988)). More particularly, preferred computer programs for determining sequence identity include but are not limited to the Basic Local Alignment Search Tool (BLAST) programs which are publicly available from National Center Biotechnology Information (NCBI) at the National Library of Medicine, National Institute of Health, Bethesda, Md. 20894; see BLAST Manual, Altschul et al., NCBI, NLM, NIH; (Altschul et al., J. Mol. Biol. 215:403-410 (1990)); version 2.0 or higher of BLAST programs allows the introduction of gaps (deletions and insertions) into alignments; for peptide sequence BLASTX can be used to determine sequence identity; and for polynucleotide sequence BLASTN can be used to determine sequence identity.

[0116] Terms used to describe sequence relationships between two or more polynucleotides include "reference sequence", "comparison window", "sequence identity", and "percentage of sequence identity". A "reference sequence" is at least 12 but frequently at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides in length. Because two polynucleotides may each comprise (1) a sequence [i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 contiguous positions, or at least about 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000 or 3000 contiguous positions in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of less than about 20%, 15%, 10% or 5% as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment [i.e. , resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res.25: 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15.

[0117] "Tissue-specific promoter", as used herein, refers to regulated promoters that are not expressed in all cells but only in one or more cell types in specific organs (such as leaves or seeds in plants, or heart, muscle or bone in animals), specific tissues (such as embryo or cotyledon in plants, such as epithelium, connective tissue or vascular tissue) in animals, or specific cell types (such as leaf parenchyma or seed storage cells in plants, or keratinocytes, lymphocytes, erythrocytes or neuronal cells in animals). These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence.

[0118] The terms "trans-acting sequence" and "trans-acting element" refer to DNA or

RNA sequences, whose function does not require them to be on the same molecule. A non-limiting example of a trans-acting sequence is a rep gene (AC1 or AL1 in ACMV or TGMV Geminiviruses, respectively), which can function in replication without being on the replicon.

[0119] "Transactivating gene" refers to a gene encoding a transactivating protein. It can encode a replication protein(s) (Rep), which is suitably a viral replication protein, or a site- specific replicase. It can be a natural gene, for example, a viral replication gene, or a chimeric gene, for example, when plant regulatory sequences are operably-linked to the open reading frame of a site-specific recombinase or a viral replication protein. "Transactivating genes" may be chromosomally integrated or transiently expressed.

[0120] As used herein, the term "trans-activation" refers to switching on of gene expression or replicon replication by the expression of another (regulatory) gene in trans. [0121] The term "transformation" means alteration of the genotype of a host by the introduction of a heterologous nucleic acid, such as the first and/or second constructs of the invention.

[0122] As used herein, the terms "transformed" and "transgenic" refer to any organism including an animal, animal part, plant, plant cell, callus, plant tissue, or plant part that contains all or part of at least one construct of the invention. In some embodiments, all or part of at least one construct of the invention is stably introduced into a chromosome or stable extra-chromosomal element, so that it is passed on to successive generations.

[0123] The term "transgene" as used herein, refers to any nucleotide sequence used in the transformation of a plant, animal, or other organism. Thus, a transgene can be a coding sequence, a non-coding sequence, a cDNA, a gene or fragment or portion thereof, a genomic sequence, a regulatory element and the like. A "transgenic" plant is a plant into which a transgene has been delivered or introduced and the transgene can be expressed in the transgenic plant to produce a product, the presence of which can impart an effect and/or a phenotype in the plant.

[0124] As used herein, the term "transient expression" refers to expression in cells in which a transgene is introduced into a plant cell, but not selected for its stable maintenance. Non- limiting methods of introducing the transgene include viral infection, agrobacterium-mediated transformation, electroporation, and biolistic bombardment

[0125] As used herein, the term "5' untranslated region" or "5' UTR" refers to a sequence located upstream {i.e., 5') of a coding region. Typically, a 5' UTR is located downstream {i.e. , 3') to a promoter region and 5' of a coding region downstream of the promoter region. Thus, such a sequence, while transcribed, is upstream of the translation initiation codon and therefore is generally not translated into a portion of the polypeptide product.

[0126] The term "3' untranslated region" or "3' UTR" refers to a nucleotide sequence downstream {i.e., 3') of a coding sequence. It generally extends from the first nucleotide after the stop codon of a coding sequence to just before the poly(A) tail of the corresponding transcribed mRNA. The 3' UTR may contain sequences that regulate translation efficiency, mRNA stability, mRNA targeting and/or polyadenylation.

[0127] By "vector" is meant a nucleic acid molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned. A vector typically contains one or more unique restriction sites and may be capable of autonomous replication in a defined plant cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined plant such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an

extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the plant cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the plant cell into which the vector is to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are well known to those of skill in the art.

[0128] The terms "wild-type," "natural," "native" and the like with respect to an organism, polypeptide, or nucleic acid sequence, that the organism polypeptide, or nucleic acid sequence is naturally occurring or available in at least one naturally occurring organism which is not changed, mutated, or otherwise manipulated by man.

[0129] As used herein, underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated in the absence of any underscoring or italicizing. For example, "rep" shall mean the rep gene, whereas "Rep" shall indicate the protein product of the "rep" gene.

[0130] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

2. Abbreviations

[0131] The following abbreviations are used throughout the application:

nt =nucleotide

nts = nucleotides

aa =amino acid(s)

kb = kilobase(s) or kilobase pair(s)

kDa =kilodalton(s)

d =day

h =hour

s =seconds

3. Expression systems for inhibiting viability of a plant cell

[0132] The present invention is directed to expression systems for expressing a toxicant nucleic acid sequence in a plant cell in combination with silencing virus genes that are associated with virus spread and/or replication. The expression systems generally comprise at least two expression system components, in which a first expression system component expresses the toxicant nucleic acid sequence and a second expression system component expresses a VVI nucleic acid sequence. Suitably, the VVI nucleic acid sequence encodes a double stranded RNA molecule. Without wishing to be bound by any one theory or mode of operation, it is proposed that by killing any cell that is infected with a virus, together with silencing a gene of the virus that is important for virus movement between cells, the viability of a plant cell expressing the expression system will be inhibited upon infection with the virus. Accordingly, plants that comprise cells comprising the expression system described above and elsewhere herein will exhibit resistance to the relevant virus strains. Accordingly, the expression systems of the present invention provide a means of conferring virus resistance to a plant {i.e., a transgenic plant). 3.1 Toxicant nucleic acids and toxicity proteins

[0133] Any nucleic acid that encodes a protein or nucleic acid molecule that can induce injury, impairment or death of the plant cells, either directly or indirectly, can be included as the toxicant nucleic acid in the first component in the methods, transgenic plants, and plant cells described above and elsewhere herein. In its broadest form, the toxicity protein can be any protein with the ability to render a plant cell that is exposed to the toxicity protein as being incapable of replicating a heterologous nucleic acid sequence (e.g., a viral genome). Those skilled in the art will understand that while the expression of some "toxicity genes" will results in injury and/or death of all types of plant cell, other toxicant nucleic acids have a more restricted use and the expressed proteins or nucleic acids will be toxic in only a selection of plant cells. It is well within the skill of a skilled person to select a suitable toxicant nucleic acid for use in the methods and transgenic plants of the present invention.

[0134] In some embodiments, the toxicant nucleic acid encodes a toxic polypeptide. For example, the toxicant nucleic acid can encode a protease, such as typsin, chymotrypsin or elastase, a ribosome inhibiting protein, such as dianthin, pokeweed antiviral protein (PAP), ricin A, or a ribonuclease such as barnase or RNAse Tl. In one embodiment, the toxicant nucleic acid is a barnase gene that encodes a highly active RNase from Bacillus amyloliquefaciens or a functional fragment or variant thereof that retains RNase activity.

[0135] In some specific embodiments, the toxicant nucleic acid is a ribonuclease, for example, a barnase. The nucleic acid sequence of an exemplary barnase gene is set forth in GenBank accession no. X12871.1, and has the following sequence:

ATGGCCCAGGTCATCAACACCTTCGACGGCGTCGCCGACTACCTGCAGACCTACCACAAGCTGCCC GACAACTACATCACCAAGAGCGAGGCCCAGGCCTTGGGCTGGGTCGCCAGCAAGGGCAACCTGGC CGACGTCGCCCCCGGCAAGAGCATCGGCGGCGACATCTTCAGCAACAGGGAGGGCAAGCTGCCCG GCAAGAGCGGCCGCACCTGGCGCGAGGCCGACATCAACTACACCAGCGGCTTCCGCAACAGCGAC CGCATCCTGTACAGCAGCGACTGGCTGATCTACAAGACCACCGACCACTACCAGACCTTCACCAAG ATCCGCTGA [SEQ ID NO:l].

[0136] The nucleotide sequence set forth in SEQ ID NO:l encodes the polypeptide sequence as follows:

MAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVAPGKSIGGDIFSNREGKLPGKS GRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIR [SEQ ID NO:2].

[0137] In another illustrative embodiment the toxicant nucleic acid can encode a protease. Typically, proteases are selected from the PA clan (proteases of mixed nucleophile, superfamily A), and include serine proteases and cysteine proteases. Members of the PA clan are structurally homologous; they share a chymotrypsin-like fold and similar proteolysis mechanism, and are derived from plants, animals, fungi, eubacteria, archaea and viruses. In this regard, illustrative examples of suitable proteases from the PA clan, include typsin, chymotrypsin, elastase tobacco etch virus protease, nuclear-inclusion-a peptidase; rabbit hemorrhagic disease virus 3C- like peptidase, porcine transmissible gastroenteritis virus-type main peptidase, calicivirin (from Southampton virus), gill-associated virus 3C-like peptidase, iflavirus processing peptidase, chymotrypsin A, togavirin (from Sindbis virus), IgA specific serine endopeptidase, flavivirin (from yellow fever virus), hepacivirin (from hepatitis C virus), potyvirus PI peptidase (from plum pox virus), pestivirus NS2 polyprotein peptidase, sobemovirus peptidase, dipeptidyl-peptidase 7 (from Porphyromonas gingivalis), SpolVB peptidase (from Bacillus subtilis) Ssy5 peptidase (from

Saccharomyces cerevisiae), pocornain-like cysteine peptidase (from Breda-1 torovirus), and white bream virus serine peptidase.

[0138] In some preferred embodiments, the protease is a serine protease. Examples of suitable human serine proteases include trypsin-1 (as identified by UniProt accession no. P07477), trypsin-2 (as identified by UniProt accession no. P07478), chymotrypsinogen B (as identified by UniProt accession no. P17538), chymotrypsinogen B2 (as identified by UniProt accession no.

Q6GPI1), alpha-l-antitrypsin (as identified by UniProt accession no. P01009), Chymotrypsin-like elastase family member 1 (as identified by UniProt accession no. Q9UNI1), chymotrypsin-C (as identified by UniProt accession no. Q99895), neutrophil elastase (as identified by UniProt accession no. P08246), amyloid beta A4 protein (as identified by UniProt accession no. P05067), mannan- binding lectin serine protease 1 (as identified by UniProt accession no. P48740), trypsin-3 (as identified by UniProt accession no. P350300), kallikrein-6 (as identified by UniProt accession no. Q92876), ka II ikrein-8 (as identified by UniProt accession no.060259), kallikrein-11 (as identified by UniProt accession no. Q9UBX7), hepsin (as identified by UniProt accession no. P05981), complement Cls subcomponent (as identified by UniProt accession no. P09871), and complement Clr subcomponent (as identified by UniProt accession no. P00736). Examples of suitable bovine serine proteases include cationic trypsin (as identified by UniProt accession no. P00760), prothrombin (as identified by UniProt accession no. P00766), chymotrypsin-like elastase family member 1 (as identified by UniProt accession no. Q28153), chymotrypsin-like elastase family member 2A (as identified by UniProt accession no. Q29461), plasma kallikrein (as identified by UnProt accession no. Q2KJ63), chymotrypsin-C (as identified by UniProt accession no. Q7M3E1), and plasminogen (as identified by UniProt accession no.P06868).

[0139] Additional examples of serine proteases that are suitable toxicity proteins include those from rat (e.g, anionic trypsin-1 as identified by UniProt accession no. P00762, anionic trypsin-2 as identified by UniProt accession no. P00763, and chymotrypsinogen B as identified by UniProt accession no. P07338), mouse (e.g., neutrophil elastase as identified by UniProt accession no. Q3UP87, and chymotrypsin-like elastase family member 1 as identified by UniProt accession no. Q91X79), pig (e.g., trypsin as identified by UniProt accession no. P00761, and chymotrypsin- like elastase family member 1 as identified by UniProt accession no. P00772), blunt-nosed viper (e.g., chymotrypsin-like protease VLCTLP as identified by UniProt accession no. E0Y421), and Aspergillus fumigatus (e.g., alkaline protease 1 as identified by UniProt accession no. P28296).

[0140] In some embodiments, toxicant nucleic acids have a nucleic acid sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% and 100% nucleic acid sequence similarity or identity to a corresponding wild-type toxicant nucleic acid sequence (e.g., a wild-type barnase gene sequence, such as that set forth in SEQ ID NO:i). In illustrative examples of this type, the toxicant nucleic acid sequence corresponds to a barnase nucleic acid sequence and has a nucleic acid sequence that has at least 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or 100% nucleic acid sequence similarity or identity to the barnase nucleic acid sequence set forth in SEQ ID NO:l.

In another illustrative example of this type, the toxicant nucleic acid sequence is a nucleic acid sequence that encodes a polypeptide that corresponds to the amino acid sequence set forth in SEQ

ID NO:2 {i.e., an amino acid sequence that displays at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or 100% sequence similarity or identity to all or a portion of the amino acid sequence set forth in SEQ ID NO:2). Alternatively a nucleic acid sequence encoding a biologically active fragment of any toxicity protein can suitably be used .

[0141] In other examples, the toxicant nucleic acid encodes a polypeptide that elicits a hypersensitive response {i.e., a hypersensitive response-elicitor polypeptide) such as the 50 kDa fragment of the TMV replicase RdRp (P50), MLO, HRP polypeptides such as HRPN, PopAl, ParAl and other hypersensitive response-elicitor polypeptides from Erwinia, Pseudomonas, Phytophthora, and Xanthamonas species. In a further example, the toxicant nucleic acid encodes a transcript

{e.g., a ribozyme or antisense nucleic acid, such as an antisense oligonucleotide, miRNA or siRNA), which is itself toxic to the host plant cell.

[0142] In particular examples, the toxicant nucleic acid encodes an avirulence polypeptide that is recognized by a polypeptide encoded by a resistance gene expressed in the plant into which the first and second components of the expression system are introduced.

Recognition of the avirulence polypeptide by the resistance polypeptide triggers the induction of a host defense response, which results in toxicity. The resistance gene can be endogenous to the plant and thus endogenously expressed in the plant, or can be a heterologous resistance gene that is introduced into the plant cell and thus expressed heterologously in the plant. For example, the resistance gene can be contained in a third component as described above and introduced into a plant cell. Such third components can be introduced into any plant cell so as to facilitate the establishment of virus resistance when the first and second constructs are also introduced into the plant cell. The resistance gene on the third construct can be constitutively expressed or can be expressed only upon infection of the plant cell with a virus.

[0143] In some examples, the avirulence polypeptide is a hypersensitive response- elicitor polypeptide, and interaction of this polypeptide with a resistance polypeptide in the plant cell elicits a hypersensitive response. Non-limiting examples of toxicant nucleic acids that encode avirulence polypeptides and exemplary corresponding resistance genes include, but are not limited to, the TMV replicase (or P50) gene (as set forth, for example, in SEQ ID NO:3 or SEQ ID NO:7) and the N gene; the potato virus X (PVX) coat protein gene (as identified, for example, by

GenBank accession no. X88788, encoding the PVX coat protein polypeptide sequence identified by UniProt accession no. P07699) and the Rx locus of potato (Bendahmane eta/, 1999, Plant Cell. 11(5): 781-792); the TMV replicase (or P50) gene (as set forth, for example, in SEQ ID NO:3 or SEQ ID NO:7) and the tomato Tm-1, Trm-2 and Tm-2a loci; and the tospoviruses movement protein gene (as identified, for example, by GenBank accession no. AF213674) and the tomato Sw- 5 gene {e.g., as identified by GenBank accession no. F.1686044.1) (Brommonschenkel etal., 2000, Mol. Plant. Microbe. Interact. 13(10): 1130-8).

[0144] In some embodiments, toxicant nucleic acids have a nucleic acid sequence that has at least 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or 100% nucleic sequence similarity or identity to a corresponding wild-type toxicant nucleic acid sequence {e.g., a nucleic acid sequence encoding an avirulence polypeptide, for example, a nucleic acid sequence identified by GenBank accession no. X88788). In illustrative examples of this type, the toxicant nucleic acid sequence is a avirulence polypeptide-encoding nucleic acid seq uence and has a nucleic acid seq uence that has at least 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or 100% nucleic acid sequence si mi la rity or identity to the corresponding wi ld-type avi rulence polypeptide-encodi ng nucleic acid seq uence as set forth in GenBank accession no. X88788. Alternatively a nucleic acid sequence encoding a biologically active fragment of any avirulence polypeptide can suitably be used .

[0145] In a n exemplary embodiment, the toxicant nucleic acid encodes a Tobacco mosaic virus (TMV) repl icase or the 50 kDa fragment thereof, P50, which interacts with the N protein encoded by the N gene in certai n tobacco cultivars. This interaction elicits a hypersensitive response and progra mmed cel l death, resulting i n loca lised necrotic lesions in the pla nt (Erickson ef a/., 1999, Pla nt J, 18 : 67-75). According ly, in some embodiments, the second component contains a TMV replicase gene or a polynucleotide encodi ng P50. The expression system ca n be introduced i nto a tobacco cultivar that is known to conta ins and express the N gene, thereby esta blishi ng resistance to the one or more viruses whose infection of the plant cell induces expression of Rep from the fi rst component. In other exa mples, the N gene is heterologous to the plant cell, a nd is i ntrod uced into the sa me plant cell as the expression system. For exa mple, the N gene can be i ntrod uced into a tomato pla nt cell (see, e.g., Whitham et al, 1996, PNAS, 93: 8876-8781), to esta blish resistance to the one or more viruses whose infection of the plant cel l induces expression of Rep from the fi rst component. The nucleic acid sequence encod ing an exempla ry 50 kDa frag ment of TMV replicase is set forth in SEQ ID NO : 7. . The polynucleotide sequences set forth in SEQ ID NO 3 a nd 7 respectively are as follows :

TMV replicase (SEQ ID NO: 3)

ATGGCATACACACAGACAGCTACCACATCAGCTTTGCTGGACACTGTCCGAGGAAACAACTCCTTGGTCAAT GATCTAGCAAAGCGTCGTCTTTACGACACAGCGGTTGAAGAGTTTAACGCTCGTGACCGCAGGCCCAAGGT GAACTTTTCAAAAGTAATAAGCGAGGAGCAGACGCTTATTGCTACCCGGGCGTATCCAGAATTCCAAATTACA TTTTATAACACGCAAAATGCCGTGCATTCGCTTGCAGGTGGATTGCGATCTTTAGAACTGGAATATCTGATGA TGCAAATTCCCTACGGATCATTGACTTATGACATAGGCGGGAATTTTGCATCGCATCTGTTCAAGGGACGAG CATATGTACACTGCTGCATGCCCAACCTGGACGTTCGAGACATCATGCGGCACGAAGGCCAGAAAGACAGTA TTGAACTATACCTTTCTAGGCTAGAGAGAGGGGGGAAAACAGTCCCCAACTTCCAAAAGGAAGCATTTGACA GATACGCAGAAATTCCTGAAGACGCTGTCTGTCACAATACTTTCCAGACAATGCGACATCAGCCGATGCAGC AATCAGGCAGAGTGTATGCCATTGCGCTACACAGCATATATGACATACCAGCCGATGAGTTCGGGGCGGCAC TCTTGAGGAAAAATGTCCATACGTGCTATGCCGCTTTCCACTTCTCTGAGAACCTGCTTCTTGAAGATTCATA CGTCAATTTGGACGAAATCAACGCGTGTTTTTCGCGCGATGGAGACAAGTTGACCTTTTCTTTTGCATCAGAG AGTACTCTTAATTATTGTCATAGTTATTCTAATATTCTTAAGTATGTGTGCAAAACTTACTTCCCGGCCTCTAAT AGAGAGGTTTACATGAAGGAGTTTTTAGTCACCAGAGTTAATACCTGGTTTTGTAAGTTTTCTAGAATAGATA CTTTTCTTTTGTACAAAGGTGTGGCCCATAAAAGTGTAGATAGTGAGCAGTTTTATACTGCAATGGAAGACGC ATGGCATTACAAAAAGACTCTTGCAATGTGCAACAGCGAGAGAATCCTCCTTGAGGATTCATCATCAGTCAAT TACTGGTTTCCCAAAATGAGGGATATGGTCATCGTACCATTATTCGACATTTCTTTGGAGACTAGTAAGAGGA CGCGCAAGGAAGTCTTAGTGTCCAAGGATTTCGTGTTTACAGTGCTTAACCACATTCGAACATACCAGGCGA AAGCTCTTACATACGCAAATGTTTTGTCCTTTGTCGAATCGATTCGATCGAGGGTAATCATTAACGGTGTGAC AGCGAGGTCCGAATGGGATGTGGACAAATCTTTGTTACAATCCTTGTCCATGACGTTTTACCTGCATACTAAG CTTGCCGTTCTAAAGGATGACTTACTGATTAGCAAGTTTAGTCTCGGTTCGAAAACGGTGTGCCAGCATGTGT GGGATGAGATTTCGCTGGCGTTTGGGAACGCATTTCCCTCCGTGAAAGAGAGGCTCTTGAACAGGAAACTTA TCAGAGTGGCAGGCGACGCATTAGAGATCAGGGTGCCTGATCTATATGTGACCTTCCACGACAGATTAGTGA CTGAGTACAAGGCCTCTGTGGACATGCCTGCGCTTGACATTAGGAAGAAGATGGAAGAAACGGAAGTGATG TACAATGCACTTTCAGAGTTATCGGTGTTAAGGGAGTCTGACAAATTCGATGTTGATG I I I I I I CCCAGATGT GCCAATCTTTGGAAGTTGACCCAATGACGGCAGCGAAGGTTATAGTCGCGGTCATGAGCAATGAGAGCGGT CTGACTCTCACATTTGAACGACCTACTGAGGCGAATGTTGCGCTAGCTTTACAGGATCAAGAGAAGGCTTCA GAAGGTGCTTTGGTAGTTACCTCAAGAGAAGTTGAAGAACCGTCCATGAAGGGTTCGATGGCCAGAGGAGA GTTACAATTAGCTGGTCTTGCTGGAGATCATCCGGAGTCGTCCTATTCTAAGAACGAGGAGATAGAGTCTTTA GAGCAGTTTCATATGGCAACGGCAGATTCGTTAATTCGTAAGCAGATGAGCTCGATTGTGTACACGGGTCCG ATTAAAGTTCAGCAAATGAAAAACTTTATCGATAGCCTGGTAGCATCACTATCTGCTGCGGTGTCGAATCTCG TCAAGATCCTCAAAGATACAGCTGCTATTGACCTTGAAACCCGTCAAAAGTTTGGAGTCTTGGATGTTGCATC TAGGAAGTGGTTAATCAAACCAACGGCCAAGAGTCATGCATGGGGTGTTGTTGAAACCCACGCGAGGAAGT ATCATGTGGCGCTTTTGGAATATGATGAGCAGGGTGTGGTGACATGCGATGATTGGAGAAGAGTAGCTGTCA GCTCTGAGTCTGTTGTTTATTCCGACATGGCGAAACTCAGAACTCTGCGCAGACTGCTTCGAAACGGAGAAC CGCATGTCAGTAGCGCAAAGGTTGTTCTTGTGGACGGAGTTCCGGGCTGTGGGAAAACCAAAGAAATTCTTT CCAGGGTTAATTTTGATGAAGATCTAATTTTAGTACCTGGGAAGCAAGCCGCGGAAATGATCAGAAGACGTG CGAATTCCTCAGGGATTATTGTGGCCACGAAGGACAACGTTAAAACCGTTGATTCTTTCATGATGAATTTTGG GAAAAGCACACGCTGTCAGTTCAAGAGGTTATTCATTGATGAAGGGTTGATGTTGCATACTGGTTGTGTTAAT TTTCTTGTGGCGATGTCATTGTGCGAAATTGCATATGTTTACGGAGACACACAGCAGATTCCATACATCAATA GAGTTTCAGGATTCCCGTACCCCGCCCATTTTGCCAAATTGGAAGTTGACGAGGTGGAGACACGCAGAACTA CTCTCCGTTGTCCAGCCGATGTCACACATTATCTGAACAGGAGATATGAGGGCTTTGTCATGAGCACTTCTTC GGTTAAAAAGTCTGTTTCGCAGGAGATGGTCGGCGGAGCCGCCGTGATCAATCCGATCTCAAAACCCTTGCA TGGCAAGATCCTGACTTTTACCCAATCGGATAAAGAAGCTCTGCTTTCAAGAGGGTATTCAGATGTTCACACT GTGCATGAAGTGCAAGGCGAGACATACTCTGATGTTTCACTAGTTAGGTTAACCCCTACACCAGTCTCCATCA TTGCAGGAGACAGCCCACATGTTTTGGTCGCATTGTCAAGGCACACCTGTTCGCTCAAGTACTACACTGTTGT TATGGATCCTTTAGTTAGTATCATTAGAGATCTAGAGAAACTTAGCTCGTACTTGTTAGATATGTATAAGGTCG ATGCAGGAACACAATAGCAATTACAGATTGACTCGGTGTTCAAAGGTTCCAATCTTTTTGTTGCAGCGCCAAA GACTGGTGATATTTCTGATATGCAGTTTTACTATGATAAGTGTCTCCCAGGCAACAGCACCATGATGAATAAT TTTGATGCTGTTACCATGAGGTTGACTGACATTTCATTGAATGTCAAAGATTGCATATTGGATATGTCTAAGTC TGTTGCTGCGCCTAAGGATCAAATCAAACCACTAATACCTATGGTACGAACGGCGGCAGAAATGCCACGCCA GACTGGACTATTGGAAAATTTAGTGGCGATGATTAAAAGGAACTTTAACGCACCCGAGTTGTCTGGCATCATT GATATTGAAAATACTGCATCTTTAGTTGTAGATAAG I I I I I I GATAGTTATTTGCTTAAAGAAAAAAGAAAACC AAATAAAAATGTTTCTTTGTTCAGTAGAGAGTCTCTCAATAGATGGTTAGAAAAGCAGGAACAGGTAACAATA GGCCAGCTCGCAGATTTTGATTTTGTAGATTTGCCAGCAGTTGATCAGTACAGACACATGATTAAAGCACAAC CCAAGCAAAAATTGGACACTTCAATCCAAACGGAGTACCCGGCTTTGCAGACGATTGTGTACCATTCAAAAAA GATCAATGCAATATTTGGCCCGTTGTTTAGTGAGCTTACTAGGCAATTACTGGACAGTGTTGATTCGAGCAGA I I I I I GTTTTTCACAAGAAAGACACCAGCGCAGATTGAGGATTTCTTCGGAGATCTCGACAGTCATGTGCCGA TGGATGTCTTGGAGCTGGATATATCAAAATACGACAAATCTCAGAATGAATTCCACTGTGCAGTAGAATACGA GATCTGGCGAAGATTGGGTTTTGAAGACTTCTTGGGAGAAGTTTGGAAACAAGGGCATAGAAAGACCACCCT CAAGGATTATACCGCAGGTATAAAAACTTGCATCTGGTATCAAAGAAAGAGCGGGGACGTCACGACGTTCAT TGGAAACACTGTGATCATTGCTGCATGTTTGGCCTCGATGCTTCCGATGGAGAAAATAATCAAAGGAGCCTTT TGCGGTGACGATAGTCTGCTGTACTTTCCAAAGGGTTGTGAGTTTCCGGATGTGCAACACTCCGCGAATCTT

ATGTGGAATTTTGAAGCAAAACTGTTTAAAAAACAGTATGGATACTTTTGCGGAAGATATGTAATACATCACG

ACAGAGGATGCATTGTGTATTACGATCCCCTAAAGTTGATCTCGAAACTTGGTGCTAAACACATCAAGGATTG

GGAACACTTGGAGGAGTTCAGAAGGTCTCTTTGTGATGTTGCTGTTTCGTTGAACAATTGTGCGTATTACACA

CAGTTGGACGACGCTGTATGGGAGGTTCATAAGACCGCCCCTCCAGGTTCGTTTGTTTATAAAAGTCTGGTG AAGTATTTGTCTGATAAAGTTCTTTTTAGAAGTTTGTTTATAGATGGCTCTAGTTGTTAA TMV replicase p50 (SEQ ID NO: 7)

GAGATAGAGTCTTTAGAGCAGTTTCATATGGCAACGGCAGATTCGTTAATTCGTAAGCAGATGAGCTCGATT GTGTACACGGGTCCGATTAAAGTTCAGCAAATGAAAAACTTTATCGATAGCCTGGTAGCATCACTATCTGCTG CGGTGTCGAATCTCGTCAAGATCCTCAAAGATACAGCTGCTATTGACCTTGAAACCCGTCAAAAGTTTGGAGT CTTGGATGTTGCATCTAGGAAGTGGTTAATCAAACCAACGGCCAAGAGTCATGCATGGGGTGTTGTTGAAAC CCACGCGAGGAAGTATCATGTGGCGCTTTTGGAATATGATGAGCAGGGTGTGGTGACATGCGATGATTGGA GAAGAGTAGCTGTCAGCTCTGAGTCTGTTGTTTATTCCGACATGGCGAAACTCAGAACTCTGCGCAGACTGC TTCGAAACGGAGAACCGCATGTCAGTAGCGCAAAGGTTGTTCTTGTGGACGGAGTTCCGGGCTGTGGGAAA ACCAAAGAAATTCTTTCCAGGGTTAATTTTGATGAAGATCTAATTTTAGTACCTGGGAAGCAAGCCGCGGAAA TGATCAGAAGACGTGCGAATTCCTCAGGGATTATTGTGGCCACGAAGGACAACGTTAAAACCGTTGATTCTT TCATGATGAATTTTGGGAAAAGCACACGCTGTCAGTTCAAGAGGTTATTCATTGATGAAGGGTTGATGTTGCA TACTGGTTGTGTTAATTTTCTTGTGGCGATGTCATTGTGCGAAATTGCATATGTTTACGGAGACACACAGCAG ATTCCATACATCAATAGAGTTTCAGGATTCCCGTACCCCGCCCATTTTGCCAAATTGGAAGTTGACGAGGTGG AGACACGCAGAACTACTCTCCGTTGTCCAGCCGATGTCACACATTATCTGAACAGGAGATATGAGGGCTTTG TCATGAGCACTTCTTCGGTTAAAAAGTCTGTTTCGCAGGAGATGGTCGGCGGAGCCGCCGTGATCAATCCGA TCTCAAAACCCTTGCATGGCAAGATCCTGACTTTTACCCAATCGGATAAAGAAGCTCTGCTTTCAAGAGGGTA TTCAGATGTTCACACTGTGCATGAAGTGCAAGGCGAGACATACTCTGATGTTTCACTAGTTAGGTTAACCCCT ACACCAGTCTCCATCATTGCAGGAGACAGCCCACATGTTTTGGTCGCATTGTCAAGGCACACCTGTTCGCTC AAGTACTACACTGTTGTTATGGATCCTTTAGTTAGTATCATTAGAGATCTAGAGAAACTTAGCTCGTACTTGTT AGATATGTATAAGGTCGATGCAGGAACACAATAG.

[0146] In a further exemplary embodi ment, the toxicant nucleic acid in the fi rst component encodes the PVX coat protein gene, a nd the expression system of the invention is introd uced into a potato plant that has the Rx locus. Alternatively, a third component comprising the Rx locus from potato is introduced into the same plant cell as the expression system of the present invention. Thus, upon i nfection of the pla nt cel l with a virus, the rep gene i n the first component is expressed, which induces expression of the PVX coat protein gene, and the PVX coat protein interacts with the expression product of the Rx locus to el icit cel l toxicity.

3.2 VVI nucleic acids

[0147] In accordance with the present invention, the VVI nucleic acids may be in the form of a double stranded RNA molecule that is capable silencing the expression of a vira l gene that encodes polypeptide that is essential for virus viability and/or virus spread . The double stra nded RNA, which is also referred to herein as "dsRNA", may be a sing le nucleotide polymer with one or more region(s) of self-complementarity such that nucleotides i n one segment of the polymer base pair with nucleotides in another segment of the polymer. Alternatively, the dsRNA may include two nucleotide polymers that have one or more reg ion(s) of complementarity to each other.

[0148] VVI nucleic acids function to prevent, red uce or i mpair the spread of a vi rus from the point of initial i nfection in a plant host. Importantly, any vi ral gene that is essential for the spread of the vi rus in the host can be targeted.

[0149] Pla nt vi ruses have evolved severa l mechanisms by which to spread throughout the plant host. Accordi ngly, in some embodiments the VVI nucleic acids si lence a virus gene selected from the group comprising or consisti ng of, a gene associated with movement of the vi rus (e.g. , a virus movement protein gene), a si lencing suppressor gene, a coat protein gene, a nuclea r shuttle protein gene, a transactivator gene, and a gene associated with the cell cycle, and a gene associated with replication initiation.

[0150] The double stranded RNA will typically comprise a duplex region comprising two anti-parallel nucleic acid strands that are partially, substantially or fully complementary, as defined herein. In specific embodiments, these anti-parallel nucleic acid strands define inverted repeats of a nucleic acid sequence. Where the nucleic acid strands are part of a single nucleotide polymer, and therefore are connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of the respective other strand, the connecting RNA chain is referred to as a "hairpin loop", "loop", "unpaired region" or "unpaired loop region" and the two anti-parallel nucleic acid strands that define the duplex region are generally referred to as a "stem". Double stranded RNA molecules comprising a loop and stem are often referred to as "hairpin" or "panhandle structures". In illustrative examples of this type, the dsRNA comprises a duplex region formed by base pairing of complementary RNA sequences, and a single stranded region that forms a loop connecting the complementary RNA sequences.

[0151] The strands of a dsRNA may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the double stranded RNA minus any overhangs that are present in the duplex. In addition to the duplex region, a double stranded RNA may comprise one or more nucleotide overhangs. The double stranded RNA may be a conventional siRNA, shRNA or miRNA (including primary transcript or pri-miRNA, pre-miRNA, or functional miRNA) or a RNA that contains more than one hairpin or panhandle structure.

[0152] Desirably, the region of the dsRNA that is present in a double stranded conformation {i.e., duplex region) includes at least about 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000 or 3000 nucleotides participating in one strand of the double stranded or duplex region, or includes all of the nucleotides being represented in the double stranded RNA. In some embodiments, the double stranded RNA is fully complementary, and does not contain any single stranded regions, such as single stranded ends.

[0153] In other embodiments, such as miRNA-type dsRNA molecules, the double stranded regions may be interspersed with one or more single stranded nucleotides or areas. In some embodiments the double stranded RNA is a shRNA.

[0154] Suitably, the dsRNA molecule is selected from long dsRNA siRNA, shRNA and miRNA. In specific embodiments, the dsRNA molecule has a length of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000 or 3000 nucleotides.

[0155] The dsRNA molecules of the present invention are suitably sufficiently distinct in sequence from the RNA expression product of a toxicant nucleic acid sequence that is expressed or desired to be expressed upon viral infection, in a host cell. They will often also be sufficiently distinct in sequence from the RNA expression products of any host polynucleotide sequences for which function is intended to be undisturbed after any of the methods of this invention are performed. Computer algorithms may be used to define the essential lack of homology between the RNA molecule and the expression product of the toxicant nucleic acid polynucleotide sequence and/or the expression products of host, essential, normal sequences. However, in some embodiments, the dsRNA molecules of the present invention have a sequence corresponding to and specific for an endogenous nucleic acid sequence.

[0156] In some embodiments, the dsRNA molecule, particularly its antisense or guide strand, will have no more than 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% sequence identity, over a suitable comparison window, to any sequence of nucleotides located within a RNA expression product of the toxicant nucleic acid sequence. In certain embodiments, the dsRNA molecule, particularly its antisense or guide strand, will have no more than 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% sequence identity, over a suitable comparison window, to any sequence of nucleotides located within endogenous RNA expression products (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 95%, 99% of endogenous expression products) of the host cell. Suitably, the comparison window is at least about 17, 18, 19, 20, 21, 22, 23, 24, 25 contiguous nucleotides, or at least about 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000 or 3000 contiguous nucleotides.

[0157] Alternatively, or in addition, the dsRNA molecule, particularly its antisense or guide strand, is generally unable to hybridize {i.e., above background) under high stringency conditions, as defined for example herein, to any sequence of nucleotides located within a RNA expression product of the toxicant nucleic acid sequence and/or to any sequence of nucleotides located within endogenous RNA expression products (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 95%, 99% of endogenous expression products) of the host cell. Suitably, the dsRNA molecule, particularly its antisense or guide strand, is unable to hybridize under medium or low stringency conditions, as defined for example herein, to any sequence of nucleotides located within a RNA expression product of the toxicant nucleic acid sequence and/or to any sequence of nucleotides located within endogenous RNA expression products (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 95%, 99% of endogenous expression products) of the host cell.

[0158] In certain embodiments, the VVI nucleic acid sequence that silences the virus gene that is targeted for RNA silencing is a nucleic acid sequence that is heterologous to the plant cell. Preferably, this gene sequence is part of the virus genome to which resistance is to be conferred.

[0159] In such embodiments, the dsRNA molecule, particularly its antisense or guide strand, has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity, over a suitable comparison window (typically corresponding to a subsequence of the endogenous nucleic acid sequence), to the gene sequence of the virus to which resistance is to be conferred. Suitably, the comparison window is at least about 17, 18, 19, 20, 21, 22, 23, 24, 25 contiguous nucleotides, or at least about 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000 or 3000 contiguous nucleotides.

[0160] In some embodiments, the dsRNA molecule of the invention is a hairpin RNA (hpRNA) or short-hairpin RNA (shRNA). Illustrative examples of such RNA molecules include RNA molecules of no more than about 1000 nucleotides, no more that about 500 nucleotides, no more than about 400 nucleotides, no more than 300 nucleotides, nor more than 200 nucleotides, or no more than 100 nucleotides, in which at least one region of about 15 to about 100 nucleotides (e.g., about 17 to about 50 nucleotides, or about 19 to about 29 nucleotides, inclusive of integer nucleotide lengths in between) is base paired with a complementary sequence located on the same RNA molecule (single RNA strand) (thus defining "complementary sequences"), and wherein the complementary sequences are separated by an unpaired region of: at least about 4 to 7 nucleotides (e.g., about 9 to about 15 nucleotides; about 15 to about 100 nucleotides; about 100 to about 1000 nucleotides; inclusive of all integer nucleotide lengths in between) which forms a single-stranded loop adjacent to the stem structure created by the two complementary sequences. The shRNA molecules suitably comprise at least one stem-loop structure comprising a double stranded stem region of: about 17 to about 300 base pairs; about 17 to about 200 base pairs; about 17 to about 100 base pairs; about 17 to about 50 base pairs; about 18 to about 40 base pairs; or from about 19 to about 29 base pairs; inclusive of all integer base pair lengths in between, homologous and complementary to a target sequence to be inhibited ; and an unpaired loop region of: at least about 4 to 7 nucleotides (or about 9 to about 15 nucleotides, about 10 to about 50 nucleotides, about 15 to about 100 nucleotides, inclusive of all integer nucleotide lengths in between), which form a single-stranded loop adjacent to the stem structure created by the two complementary sequences. It will be recognized, however, that it is not strictly necessary to include a "loop region" or "loop sequence" because a RNA molecule comprising a sequence followed immediately by its reverse complement will tend to assume a stem-loop conformation even when not separated by an irrelevant "stuffer" sequence.

[0161] The VVI nucleic acid sequence that encodes the dsRNA is typically operably connected in an expression cassette to at least one regulatory element, including transcriptional regulatory elements such as promoters. The choice of promoter will vary depending on the temporal and spatial requirements for expression of the VVI nucleic acid sequence, and also depending on the plant cell in which this sequence is desired to be expressed. In some cases, expression in multiple tissues is desirable. While in others, tissue-specific expression is desirable. The promoter may be constitutive or inducible, as discussed for example below. Expression of the VVI nucleic acid sequence can also be controlled at the level of replication.

[0162] The VVI nucleic acid sequence may be in the form of a contiguous nucleic acid entity that encodes an intact or uninterrupted dsRNA molecule. Alternatively, the VVI nucleic acid sequence may be in the form of a non-contiguous nucleic acid entity or split gene which comprises a plurality of spaced nucleic acid subsequences, each encoding different portions of the dsRNA molecule, wherein the spaced nucleic acid subsequences are capable of rearranging (e.g., by replication or recombination) to form a contiguous nucleic acid entity that encodes an intact dsRNA molecule.

Movement proteins

[0163] Plant viruses migrate through their hosts using a number of pathways, the most common being movement cell to cell through plasmodesmata. Plasmodesmata are unique intercellular organelles specific to plants, which function as pores between cell walls that allow plant cells to communicate with one another. Usually, plasmodesmata only allow the passage of small diffusible molecules, such as various metabolites. Neither virus particles nor viral genomic nucleic acids can pass through plasmodesmata unaided. [0164] In some embodiments the viral viability impairment nucleic acids of the present invention silence a viral gene encoding a movement protein. Plant virus movement proteins are well established as a class of non-structural proteins encoded by plant viruses to enable their movement from one infected cell to the neighbouring cells. Some viruses encode more than one movement protein, with the movement protein of Tobacco mosaic virus (TMV) being the most extensively studied.

[0165] Movement proteins modify the plasmodesmata by one of two well-defined mechanisms. The movement proteins of many plant viruses form a transport tubule within the pore of the plasmodesmata that allow the transport of mature virus particles. Examples of viruses that use this mechanism are Cowpea mosaic virus (CPMV) and Tomato spotted wilt virus (TSWV). The second mechanism by which movement proteins often work is by associating with and coating the genome of the virus, causing the ribonucleoprotein complexes to be transported through plasmodesmata into neighbouring cells. The 30 kDa movement protein identified from TMV acts through this mechanism.

[0166] In some embodiments, the VVI nucleic acid silences a Geminivirus movement protein gene. For example, the movement protein-silencing ("MP-silencing") nucleic acid may suitably target a viral 1/2 gene. Suitable movement proteins that are encoded by Geminivirus 1/2 genes include those expressed by the bean yellow dwarf virus (BeYDV) (encoding the polypeptide identified by UniProt accession no. 039519) ; maize streak virus (MSV) (genotype A, encoding the polypeptides identified by UniProt accession no. P0C648, P0C649, P14992; genotype B, encoding the polypeptide identified by UniProt accession no. Q9IGY9; genotype C, 040984; genotype D, encoding the polypeptide identified by UniProt accession no. Q91MG0; and genotype E, encoding the polypeptide identified by UniProt accession no. Q91MG4) ; miscanthus streak virus (MiSV) (encoding the polypeptide identified by UniProt accession no. Q67593) ; Panicum streak virus (PanSV) (encoding the polypeptide identified by UniProt accession no. Q00336); sugarcane streak virus (SSV) (Sugarcane streak virus) (encoding the polypeptide identified by UniProt accession no. Q89448); tobacco yellow dwarf virus (strain Australia) (TYDV) (encoding the polypeptide identified by UniProt accession no. P31619) ; and wheat dwarf virus (isolate Sweden) (WDV) (encoding the polypeptide identified by UniProt accession no. P06849).

[0167] In some embodiments the virus impairment nucleic acid silences a viral VI gene, encoding for a movement protein. By way of an example, suitable movement proteins that are encoded by Geminivirus VI genes, include those expressed by the Chickpea chlorotic dwarf virus (encoding the polypeptides identified by UniProt accession no. B4ERB7, B4ER99, B4ERA3, B0BL30, I6NUT9, B0BL26, and I6NY38) ; cotton mastrevirus (encoding the polypeptide identified by UniProt accession no. W0U1B1); panicum streak virus - Karino (encoding the polypeptide identified by

UniProt accession no. Q84368); maize streak virus (MSV) (encoding the polypeptides identified by UniProt accession no. Q83464, Q77JB8, Q77JB7, Q77JC0, Q77JC1, Q910J2, Q91MI9, Q77JC4, Q9IGZ3, Q91MH4, Q9IGZ7, Q91MG8, and Q77JB6); sugar beet mastrevirus (encoding the polypeptide identified by UniProt accession no. B4XFH0); sugarcane streak Reunion virus (encoding the polypeptide identified by UniProt accession no. Q9QSV4); sugarcane streak Egypt virus (SSEV)

(encoding the polypeptides identified by UniProt accession no. 056310, Q9YXD8, Q9YXE2, Q9YXD5, and Q9IH25); sweet potato symptomless mastrevirus 1 (encoding the polypeptide identified by

UniProt accession no. C4NFN2); wheat dwarf virus (encoding the polypeptides identified by UniProt accession no. B0JDB4, A0A0F6N339, H7BTB7, H7BTH3, H7BT93, A0A0F6N4A9, Q89237, Q5W1D7, B0JDA5, B0JD97, Q5W1D3, B0JDA9, B0JDB1, B0JD89, Q8UYK4, B2FH31, B0JD95, B0JDA3, B0JD81, Q8UYK2, Q8UYJ8, B0JD83, Q5W1C9, Q8UYK0, B2FH29, Q18LC5, B0JD93, Q5W1D5, A0A0F6N358, B0JD99, B0JDB3, B0JD85, B0JD87, A0A0F6N492, Q5W1D2, Q5W1D9, B0JDA7, B0JD91, B0JDA1, A0A0F6N2P3, A0A0F6N3B0, H7BTK1, A0A0F6N3C2, D5H3S9, H6V579, H6V583, A0A0F6N2S1, H7BTP5, A0A0F6N2M8, H7BTG1, H7BTK9, H7BTA5, A7KQV7, A7K 37, Q8UYL2, A7KQY5, A7KQV3, Q0GFC3, A4F5E9, A7KR01, A7KQZ3, A7KR17, A7KR25, A7KR13, A7KR05, A7KQU1); and wheat dwarf India virus (encoding the polypeptide identified by UniProt accession no. I0E0B3).

[0168] In other embodiments, the MP-silencing nucleic acid targets a viral BC1 gene. By way of an example, suitable movement proteins that are encoded by Geminivirus BC1 genes, include those expressed by the Abutilon mosaic virus (AbMV) (Q6W5B2, P21946); Abutilon mosaic virus-HW (Q96615); African cassava mosaic virus (ACMV) (Q9WR14); Bean golden yellow mosaic virus (BGYMV) (Q9QGH1); Blechum interveinal chlorosis virus (K4NX34); Cabbage leaf curl virus (isolate Jamaica) (CaLCuV) (Q96707); Chino del tomate virus (Q9J046); Corchorus yellow vein virus - [Hoa Binh] (Q645G7); Cowpea golden mosaic virus (CPGMV) (Q9E0Z9); Cucurbit leaf crumple virus (CuLCrV) (Q9J0D9); East African cassava mosaic Cameroon virus (Q9WR06);

Mungbean yellow mosaic India virus (Mungbean: Q917N7, Cowpea: Q8JRQ0, Soybean, Q913F2); Pepper Geminivirus (Q67602); Sida golden mosaic virus (056374); Squash leaf curl China virus (A0PDU6); Squash leaf curl virus (SLCV) (P21936); Tomato dwarf leaf curl virus (012667); Tomato Geminivirus (Q67607); Tomato leaf curl New Delhi virus-Severe (Q809H0); Tomato mosaic

Barbados virus (Q9Q2W2); Tomato mottle virus (ToMoV) (041445, 041443, 041444); and Tomato mottle virus (isolate Florida) (ToMoV) (Q06660).

[0169] In some embodiments the MP-silencing nucleic acid encodes at least part of a MP gene that codes for a movement protein. By way of an example, suitable movement proteins that are encoded by Geminivirus MP genes, include those expressed by the Barley dwarf virus

(encoding the polypeptide identified by UniProt accession no. U4KTQ0); chickpea chlorotic dwarf virus (encoding the polypeptides identified by UniProt accession no. F8K7V3, F8K7V7); maize streak virus (encoding the polypeptides identified by UniProt accession no. A5JLB8, A5JLM4, A5JLH7, A5JLY3, A5JLW4, A5JLZ1, A5JLE2, and A5JLQ9); wheat dwarf virus (encoding the polypeptides identified by UniProt accession no. B2RER4, C3UK74, C3UKA2, C3UK98, C3UK60, and U4KTM3).

[0170] Other movement proteins that may be encoded by the MP-silencing nucleic acid of the present invention include those expressed by the axonopus compressus streak virus (encoding the polypeptide identified by UniProt accession no. X2EXT4); barley dwarf virus (encoding the polypeptide identified by UniProt accession no. B7FDB3); bromus catharticus striate mosaic virus (encoding the polypeptide identified by UniProt accession no. E7D4Y0); chickpea chlorosis Australia virus (encoding the polypeptides identified by UniProt accession no. H9BAA4, H9BAA0, and H9BAC0); chickpea chlorotic dwarf virus (encoding the polypeptides identified by UniProt accession no. W0U170, T1SJJ9, T1SHL0, T1SJF6, A0A0A1EPA7, A0A0A1ENJ8,

A0A0A0U9W3, I7B2V9, A0A0A0U9V3, T1SJI5, V5RCW8, T1SI12, A0A0F6QQIN7, T1SHIM3,

A0A0A0UCX6, A0A0A0U8P9, A0A0A1EPG8, A0A0A1ENQ1, A0A0A1EN75, T1SHV2, T1SHL4,

A0A0A1ER01, A0A0A1EQX1, A0A0A1ET70, T1SHM6, A0A0A1EPF0, T1SHW2, T1SHJ8, T1SHX2,

T1SI31, T1SHQ5, A0A0A1EN79, A0A0A1EQN2, A0A0A1EMZ3, A0A0A1EMH5, T1SI44, A0A0A1ES97,

A0A0A1ELY9, A0A0A1EMM5, A0A0A1EQL7, T1SHZ9, A0A0A1EMM2, A0A0A1ENL1, A0A0A1ET37, A0A0A1EME8, A0A0A1EMS3, A0A0A1EN88, and A0A0A1ET92); chickpea chlorotic dwarf virus D (encoding the polypeptide identified by UniProt accession no. U3MJS8); chickpea chlorosis virus (encoding the polypeptide identified by UniProt accession no. T1SJR7); chickpea chlorosis virus-A (encoding the polypeptides identified by UniProt accession no. H9BA80, H9BA88, E5DE80, T1SHR3, and T1SJL6); chickpea chlorosis virus-B (encoding the polypeptide identified by UniProt accession no. T1SJN5); chickpea chlorosis virus-C (encoding the polypeptides identified by UniProt accession no. H9BA92, and H9BA96); chickpea chlorosis virus-E (encoding the polypeptides identified by UniProt accession no. H9BAE0, H9BAE8, H9BAF2, T1SI50, and T1SJQ1); chickpea yellow dwarf virus (encoding the polypeptides identified by UniProt accession no. A0A0A0UCY7, and

A0A0A0U9W7); chickpea yellows virus (encoding the polypeptide identified by UniProt accession no. H9BAI4); chloris striate mosaic virus (CSMV) (encoding the polypeptides identified by UniProt accession no. J7FHM0, J7FGY5, J7FGH5, J7FG53, J7FGY1, J7FHM4, and J7FG48); digitaria ciliaris striate mosaic virus (encoding the polypeptides identified by UniProt accession no. J7FGV5, J7FHJ8, and J7FGE1); dragonfly-associated mastrevirus (encoding the polypeptide identified by UniProt accession no. K7S1S4); Eragrostis minor streak virus (encoding the polypeptide identified by UniProt accession no. F6M061); Eragrostis streak virus (encoding the polypeptide identified by UniProt accession no. B0Z3X6); maize streak Reunion virus (encoding the polypeptides identified by UniProt accession no. I1Z768, I1Z772, and X2EZS1); maize streak virus (encoding the polypeptides identified by UniProt accession no. B2BX37, B2BX48, D2E0Z5, B6CZA5, G0YI25, B6CZR4, D2E128, B6CZ99, D2E107, B6CZR1, D2E167, D2E0V9, B6CZE7, B6CZK1, B6CZM5, G0YI37, B6CZ84, G0YHK7, G0YHE1, X2EXU9, X2F2E6, X2EXV9, B6CZV0, B6CZV0, B6CZQ2, B6CZN7, B6CZT5, B6CZU1, A0A088D7Z4, A0A088D7U8, A0A088D7R6, B6CZV3, B6CZU4, B6CZU7, B6CZT8, B6CZP3, A0A088D8X1, B6CZT2, B6CZP6, A0A088D7A8, G0YI04, G0YI85, G0YHK1, G0YHJ5, G0YHX7, G0YH18, G0YH21, G0YH30, G0YHU1, B6CZQ5, B6CZL6, B6CZF9, B6CZA2, B6CZL9, D2E137, B6CZP0, A0A088D786, B6CZF3, B6CZH1, D2E0Z2, D2E164, B6CZA8, D2E0Z8, B6CZL3, B6CZC3, D2E188, B6CZ96, B6CZE4, B6CZB7, B6CZG2, B6CZV6, B6CZS0, B6CZE1, D2E155, B6CZB4, D2E185, D2E1A0, B6CZL0, B6CZC6, D2E1A6, D2E110, D2E122, D2E0X7, B6CZD2, B6CZ93, B2BX45, B7SB69, G0YHR1, G0YH54, G0YH75, G0YGS2, G0YH00, G0YGW7, G0YHC9, G0YHU4, and G0YGX0); Maize streak virus - A (encoding the polypeptide identified by UniProt accession no.036261); Panicum streak virus (encoding the polypeptides identified by

UniProt accession no. B0Z3V4, B0Z3W0, B0Z3W3, B0Z3W6, B0Z3V7, D2IW23, D2IW11, D2IW14, D2IW35, D2IW29, D2IW05, D2IW26, D2IW17, D2IW38, D2IW20, D2IW47, and D2IW41);

paspalum dilatatum striate mosaic virus (encoding the polypeptides identified by UniProt accession no. J7FHL4, J7FG31, J7FGG7, J7FHL8, and J7FHB0); paspalum striate mosaic virus (PSMV) (encoding the polypeptides identified by UniProt accession no. G1CSA4, J7FG19, J7FGW7, J7FHK2, J7FHK6, J7FFY8, J7FFZ8, J7FGW3, J7FGE5, J7FH95, J7FHA2, J7FGF2, and J7FHA6); saccharum streak virus (encoding the polypeptide identified by UniProt accession no. D0U2D6); sporobolus striate mosaic virus 1 (J7FHN0); sporobolus striate mosaic virus 2 (J7FGJ8); sugarcane streak Reunion virus (SSRV) (encoding the polypeptides identified by UniProt accession no. X2F2G8, B0Z3X9, and B0Z3X0); sugarcane streak virus (SSV) (encoding the polypeptides identified by

UniProt accession no. B0Z3X3); sugarcane white streak virus (encoding the polypeptides identified by UniProt accession no. X5CY96, A0A023SHB9, and A0A023SGG2); sweetpotato symptomless mastrevirus 1 (encoding the polypeptides identified by UniProt accession no. M1J871, and

V5UY96); switchgrass mosaic-associated virus 1 (encoding the polypeptides identified by UniProt accession no. A0A0A0QTH0, A0A0A0QTH2); tobacco yellow dwarf virus (TYDV) (encoding the polypeptides identified by UniProt accession no. Q6T335, Q6T331, Q6T332, Q6T330, and Q6T337); tobacco yellow dwarf virus-A (encoding the polypeptides identified by UniProt accession no.

H9BAJ6, H9BAK0, H9BAK8, and H9BAL2); urochloa streak virus (UroSV) (encoding the

polypeptides identified by UniProt accession no. B3FVZ6, and B3FVY4, B3FVZ9, B3FVY7, B3FVZ3, and X2EZS7); and wheat dwarf virus (WDV) (encoding the polypeptides identified by UniProt accession no. Q5K2S1, Q4LAS2, A6GVI0, A6GVH5, B2RG60, and B5U7V3).

[0171] In some embodiments, the MP-silencing nucleic acid silences a Nanovirus gene that encodes for a movement protein. By way of an example, suitable movement proteins that are encoded by Nanovirus movement protein are expressed by Banana bunchy top virus; Faba bean necrotic stunt virus (encoding the polypeptides identified by UniProt accession no. C7DLN5, D2N100, V9TNA1, V9TSP7, and V9TSQ4); Faba bean necrotic yellows virus (encoding the polypeptides identified by UniProt accession no. Q9WIJ7, 039830, D2WHU4, V9TQG6, Q9QIZ5, V9TRU0, and V9TRV8); Faba bean yellow leaf virus (encoding the polypeptides identified by UniProt accession no. K4Q5W3) Milk vetch dwarf virus (MDV) (encoding the polypeptides identified by UniProt accession no. Q9Z0C8); Pea necrotic yellow dwarf virus (I6QHN8); and Subterranean clover stunt virus (SCSV) (encoding the polypeptides identified by UniProt accession no. Q87008).

Nuclear shuttle proteins

[0172] Nuclear shuttle proteins ("NSP", and commonly referred to as BV1 and BR1) are essential for the transport of viral DNA across the nuclear envelope of plant cells. Nuclear shuttle proteins bind newly replicated viral ssDNA genomes and move these between the nucleus and the cytoplasm. These NSP-genome complexes are then directed to the cell periphery through interactions between NSP and movement proteins where, as the result of movement protein action, the complexes are moved to adjacent uninfected cells. Thus, in some embodiments, the VVI nucleic acids silence a nuclear shuttle gene.

[0173] As such, in some embodiments, the VVI nucleic acid silences to at least a portion of the nuclear shuttle protein genes included in Table 1:

Table 1. Exemplary nuclear shuttle protein genes

Virus GenBank Accession

No.

Tomato leaf curl New Delhi virus NP_803226

Cabbage leaf curl virus NP_624352

Sri Lankan cassava mosaic virus-[Colombo] NP_620869

Tomato chlorotic mottle virus NP_620014

Tomato rugose mosaic virus NP_066376

Blechum interveinal chlorosis virus YP_006902888

Tomato chlorotic leaf distortion virus- YP_004821540

[Venezuela:Zulia:2004]

Tomato dwarf leaf virus YPJD04958251

Abutilon Brazil virus YP_003622546 Tomato leaf curl Sinaloa virus YP_001294912

Pepper yellow leaf curl Indonesia virus YP_717095

Sida micrantha mosaic virus NP_957676

West African Asystasia virus 1 YPJD04958235

Rhynchosia yellow mosaic India virus YP_004123937

Tomato leaf curl Palampur virus YP_001960966

Melochia yellow mosaic virus YP_009175087

Melochia mosaic virus YP_009175080

Bean chlorotic mosaic virus YP_008400119

Bean chlorosis virus YP_007024778

Dalechampia chlorotic mosaic virus] YP_006860606

Datura leaf distortion virus YP_006860594

Soybean chlorotic blotch virus YPJD03622561

Solanum mosaic Bolivia virus YP_009042060

Abutilon mosaic Bolivia virus YP_004207831

Sida mosaic Bolivia virus 2 YPJD04207829

Sida mosaic Bolivia virus 1 YP_004207817

Asystasia mosaic Madagascar virus YP_009121934

Coccinia mosaic Tamil Nadu virus YP_009056852

Bitter gourd yellow vein virus YP_009058926

Euphorbia mosaic virus YP_717931

Tomato yellow mottle virus YP_007250563

Tomato yellow leaf distortion virus YPJD06331058

Passionfruit severe leaf distortion virus YP_002941857

Rhynchosia golden mosaic Yucatan virus YP_002753152

Gossypium punctatum mild leaf curl virus YP_002608412

Okra mottle virus YP_002154621

Macroptilium golden mosaic virus YP_001974417

Wissadula golden mosaic virus YP_001974411

Rhynchosia golden mosaic virus YP_001655003

Tomato severe rugose virus YP_001294933

Tomato mild yellow leaf curl Aragua virus YP_001249282

Sida mosaic Sinaloa virus YP_619876

Squash leaf curl Philippines virus YP_006444 Sida mosaic Alagoas virus YPJD04958244

Abutilon mosaic Brazil virus YP_004958237

Cleome leaf crumple virus YPJD04958217

Cassava mosaic Madagascar virus YP_005352919

Cucurbit leaf crumple virus NP_148989

Tomato golden mottle virus YP_619878

Euphorbia yellow mosaic virus YP_002791015

Corchorus yellow vein virus - [Hoa Binh] YP_115513

Tomato mottle wrinkle virus YP_009091995

East African cassava mosaic Malawi virus YP_008719942

Tomato mottle virus NPJD47253

Sida golden mosaic virus PJD49350

East African cassava mosaic Zanzibar virus NP_808889

Cotton leaf crumple virus P_795341

Cowpea golden mosaic virus AF486834

Bean calico mosaic virus NP_612599

Sida golden mosaic Costa Rica virus NP_808892

South African cassava mosaic virus NP_620667

East African cassava mosaic virus NP_817128

Mungbean yellow mosaic India virus NP_803152

Sida yellow vein virus NP_808905

Sida golden mosaic Honduras virus NP_808898

Luffa yellow mosaic virus NP_852655

Tomato yellow leaf curl Thailand virus NP_049920

Chino del tomate virus NP_620750

Watermelon chlorotic stunt virus NP_620299

Tomato mosaic Havana virus NP_620894

East African cassava mosaic Cameroon NP_803873 virus

Macroptilium yellow mosaic Florida virus NP_671470

Macroptilium mosaic Puerto Rico virus NP_671463

Potato yellow mosaic Trinidad virus NP_808798

Potato yellow mosaic Panama virus NPJD49357

Peristrophe mosaic virus YP_006908978

Mungbean yellow mosaic virus NP_620377 Melon chlorotic mosaic virus YP_003828909

Dolichos yellow mosaic virus YP_009055065

African cassava mosaic virus NPJD77102

Squash yellow mild mottle virus NP_620861

Tomato golden mosaic virus NP_077114

Pepper golden mosaic virus NP_671456

Bean golden yellow mosaic virus NP_040768

Tomato mottle Taino virus NPJD47198

Dicliptera yellow mottle virus NP_620853

Squash mild leaf curl virus NP_808805

East African cassava mosaic Kenya virus YP_002317400

Potato yellow mosaic virus NP_047241

Pepper huasteco yellow vein virus NPJD40354

Tomato leaf curl Gujarat virus NP_783158

Indian cassava mosaic virus NP_047235.

Bean golden mosaic virus NP_660085

Bean dwarf mosaic virus NP_047221

Banana bunchy top virus NP 604479

Pea yellow stunt virus YP_008997808

Black medic leafroll virus YPJD08997799

Faba bean necrotic yellows virus NP_619573

Milk vetch dwarf virus NP_619764

Silencing suppressor genes

[0174] Plants have evolved to use RNA silencing as a fundamental process for cellular defense against viruses. Key players in the plant RNA silencing machinery include the ribonuclease Dicer, RNA-dependent RNA polymerase (RDR), and Argonaute.

[0175] However, as a means to evade the plant silencing defense system, viruses have evolved to comprise viral suppressors. Viruses from different families have acquired a variety of suppressors that affect different (and/or multiple) steps in the plant silencing pathway. Silencing suppressor genes allow efficient virus replication in a single cell of a plant, before the systemic spread of the infection. Accordingly, in some embodiments, the VVI nucleic acid silences a silencing suppressor gene.

[0176] Illustrative examples of silencing suppressor genes that would suitably impair the infecting virus, include the coat protein of the turnip crinkle virus, pl9 from the tombusviruses, movement protein P25 from potato virus X. For example, HC-Pro gene encoded by many potyviruses (for example, the HC-Pro gene sequence from the tobacco etch virus is set forth in GenBank accession no. DQ365889) is able to reverse established silencing in plants, and block local silencing in transient assays. HC-Pro is also known to partially inhibit dsRNA processing by Dicer, and interfere with the unwinding of duplex siRNA. In yet another example, the 2b protein expressed from the cucumber mosaic virus (gene sequence set forth in GenBank accession no. AY512659) is also known to suppress transgene silencing.

[0177] Another silencing suppressor gene that is suitably the target of the viral impairment nucleic acids is the AL2 gene (also commonly referred to as the AC2 gene , C2 gene or TrAP gene) is found in all begomoviruses. Specifically, the AL2 gene encodes a transcription factor that was initially found to be required for expression in late viral genes, and has been shown to capable of reversing established gene silencing. Illustrative examples of begomovirus AC2 genes include those from the African cassava mosaic virus (GenBank accession no. FN 43528 ), mung bean yellow mosaic virus, tomato yellow leaf curl virus (GenBank accession no. ,3X239482), tomato golden mottle virus (GenBank accession no. YP_619882.1), and beet curly top virus.

Coat protein genes

[0178] The coat protein (encoded by genes typically named VI, 1/2, or AVI (also known asARl) also has an important function in cell to cell movement in many plant viruses, including geminiviruses and nanoviruses. For monopartite viruses, the coat protein is essential for systemic spread through a plant host. In bipartite viruses, although the coat protein may not be absolutely essential for this task, it is found that silencing the coat protein even in bipartite viruses is sufficient to impair the virus and reduce or slow viral spread.

[0179] Accordingly, in some embodiments the viral impairment nucleic acid silences a coat protein (also referred to as capsid protein). Some non-limiting examples of such coat proteins include those present in tobacco yellow draft virus (as set forth in GenBank accession no.

NP_620725). Exemplary coat proteins from exemplary begomoviruses, mastreviruses and nanoviruses include, but are not limited to, those provided in Table 2:

Table 2. Exemplary Coat protein genes

Tomato yellow leaf curl Guangdong virus YP_764450

Tomato chlorotic mottle virus NP_620009

Tomato rugose mosaic virus NPJD66373

Tomato leaf curl Iran virus YP_006423

Melon chlorotic leaf curl virus-[Guatemala] NP_835272

Ageratum yellow vein Hualian virus YP_001950234

Tomato leaf curl Hsinchu virus YP_956838

Sida mosaic Sinaloa virus YP_619885

Tomato golden mottle virus YP_619880

Squash leaf curl virus NP_047244

Mastrevirus Accession No.

Maize streak virus YP_009154762

Chickpea chlorosis virus YP_004046666

Chickpea redleaf virus YP_004046662

Tobacco yellow dwarf virus NP_620725

Nanovirus Accession No.

Banana bunchy top virus NP 604477

Abaca bunch top virus YP_001661657

Faba bean necrotic yellows virus NP_619570

Milk vetch dwarf virus NP_619767

Faba bean necrotic stunt virus NC_013095

Pea necrotic yellow dwarf virus YP_008992019

Pea yellow stunt virus YP_008997806

Black medic leafroll virus YP_008997802

Transactivator genes

[0180] A trasactivation signal is essential for the expression of the coat protein gene in many geminiviruses and nanoviruses. Thus, efficient expression of the virus coat protein is dependent upon the presence of a functional AL2 gene (or the encoded AL2 expression product). Accordingly, in some embodiments the VVI nucleic acid silences a virus transactivator gene (for example, wheat dwarf virus AL2 gene or the tomato golden mosaic virus AL2 gene).

Cell cycle control proteins (retinoblastoma-like binding protein)

[0181] Single stranded DNA viruses typically encode a protein with cell cycle control activity. For example, the wheat dwarf virus (a geminivirus) encodes two cell cycle control proteins, CI and C2. In mastrevi ruses, the cell cycle control proteins generally contain the consensus retinoblastoma (Rb)-binding motif LeuXCysXGiu. Rb is part of a conserved pathway that controls the activation of cel l division, by mani pulating the host cell cycle into S phase (which is favourable for virus repl ication) . It is wel l esta blished in the art that the cell cycle control proteins may also play other roles, and even belong to other classes or proteins. For example, CI and C2 cell cycle control proteins correspond to RepA and Rep, respectively.

[0182] Other genera of geminiviruses (e.g. , begomoviruses) do not contain a

LeuXCysXGIu motif, but are known to mani pulate the cell cycle usi ng a similar strategy.

Na noviruses (e.g., banana bunchy top virus (BBTV)) have multiple ssDNA components, each encod ing a different gene. In the case of BBTV it is the DNA-C component that encodes a protein that comprises a LeuXCysXGIu motif, a nd which is therefore considered to be a cell cycle control protein. Accordingly, in some embodiments the virus impairment gene encodes a cell cycle control protein. An exa mple of a suitable cel l cycle control protein is that encoded by BBTV DNA-C, as set forth below:

M EFWESSAM PDDVKREIKEIYWEDRKKLLFCQKLKSYVRRILVYGDQEDALAGVKDMKTSIIRYSEYLK KPCVVICCVSN KSIVYRLN SMVFFYHEYLEELGGDYSVYODLYCDEVLSSSSTEEEDVGVIYRNVIMAST QEKFSWSDCQQIVISDYDVTLL

3.1 Site-specific replicase activation

[0183] In specific embodiments, expression of the toxicant nucleic acid sequence of the invention is reg ulated usi ng replicase-mediated gene activation. In these embodi ments, the toxica nt nucleic acid sequence, which may be in the form of a contig uous nucleic acid entity or a non-contiguous nucleic acid entity, is expressed using a binary expression system that comprises a proreplicon and a regulated transactivating replication gene (rep) . The proreplicon generally comprises c/s-acting sequences (e.g. , vira l sequences) fla nking the toxica nt nucleic acid sequence, which are required for replication, but is incapable of episomal replication in cells (e.g. , pla nt cells) because it lacks a functional rep gene(s) essential for repl ication. U nder a ppropriate stimulus, the transactivati ng rep gene expresses the replication protein (Rep) (e.g. , vira l Rep) missing in the proreplicon and al lows the release of a replicon from the proreplicon and its episomal repl ication in a cell a utonomous manner. Typical ly the repl ication elements a re derived from viruses, as described for example below. Non-l iming examples of such binary expression systems are described by Dale et al. (U. S. Pat. No. 7,863,430), Dugda le et al. (2013), Yadav (U .S. Pat. No. 6,077,992) and Yadav et al. (U.S. Pat. No. 6,632980 and U.S. Pat. Appl. Pub. No. 2004/0092017), each of which is incorporated by reference herei n i n their entirety.

[0184] In some embod iments, expression of both the toxicant nucleic acid a nd the viral impa irment nucleic acid is by way of a binary expression system as described above and elsewhere herei n.

[0185] Thus, replicon replication can be ta rgeted to specific cells by controlli ng the expression of replication protein(s) to those cells. The proreplicon embodi ments of the present invention are particularly advantageous for expressing toxicant nucleic acid sequences in plant hosts. Plants a re genera lly sensitive to cellular toxicity and/or the detrimental effect of viral replication a nd/or repl ication protein(s) in early stages of pla nt growth and d ifferentiation that involve cell division and differentiation. Thus, controlling the expression of the replication protei n and repl icon replication entirely or largely to non-dividing, terminal ly-differentiated cells will red uce the detrimental effect of replicon replication on pla nt growth a nd development. Exa mples of such terminally-d ifferentiated cells include, but are not limited to, the storage cel ls of seed a nd root tissues and mature leaf cells. Furthermore, the proreplicon when introd uced into a plant host serves as a master copy for replicons not only in different generations but also i n the same generation if cell d ivisions occur after the onset of episomal repl ication. This strategy will a lso solve the problem of episomal instabi lity throug h cell divisions, since episomes a re unstable in the absence of selection. Furthermore, replicon replication is expected to achieve high level expression of toxicant nucleic acid sequences throug h gene amplification that is herita ble when stably integrated into the host chromosome a nd cell autonomous.

[0186] eplicase genes a re selected so that they recognize the Rep recognition elements required for release of a replicon from the proreplicon and autonomous episomal replication of the replicon. Exemplary rep genes include those from ssDNA pla nt vi ruses, such as Geminiviruses and IMa novi ruses, as wel l as those from bacteria, incl uding phytoplasmal rep genes. For example, a Mastrevi rus rep gene encod ing both Rep and RepA proteins can be included in a construct for expressing a toxica nt nucleic acid sequence. In other examples, a Curtovirus, Topocuvirus or Begomovirus rep gene is incl uded . In further exa mples, a Nanovirus rep gene encod ing the master replication initiation protei n (M-Rep) is included. Non-l imiti ng exa mples of rep genes for use in the expression system described herein include those set forth in Table 3.

Table 3. Exemplary rep genes

Virus

Banana bunchy top virus ( DNA- 1 Rep)

Tobacco yellow dwarf virus (Rep/RepA)

Maize streak vi rus (Rep/RepA)

Tomato leaf curl virus (A2 Rep)

Bean golden mosaic vi rus (AC2 Rep)

Beet curly top virus (C2)

Tomato pseudo-curly top vi rus (C2)

Banana bunchy top virus (DNA-1 Rep)

ATGGCGCGATATGTGGTATGCTGGATGTTCACCATCAACAATCCCACAACACTACCAGTGATGAGGGATGAG ATAAAATATATGGTATATCAAGTGGAGAGGGGACAGGAGGGTACTCGTCATGTGCAAGGTTATGTCGAGATG AAGAGACGAAGCTCTCTGAAGCAGATGAGAGGCTTCTTCCCAGGCGCACACCTTGAGAAACGAAAGGGAAG CCAAGAAGAAGCGCGGTCATACTGTATGAAGGAAGATACAAGAATCGAAGGTCCCTTCGAGTTTGGTTCATT TAAATTGTCATGTAATGATAATTTATTTGATGTCATACAGGATATGCGTGAAACGCACAAAAGGCCTTTGGAG TATTTATATGATTGTCCTAACACCTTCGATAGAAGTAAGGATACATTATACAGAGTACAAGCAGAGATGAATA AAACGAAGGCGATGAATAGCTGGAGAACTTCTTTCAGTGCTTGGACATCAGAGGTGGAGAATATCATGGCGC AGCCATGTCATCGGAGAATAATTTGGGTCTATGGCCCAAATGGAGGAGAAGGAAAGACAACGTATGCAAAAC ATCTAATGAAGACGAGAAATGCGTTTTATTCTCCAGGAGGAAAATCATTGGATATATGTAGACTGTATAATTA CGAGGATATTGTTATATTTGATATTCCAAGATGCAAAGAGGATTATTTAAATTATGGGTTATTAGAGGAATTTA AGAATGGAATAATTCAAAGCGGGAAATATGAACCCGTTTTGAAGATAGTAGAATATGTCGAAGTCATTGTAAT GGCTAACTTCCTTCCGAAGGAAGGAATCTTTTCTGAAGATCGAATAAAGTTGGTTTCTTGCTGA

[0187] Modified or variant rep genes can also be used in the expression system described herein, provided the encoded Rep protein retains the required activity to i nitiate rol li ng circle replication. The structure-function relationships of Geminivirus and Nanovirus rep genes are well known to those skilled in the art (see, e.g. Laufs et al. (1995) Biochimie 77: 765-773;

Gronenborn (2004) Vet Micro 98 : 103-109; Dasgupta et al. (2004) Plant Sci 166 : 1063-1067; and

Vadivuka rasi et al. (2007) J Biosc 32 : 17-29). Accord ing ly, those skil led in the art would understa nd which regions of the rep gene can be mod ified while still reta ining the required activities, a nd which regions are less tolerant to modification. For example, those skilled in the art would understand that conserved regions of Geminivirus and Nanovirus Rep protein are less tolerant to change. Such conserved regions include several conserved protein motifs: motif I, which is required for Rep/DNA binding ; motif II, which is involved in metal ion binding and activity of Rep; motif III, which contains a conserved tyrosine residue that participates in phosphodiester bond cleavage and in the covalent linkage of Rep to the 5' terminus of the nicked nonanucleotide motif at the origin of replication; and the Walker A motif (or P-loop) and Walker B motif, which are ATP binding and hydrolysis motifs, respectively, and are involved in helicase activity of the protein. Modified rep genes can be functionally analyzed using standard assays to confirm that the encoded modified Rep protein has retained the required activity (see e.g. Jin er a/. (2008) J Gen Virol 89: 2636- 2641).

3.2 Exemplary Rep recognition elements

[0188] In particular embodiments, the Rep recognition elements used in the expression system described herein are Geminivirus or Nanovirus intergenic regions (IRs), which are the non- coding regions of the Geminivirus or Nanovirus genomes and which contain the iterons for Rep binding, the inverted repeats and the consensus nonanucleotide with the rolling circle replication initiation site. Fragments or variants of IRs that retain the necessary features for rolling circle replication, (e.g. iterons, inverted repeats and consensus nonanucleotide) also can be used as Rep recognition elements in the methods and transgenic plants described herein. In other

embodiments, origins of replication from bacterial rolling circle plasmids are used as the Rep recognition elements.

[0189] Exemplary IRs or fragments thereof that can be used in the first and/or second constructs are the long intergenic regions (LIRs) from viruses of the Mastrevirus genus, the IRs from viruses of the Curtovirus genus, the common regions (CRs) from viruses of the Topocuvirus or Begomovirus genus (which are highly conserved regions of approximately 200 nucleotides within the Topocuvirus or Begomovirus IR), the Nanovirus IRs, and IRs from Begomovirus betasatellites (DNA-β satellites) or alphasatellites.

[0190] In particular embodiments, the Rep recognition elements are Mastrevirus LIRs, including fragments or variants thereof that retain the necessary features for rolling circle replication. Inclusion of LIRs from a Mastrevirus in the first construct facilitates expression of the rep gene, thereby facilitating expressing of the toxicant nucleic acid sequence and resistance of the plant to the particular virus from which the LIR is derived. For example, transgenic plants of the present invention that contain a first construct with a TYDV LIR will be resistant to TYDV.

[0191] Mastrevirus genomes also contain a short intergenic region (SIR), which is the origin of second strand synthesis and thus required for efficient rolling circle replication.

Accordingly, in embodiments of the present invention where one or more of the Rep recognition elements are Mastrevirus LIRs or fragments or variants thereof, a Mastrevirus SIR or fragment or variant thereof is also included in the construct. For example, where Mastrevirus LIRs or fragments or variants thereof are used as the Rep recognition elements in the first and/or second construct (or optionally the third construct), the construct can contain a Mastrevirus SIR between the 5' and 3' portion of the rep gene or the toxicant nucleic acid sequence, such as between the terminator that is operably linked to the 3' portion of the rep gene or toxicant nucleic acid sequence and the promoter that is operably linked to the 5' portion of the rep gene or the toxicant nucleic acid sequence. In some examples, the SIR is from the same Mastrevirus as the LIR. In other instances, the SIR and LIR used in the construct are from different Mastrevi ruses. In such instances, however, the SIR is typically from a Mastrevirus that is known to infect the plant into which the construct is stably introduced.

[0192] Non-limiting examples of Mastrevirus LIRs and SIRs for use in the invention are those from Bean yellow dwarf virus (BeYDV); Bromus striate mosaic virus (BrSMV); Chickpea chlorosis virus; Chickpea chlorotic dwarf Pakistan virus (CpCDV); Chickpea chlorotic dwarf Sudan virus (CpCDSV); Chickpea chlorotic dwarf Syria virus; Chickpea redleaf virus; Chloris striate mosaic virus (CSMV); Digitaria streak virus (DSV); Digitaria striate mosaic virus (DiSMV); Eragrostis curvula streak virus; Eragrostis minor streak virus; Eragrostis streak virus (ESV); Maize streak virus (MSV); Millet streak virus (MilSV); Miscanthus streak virus (MiSV); Panicum streak virus (PanSV); Paspalum striate mosaic virus (PSMV); Saccharum streak virus; Setaria streak virus (SetSV); Sugarcane streak Egypt virus (SSEV); Sugarcane streak Reunion virus (SSRV);

Sugarcane streak virus (SSV); Sweetpotato symptomless mastrevirus 1; Tobacco yellow dwarf virus (TYDV); Sugar beet mastrevirus; Urochloa streak virus (UroSV); and Wheat dwarf virus

(WDV). Exemplary Mastrevirus LIRs and SIRs that can be used in the expression system described herein are set forth in Table 4.

Table 4. Exemplary Mastrevirus LIRs and SIRs

Virus

Tobacco yellow dwarf virus

Maize streak virus

Tobacco yellow dwarf virus LIR

ATTAAGGCTCAAGTACCGTACGATGAAACTCTATTCTTAACTAGTGAGTGAGCCAGTGAGCCAGGTTTATGTG GCATTTATATTGGTAGGTGGACCGTTAGGATCTTGACGCGTGGGGCACACTCACGGATTTTAATATTACCCGT GAGTGCTCTCTTGGCCCCACGCGAGCCCTTTAGGGCGAGCGAAAGTGCGCCGTAGTTTCCTTTAGTGGTTCA TGAGTCATTATAGTGCTATATAAAGATGATGTGACATCCCAAAGTTGAAA

Tobacco yellow dwarf virus SIR

TAAAAATGTCGTTATTTTGATTTCATATTAATGAGCTTCAGTGTAGAGAAATTCAAATCTTATTAATAAAAACCC GGAATACAAAAACACACGAAAACGAAAAAAAGACACCTTACAATCATTACACACTATATACCCTCCTATGAGG AGAGGCACGTTCA

Maize streak virus LIR

AGCAGACGACGGAGGCTGAGGCTGAGGGATGGCAGACTGGGAGCTCCAAACTCTATAGTATACCCGTGCGC CTTCGAAATCCGCCGCTCCCTTGTCTTATAGTGGTTGTAAATGGGCCGGACCGGTCCGGCCCAGCAGGAAAA GAAGGCGCGCACTAATATTACCGCGCCTTCTTTTCCTGCGAGGGCCCGGTAGGGACCGAGCGCTTTGATTTA AAGCCTGGTTCTGCTTTGTATGATTTATCTAAAGCAGCCCAATCTAAAGAAACCGGTCCCGGGCACTATAAAT TGCCTAACAAGTGCGATTCATTC

Maize streak virus SIR

TAATGAATAAAAACGCCCGTTTTATTATATCTGATGAATGCTGAAAGCTTACATTAATATGTCGTGCGATGGC ACGAAAAAACACACGCAATCAATACAGGGGGGTAGTCGGCGGGCGGCTAAGGGTGGTGCTCGGCGGGCAA AACATCGAAAAATCAAGATCTATATGAA

[0193] In other embodiments, the Rep recognition elements are Begomovirus IRs, including fragments or variants thereof, such as the constant regions, that retain the necessary features for rolling circle replication. Exemplary Begomovirus IRs that can be utilised in the constructs, methods and transgenic plants of the present invention are those from the Abutilon mosaic virus (AbMV); African cassava mosaic virus (ACMV); Ageratum enation virus (AEV);

Ageratum leaf curl virus (ALCuV); Ageratum yellow vein Hualian virus (AYVHuV); Ageratum yellow vein Sri Lanka virus (AYVSLV); Ageratum yellow vein virus (AYVV); Alternanthera yellow vein virus (AIYVV); Bean calico mosaic virus (BCaMV); Bean dwarf mosaic virus (BDMV); Bean golden mosaic virus (BGMV); Bean golden yellow mosaic virus (BGYMV); Bhendi yellow vein mosaic virus (BYVMV); Bitter gourd yellow vein virus (BGYVV); Boerhavia yellow spot virus (BoYSV); Cabbage leaf curl Jamaica virus (CabLCJV); Cabbage leaf curl virus (CabLCV); Chayote yellow mosaic virus (ChaYMV); Chilli leaf curl virus (ChiLCuV); Chino del tomate virus (CdTV); Clerodendron golden mosaic virus (CIGMV); Corchorus golden mosaic virus (CoGMV); Corchorus yellow spot virus (CoYSV); Corchorus yellow vein virus (CYVV); Cotton leaf crumple virus (CLCrV); Cotton leaf curl Alabad virus (CLCuAV); Cotton leaf curl Bangalore virus (CLCuBV); Cotton leaf curl Gezira virus (CLCuGV); Cotton leaf curl Kokhran virus (CLCuKV); Cotton leaf curl Multan virus (CLCuMV); Cowpea golden mosaic virus (CPGMV); Croton yellow vein mosaic virus (CYVMV); Cucurbit leaf crumple virus (CuLCrV); Desmodium leaf distortion virus (DesLDV); Dicliptera yellow mottle Cuba virus (DiYMoCV); Dicliptera yellow mottle virus (DiYMoV); Dolichos yellow mosaic virus (DoYMV); East African cassava mosaic Cameroon virus (EACMCV); East African cassava mosaic Kenya virus (EACMKV); East African cassava mosaic Malawi virus (EACMMV); East African cassava mosaic virus (EACMV); East African cassava mosaic Zanzibar virus (EACMZV); Erectites yellow mosaic virus (ErYMV); Eupatorium yellow vein mosaic virus (EpYVMV); Eupatorium yellow vein virus (EpYVV); Euphorbia leaf curl Guangxi virus (EuLCGxV); Euphorbia leaf curl virus (EuLCV); Euphorbia mosaic virus (EuMV); Hollyhock leaf crumple virus (HLCrV); Honeysuckle yellow vein Kagoshima virus (HYVKgV); Honeysuckle yellow vein mosaic virus (HYVMV); Honeysuckle yellow vein virus (HYVV); Horsegram yellow mosaic virus (HgYMV); Indian cassava mosaic virus (ICMV); Ipomoea yellow vein virus (IYVV); Kudzu mosaic virus (KuMV); Lindernia anagallis yellow vein virus (LAYVV); Ludwigia yellow vein Vietnam virus (LuYVVNV); Ludwigia yellow vein virus (LuYVV); Luffa yellow mosaic virus (LYMV); Macroptilium mosaic Puerto Rico virus (MacMPRV); Macroptilium yellow mosaic Florida virus (MacYMFV); Macroptilium yellow mosaic virus (MacYMV); Malvastrum leaf curl Guangdong virus (MaLCGdV); Malvastrum leaf curl virus (MaLCV); Malvastrum yellow leaf curl virus (MaYLCV); Malvastrum yellow mosaic virus (MalYMV); Malvastrum yellow vein virus (MYVV); Malvastrum yellow vein Yunnan virus (MYVYV); Melon chlorotic leaf curl virus (MeCLCV); Merremia mosaic virus (MerMV); Mesta yellow vein mosaic virus (MYVMV); Mimosa yellow leaf curl virus (MiYLCV); Mungbean yellow mosaic India virus (MYMIV); Mungbean yellow mosaic virus (MYMV); Okra yellow crinkle virus (OYCrV); Okra yellow mosaic Mexico virus (OYMMV); Okra yellow mottle Iguala virus (OYMolgV); Okra yellow vein mosaic virus (OYVMV); Papaya leaf curl China virus (PaLCuCNV); Papaya leaf curl Guandong virus (PaLCuGDV); Papaya leaf curl virus (PaLCuV); Pedilenthus leaf curl virus (PedLCuV); Pepper golden mosaic virus (PepGMV); Pepper huasteco yellow vein virus (PHYVV); Pepper leaf curl Bangladesh virus (PepLCBV); Pepper leaf curl virus (PepLCV); Pepper yellow leaf curl Indonesia virus (PepLCIV); Pepper yellow vein Mali virus (PepYVMLV); Potato yellow mosaic Panama virus (PYMPV); Potato yellow mosaic virus (PYMV); Pumpkin yellow mosaic virus (PuYMV); Radish leaf curl virus (RaLCV); Rhynchosia golden mosaic Sinaloa virus (RhGMSIV); Rhynchosia golden mosaic virus (RhGMV); Senecio yellow mosaic virus (SeYMV); Sida golden mosaic Costa Rica virus (SiGMCRV); Sida golden mosaic Florida virus

(SiGMFIV); Sida golden mosaic Honduras virus (SiGMHNV); Sida golden mosaic virus (SiGMV); Sida golden yellow vein virus (SiGYVV); Sida leaf curl virus (SiLCuV); Sida micrantha mosaic virus

(SiMMV); Sida mottle virus (SiMoV); Sida yellow mosaic China virus (SiYMCNV); Sida yellow mosaic virus (SiYMV); Sida yellow mosaic Yucatan virus (SiYMYuV); Sida yellow vein Madurai virus (SiYVMaV); Sida yellow vein Vietnam virus (SiYVVNV); Sida yellow vein virus (SiYVV); Siegesbeckia yellow vein Guangxi virus (SbYVGxV); Siegesbeckia yellow vein virus (SbYVV); South African cassava mosaic virus (SACMV); Soybean blistering mosaic virus (SbBMV); Soybean crinkle leaf virus (SbCLV); Spilanthes yellow vein virus (SpYW); Squash leaf curl China virus (SLCCNV); Squash leaf curl Philippines virus (SLCPHV); Squash leaf curl virus (SLCuV); Squash leaf curl Yunnan virus (SLCYNV); Squash mild leaf curl virus (SMLCuV); Sri Lankan cassava mosaic virus (SLCMV); Stachytarpheta leaf curl virus (StaLCV); Sweet potato leaf curl Canary virus (SPLCCanV); Sweet potato leaf curl China virus (SPLCCNV); Sweet potato leaf curl Georgia virus (SPLCGV); Sweet potato leaf curl Lanzarote virus (SPLCLanV); Sweet potato leaf curl Spain virus (SPLCESV); Sweet potato leaf curl virus (SPLCV); Tobacco curly shoot virus (TbCSV); Tobacco leaf curl Cuba virus (TbLCuCV); Tobacco leaf curl Japan virus (TbLCJV); Tobacco leaf curl Yunnan virus (TbLCYV); Tobacco leaf curl Zimbabwe virus (TbLCZV); Tomato chino La Paz virus (ToChLPV); Tomato chlorotic mottle virus (ToCMoV); Tomato curly stunt virus (ToCSV); Tomato golden mosaic virus (TGMV); Tomato golden mottle virus (ToGMoV); Tomato leaf curl Arusha virus (ToLCArV); Tomato leaf curl Bangalore virus (ToLCBV); Tomato leaf curl Bangladesh virus (ToLCBDV); Tomato leaf curl China virus (ToLCCNV); Tomato leaf curl Comoros virus (ToLCKMV); Tomato leaf curl Guangdong virus (ToLCGuV); Tomato leaf curl Guangxi virus (ToLCGxV); Tomato leaf curl Gujarat virus (ToLCGV); Tomato leaf curl Hsinchu virus (ToLCHsV); Tomato leaf curl Java virus (ToLCJV);

Tomato leaf curl Joydebpur virus (ToLCJoV); Tomato leaf curl Karnataka virus (ToLCKV); Tomato leaf curl Kerala virus (ToLCKeV); Tomato leaf curl Laos virus (ToLCLV); Tomato leaf curl

Madagascar virus (ToLCMGV); Tomato leaf curl Malaysia virus (ToLCMV); Tomato leaf curl Mali virus (ToLCMLV); Tomato leaf curl Mayotte virus (ToLCYTV); Tomato leaf curl New Delhi virus (ToLCNDV); Tomato leaf curl Philippines virus (ToLCPV); Tomato leaf curl Pune virus (ToLCPuV); Tomato leaf curl Seychelles virus (ToLCSCV); Tomato leaf curl Sinaloa virus (ToLCSInV); Tomato leaf curl Sri Lanka virus (ToLCSLV); Tomato leaf curl Sudan virus (ToLCSDV); Tomato leaf curl Taiwan virus (ToLCTWV); Tomato leaf curl Uganda virus (ToLCUV); Tomato leaf curl Vietnam virus (ToLCVV); Tomato leaf curl virus (ToLCV); Tomato mild yellow leaf curl Aragua virus (ToMYLCV); Tomato mosaic Havana virus (ToMHaV); Tomato mottle Taino virus (ToMTaV); Tomato mottle virus (ToMoV); Tomato rugose mosaic virus (ToRMV); Tomato severe leaf curl virus (ToSLCV); Tomato severe rugose virus (ToSRV); Tomato yellow leaf curl Axarquia virus (TYLCAxV); Tomato yellow leaf curl China virus (TYLCCNV); Tomato yellow leaf curl Guangdong virus (TYLCGuV); Tomato yellow leaf curl Indonesia virus (TYLCIDV); Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV); Tomato yellow leaf curl Malaga virus (TYLCMAIV); Tomato yellow leaf curl Mali virus (TYLCMLV); Tomato yellow leaf curl Sardinia virus (TYLCSV); Tomato yellow leaf curl Thailand virus (TYLCTHV); Tomato yellow leaf curl Vietnam virus (TYLCVNV); Tomato yellow leaf curl virus (TYLCV); Tomato yellow margin leaf curl virus (TYMLCV); Tomato yellow spot virus (ToYSV); Tomato yellow vein streak virus (ToYVSV); Vernonia yellow vein virus (VeYVV); and Watermelon chlorotic stunt virus (WmCSV). Table 5 sets forth non-limiting examples of specific Begomovirus IRs that can be used in the expression system described herein.

Table 5. Exemplary Begomovirus IRs

Virus

Tomato yellow leaf curl virus

Bean golden mosaic virus DNA-A IR

Bean golden mosaic virus DNA-B IR Tomato yellow leaf curl virus

GTTGAAATGAATTGGTGTCCCTCAAAGCTCTATGGCAATCGGTGTATCGGTGTCTTACTT60ATACCTGGACA CCTAATGGCTATTTGGTAATTTTGTAAAAGTACATTGCAAAAATCAAAA120ATCAAATCATTAAAGCGGCCAT CCGTATAATATTACCGGATGGCCGCGCCTTTTCCTTTT180ATGTGGTCCCCACGAGGGTTCCACAGACGTCA CTGTCAACCAATCAAATTGCATCCTCAA240ACGTTAGATAAGTGTTCATTTGTCTTTATATACTTGGTCCTCA AGTAGTTTGTCTTG C AC

Bean golden mosaic virus DNA-A IR

ACTTGTAAATAAGAGGGTGTACCCCGATTGAGCTCTCGTTCAAAAGTCTCTATGAATCGG60TGTAATGGTGC CAATATATAGTAAGAAGTTCTTTAAGGATCTGTAGACACGTGGCGGCCA120TCCGCTATAATATTACCGGAT GGCCGCGCGA I I I I I I

Bean golden mosaic virus DNA-B IR

TTTAAAACGCTTTGGTGATGGCATACTCGTAAATAAGAGGGTGTACCCCGATTGAGCTCT60CGTTCAAAAGT CTCTATGAATCGGTGTAATGGTGCCAATATATAGTATGAAGTTCTTTAA120GGATCTGGAGACACGTGGCGG CCATCCGTTATAATATTACCGGATGGCCGCGCGATTTTT

[0194] In other embodi ments, Topocuvi rus IRs, incl uding fragments or va riants thereof that retain the necessary features for rol ling circle repl ication, are util ized as Rep recog nition elements. An exemplary Topocuvi rus IR is the IR from Tomato pseudo-curly top virus (TPCTV) .

[0195] In further embodi ments, the Rep recognition elements used in the present i nvention are Curtovi rus IRs, includi ng frag ments or varia nts thereof, such as Curtovirus CRs, that reta in the necessary features for rol ling ci rcle replication. Exemplary Curtovirus IRs incl ude, but are not li mited to, those from Beet curly top Iran vi rus (BCTIV) ; Beet curly top virus (BCTV; includ ing the Beet curly top virus-Ca lifornia/Logan, Suga rbeet curly leaf virus, Sugarbeet curly top virus, Suga rbeet vi rus 1, Tomato yellow virus and Western yellow blight vi rus) ; Beet mild curly top virus (BMCTV); Beet severe curly top virus (BSCTV) ; Horseradish curly top virus (HrCTV) ; Pepper curly top virus (PepCTV) ; a nd Spi nach curly top virus (SpCTV) . Non-li miting examples of specific Curtovirus IRs that ca n be used in the expression system of the present invention include Beet curly top virus, with the following sequence:

TGTACTCCGATGACGTGGCTTAGCATATTAACATATCTATTGGAGTATTGGAGTATTATATATATTAGTACAAC TTTCATAAGGGCCATCCGTTATAATATTACCGGATGGCCCGAAAAAAATGGGCACCCAATCAAAACGTGACA CGTGGAAGGGGACTGTTGAATGATGTGACG I I I I I GAGCGGGAAACTTCCTGAAGAAGATTCCTGCGGGAA ACTTCCTGAAGAAGATTCCTTTCAGATAAGATTTGTTGACTGGTCAAAAGAAGGGGACAACTTTATTAAAGTA ACTTTACTTTAGTAAAAGTAAAGTAAGTGTGCCCCACAGGAAACTTGCTCAGCAAGTTTTGAATTATGTCGTTT TATATACGTTATTTTTACATGTATATGTAATTATAA

[0196] In other examples, the Rep recognition elements used in the expression system described herein are Nanovirus IRs, i ncl ud ing fragments or va riants thereof that retain the necessary features for roll ing circle replication. For exa mple, IRs from Bana na bunchy top virus (BBTV) , Faba bea n necrotic stunt virus (FBNSV), Faba bean necrotic yellows virus (FBNYV), M il k vetch dwa rf virus (MDV) ; Pea necrotic yellow dwarf virus (PNYDV), or Subterra nean clover stunt virus (SCSV) ca n be used as the Rep recog nition elements in the subject expression system. Table 7 sets forth exempla ry Nanovirus IRs for use in the invention. Table 7. Exemplary Nanovirus IRs

Virus

Banana bunchy top virus DNA-1 IR

Banana bunchy top virus DNA-2 IR

Banana bunchy top virus DNA-3 IR

Banana bunchy top virus DNA-4 IR

Banana bunchy top virus DNA-5 IR

Banana bunchy top virus DNA-6 IR

[0197] In further embodiments, a Begomovirus-associated DNA-β satellite intergenic region or fragment or variant thereof is used in the present invention. Rep proteins from most, if not all, Begomovi ruses recognise and bind DNA-β satellite intergenic regions. Accordingly, transgenic plants of the present invention that contain a first construct with DNA-β satellite intergenic regions flanking the split Rep gene will exhibit resistance to multiple Begomoviruses, including, but not limited to, two or more of Abutilon mosaic virus (AbMV); African cassava mosaic virus (ACMV); Ageratum enation virus (AEV); Ageratum leaf curl virus (ALCuV); Ageratum yellow vein Hualian virus (AYVHuV); Ageratum yellow vein Sri Lanka virus (AYVSLV); Ageratum yellow vein virus (AYVV); Alternanthera yellow vein virus (AIYVV); Bean calico mosaic virus (BCaMV); Bean dwarf mosaic virus (BDMV); Bean golden mosaic virus (BGMV); Bean golden yellow mosaic virus (BGYMV); Bhendi yellow vein mosaic virus (BYVMV); Bitter gourd yellow vein virus (BGYW); Boerhavia yellow spot virus (BoYSV); Cabbage leaf curl Jamaica virus (CabLCJV); Cabbage leaf curl virus (CabLCV); Chayote yellow mosaic virus (ChaYMV); Chilli leaf curl virus (ChiLCuV); Chino del tomate virus (CdTV); Clerodendron golden mosaic virus (CIGMV); Corchorus golden mosaic virus (CoGMV); Corchorus yellow spot virus (CoYSV); Corchorus yellow vein virus (CYVV); Cotton leaf crumple virus (CLCrV); Cotton leaf curl Alabad virus (CLCuAV); Cotton leaf curl Bangalore virus (CLCuBV); Cotton leaf curl Gezira virus (CLCuGV); Cotton leaf curl Kokhran virus (CLCuKV); Cotton leaf curl Multan virus (CLCuMV); Cowpea golden mosaic virus (CPGMV); Croton yellow vein mosaic virus (CYVMV); Cucurbit leaf crumple virus (CuLCrV); Desmodium leaf distortion virus (DesLDV); Dicliptera yellow mottle Cuba virus (DiYMoCV); Dicliptera yellow mottle virus (DiYMoV); Dolichos yellow mosaic virus (DoYMV); East African cassava mosaic Cameroon virus (EACMCV); East African cassava mosaic Kenya virus (EACMKV); East African cassava mosaic Malawi virus (EACMMV); East African cassava mosaic virus (EACMV); East African cassava mosaic Zanzibar virus (EACMZV);

Erectites yellow mosaic virus (ErYMV); Eupatorium yellow vein mosaic virus (EpYVMV); Eupatorium yellow vein virus (EpYVV); Euphorbia leaf curl Guangxi virus (EuLCGxV); Euphorbia leaf curl virus (EuLCV); Euphorbia mosaic virus (EuMV); Hollyhock leaf crumple virus (HLCrV); Honeysuckle yellow vein Kagoshima virus (HYVKgV); Honeysuckle yellow vein mosaic virus (HYVMV);

Honeysuckle yellow vein virus (HYVV); Horsegram yellow mosaic virus (HgYMV); Indian cassava mosaic virus (ICMV); Ipomoea yellow vein virus (IYVV); Kudzu mosaic virus (KuMV); Lindernia anagallis yellow vein virus (LAYVV); Ludwigia yellow vein Vietnam virus (LuYVVNV); Ludwigia yellow vein virus (LuYVV); Luffa yellow mosaic virus (LYMV); Macroptilium mosaic Puerto Rico virus (MacMPRV); Macroptilium yellow mosaic Florida virus (MacYMFV); Macroptilium yellow mosaic virus (MacYMV); Malvastrum leaf curl Guangdong virus (MaLCGdV); Malvastrum leaf curl virus (MaLCV); Malvastrum yellow leaf curl virus (MaYLCV); Malvastrum yellow mosaic virus (MalYMV);

Malvastrum yellow vein virus (MYVV); Malvastrum yellow vein Yunnan virus (MYVYV); Melon chlorotic leaf curl virus (MeCLCV); Merremia mosaic virus (MerMV); Mesta yellow vein mosaic virus (MYVMV); Mimosa yellow leaf curl virus (MiYLCV); Mungbean yellow mosaic India virus (MYMIV); Mungbean yellow mosaic virus (MYMV); Okra yellow crinkle virus (OYCrV); Okra yellow mosaic Mexico virus (OYMMV); Okra yellow mottle Iguala virus (OYMolgV); Okra yellow vein mosaic virus (OYVMV); Papaya leaf curl China virus (PaLCuCNV); Papaya leaf curl Guandong virus (PaLCuGDV); Papaya leaf curl virus (PaLCuV); Pedilenthus leaf curl virus (PedLCuV); Pepper golden mosaic virus (PepGMV); Pepper huasteco yellow vein virus (PHYVV); Pepper leaf curl Bangladesh virus

(PepLCBV); Pepper leaf curl virus (PepLCV); Pepper yellow leaf curl Indonesia virus (PepLCIV); Pepper yellow vein Mali virus (PepYVMLV); Potato yellow mosaic Panama virus (PYMPV); Potato yellow mosaic virus (PYMV); Pumpkin yellow mosaic virus (PuYMV); Radish leaf curl virus (RaLCV); Rhynchosia golden mosaic Sinaloa virus (RhGMSIV); Rhynchosia golden mosaic virus (RhGMV); Senecio yellow mosaic virus (SeYMV); Sida golden mosaic Costa Rica virus (SiGMCRV); Sida golden mosaic Florida virus (SiGMFIV); Sida golden mosaic Honduras virus (SiGMHNV); Sida golden mosaic virus (SiGMV); Sida golden yellow vein virus (SiGYVV); Sida leaf curl virus (SiLCuV); Sida micrantha mosaic virus (SiMMV); Sida mottle virus (SiMoV); Sida yellow mosaic China virus

(SiYMCNV); Sida yellow mosaic virus (SiYMV); Sida yellow mosaic Yucatan virus (SiYMYuV); Sida yellow vein Madurai virus (SiYVMaV); Sida yellow vein Vietnam virus (SiYVVNV); Sida yellow vein virus (SiYVV); Siegesbeckia yellow vein Guangxi virus (SbYVGxV); Siegesbeckia yellow vein virus (SbYVV); South African cassava mosaic virus (SACMV); Soybean blistering mosaic virus (SbBMV); Soybean crinkle leaf virus (SbCLV); Spilanthes yellow vein virus (SpYVV); Squash leaf curl China virus (SLCCNV); Squash leaf curl Philippines virus (SLCPHV); Squash leaf curl virus (SLCuV); Squash leaf curl Yunnan virus (SLCYNV); Squash mild leaf curl virus (SMLCuV); Sri Lankan cassava mosaic virus (SLCMV); Stachytarpheta leaf curl virus (StaLCV); Sweet potato leaf curl Canary virus (SPLCCanV); Sweet potato leaf curl China virus (SPLCCNV); Sweet potato leaf curl Georgia virus (SPLCGV); Sweet potato leaf curl Lanzarote virus (SPLCLanV); Sweet potato leaf curl Spain virus (SPLCESV); Sweet potato leaf curl virus (SPLCV); Tobacco curly shoot virus (TbCSV); Tobacco leaf curl Cuba virus (TbLCuCV); Tobacco leaf curl Japan virus (TbLCJV); Tobacco leaf curl Yunnan virus (TbLCYV); Tobacco leaf curl Zimbabwe virus (TbLCZV); Tomato chino La Paz virus (ToChLPV); Tomato chlorotic mottle virus (ToCMoV); Tomato curly stunt virus (ToCSV); Tomato golden mosaic virus (TGMV); Tomato golden mottle virus (ToGMoV); Tomato leaf curl Arusha virus (ToLCArV);

Tomato leaf curl Bangalore virus (ToLCBV); Tomato leaf curl Bangladesh virus (ToLCBDV); Tomato leaf curl China virus (ToLCCNV); Tomato leaf curl Comoros virus (ToLCKMV); Tomato leaf curl Guangdong virus (ToLCGuV); Tomato leaf curl Guangxi virus (ToLCGxV); Tomato leaf curl Gujarat virus (ToLCGV); Tomato leaf curl Hsinchu virus (ToLCHsV); Tomato leaf curl Java virus (ToLCJV); Tomato leaf curl Joydebpur virus (ToLCJoV); Tomato leaf curl Karnataka virus (ToLCKV); Tomato leaf curl Kerala virus (ToLCKeV); Tomato leaf curl Laos virus (ToLCLV); Tomato leaf curl

Madagascar virus (ToLCMGV); Tomato leaf curl Malaysia virus (ToLCMV); Tomato leaf curl Mali virus (ToLCMLV); Tomato leaf curl Mayotte virus (ToLCYTV); Tomato leaf curl New Delhi virus (ToLCNDV); Tomato leaf curl Philippines virus (ToLCPV); Tomato leaf curl Pune virus (ToLCPuV); Tomato leaf curl Seychelles virus (ToLCSCV); Tomato leaf curl Sinaloa virus (ToLCSInV); Tomato leaf curl Sri Lanka virus (ToLCSLV); Tomato leaf curl Sudan virus (ToLCSDV); Tomato leaf curl

Taiwan virus (ToLCTWV); Tomato leaf curl Uganda virus (ToLCUV); Tomato leaf curl Vietnam virus

(ToLCVV); Tomato leaf curl virus (ToLCV); Tomato mild yellow leaf curl Aragua virus (ToMYLCV);

Tomato mosaic Havana virus (ToMHaV); Tomato mottle Taino virus (ToMTaV); Tomato mottle virus (ToMoV); Tomato rugose mosaic virus (ToRMV); Tomato severe leaf curl virus (ToSLCV); Tomato severe rugose virus (ToSRV); Tomato yellow leaf curl Axarquia virus (TYLCAxV) ; Tomato yellow leaf curl China virus (TYLCCNV) ; Tomato yellow leaf curl Guangdong virus (TYLCGuV) ; Tomato yellow leaf curl Indonesia virus (TYLCIDV) ; Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV) ; Tomato yellow leaf curl Malaga virus (TYLCMAIV); Tomato yellow leaf curl Mali virus (TYLCMLV); Tomato yellow leaf curl Sardinia virus (TYLCSV); Tomato yellow leaf curl Thailand virus (TYLCTHV); Tomato yellow leaf curl Vietnam virus (TYLCVNV) ; Tomato yellow leaf curl virus (TYLCV) ; Tomato yellow margin leaf curl virus (TYMLCV) ; Tomato yellow spot virus (ToYSV); Tomato yellow vein streak virus (ToYVSV) ; Vernonia yellow vein virus (VeYVV) ; and Watermelon chlorotic stunt virus (WmCSV).

[0198] Table 9 sets forth exemplary DNA-β satellite IRs for use in the expression system described herein.

Table 9. Exemplary DNA-β satellite IRs

DNA-β

Ageratum leaf curl virus DNA-β IR

Ageratum yellow vein virus DNA-β IR

Chilli leaf curl virus DNA-β IR

Cotton leaf curl virus DNA-β IR

Eupatorium yellow vein virus DNA-β IR

Mimosa yellow leaf curl virus DNA-β IR

Malachra yellow vein mosaic virus DNA-β IR

Pepper yellow leaf curl virus DNA-β IR

Tobacco curly shoot virus DNA-β IR

Tomato leaf curl virus (Thailand) DNA-β IR

Tomato yellow leaf curl virus DNA-β IR

Bacterial or phytoplasmal plasmid origins of replication also can be used as the Rep recognition element in the second construct (and optionally third construct) where the rep gene in the first construct is a bacterial or phytoplasmal rep gene. Rolling circle replication of bacterial plasmids has been well characterized the necessary elements for rolling circle replication to occur are known to those in the art (see, e.g. Kahn (1997) Micr Mol Biol Rev 61 :442-455). Bacterial origins of replication typically include Rep binding and nicking sites, and inverted repeats, which can form hairpin structures. For example, the pT181 origin comprises three sets of inverted repeats, IRI, IRII and IRIII, and the IRII repeats form a hairpin structure, wherein the loop of the hairpin comprises the nicking site (Kahn (1997) Micr Mol Biol Rev 61 :442-455). Exemplary pT181 origins that can be used as Rep recognition sequences in the present invention include those set forth in SEQ ID NOS: 170 and 171 (SEQ ID NO: 170

tttagacaatttttctaaaaccggctactctaatagccggttggacgcacatactgtgtgcatatctgatc; and SEQ ID NO: 171 accggctac tctaatagcc ggttggacgc acatactgtg).

[0199] It is well within the skill of a skilled artisan to identify suitable Rep recognition elements for use in the constructs, methods and transgenic plants described herein. For example, the IR from a virus of interest can be identified by aligning the genome sequence of the virus with that of a well-characterized virus of the same genus or with the one of the IRs set forth in any one of Tables 3 to 6 using standard sequence homology programs. Furthermore, fragments and variants of IRs that retain the necessary sequences for rolling circle replication can be generated and tested using standard assays. For example, a truncated and modified Tobacco yellow dwarf virus (TYDV) LIR that retains the necessary elements for rolling circle replication but from which non-essential sequences and sequences that may interfere with intron splicing have been removed, has been generated and can be used in the methods and transgenic plants herein. An illustrative modified TYDV LIR sequence that is suitable for use for rolling circle replication has the following sequence:

ctcaagtaccgtacgatgaaactctattcttaactagtgagtgagccagtgagccaggtttatgtggcatttatattggtcggtgga ccgttcggatcttgacgcgtggggcacactcacggattttaatattacccgtgagtgctctcttggccccacgcgagccctttaggg cgagcgaaagtgcgccgtagtttcctttagt [SEQ ID NO: 172].

3.3 Expression cassettes

[0200] The toxicant and virus impairment nucleic acid sequences of the invention are operably connected to at least one regulatory element including a promoter for driving their expression. Useful promoters include those that are inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, tissue-specific, and spatio-temporally regulated. Where expression in specific tissues or organs is desired, tissue-specific promoters may be used. In contrast, where gene expression in response to a stimulus is desired, inducible promoters are the regulatory elements of choice. Where continuous expression is desired throughout the cells of a host, constitutive promoters are utilized. Additional regulatory sequences upstream and/or downstream from the core promoter sequence may be included in expression cassettes to bring about varying levels of expression of the toxicant and virus impairment nucleic acid sequences in a transgenic host.

3.4 Promoters

[0201] The choice of the promoter will vary upon the host in which the expression system of the invention is introduced and it shall be understood that the present invention contemplates any promoter that is operable in a chosen host. In specific embodiments, the hosts are selected from plants, animals and yeast.

Plant promoters

[0202] Promoters contemplated by the present invention may be native to a host plant or may be derived from an alternative source, where the promoter is functional in the host plant. Numerous promoters that are active in plant cells have been described in the literature. The choice of plant promoter will generally vary depending on the temporal and spatial requirements for expression, and also depending on the toxicant plant species. In some cases, expression in multiple tissues is desirable. While in others, tissue-specific, e.g. , leaf-specific, expression is desirable. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and wee versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons. However, there is no restriction to the provenance of selected promoters; it is sufficient that they are operational in driving the expression of the toxicant and/or VVI nucleic acid sequences in the desired plant cell.

[0203] These promoters include, but are not limited to, constitutive, inducible, temporally regulated, developmental ly regulated, spatially-regulated, chemically regulated, stress- responsive, tissue-specific, viral and synthetic promoters. Promoter sequences are known to be strong or weak. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression. An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus.

[0204] Within a plant promoter region there are several domains that are necessary for full function of the promoter. The first of these domains lies immediately upstream of the structural gene and forms the "core promoter region" containing consensus sequences, normally 70 base pairs immediately upstream of the gene. The core promoter region contains the characteristic CAAT and TATA boxes plus surrounding sequences, and represents a transcription initiation sequence that defines the transcription start point for the structural gene.

[0205] The presence of the core promoter region defines a sequence as being a promoter: if the region is absent, the promoter is non-functional. Furthermore, the core promoter region is insufficient to provide full promoter activity. A series of regulatory sequences upstream of the core constitute the remainder of the promoter. The regulatory sequences determine expression level, the spatial and temporal pattern of expression and, for an important subset of promoters, expression under inductive conditions (regulation by external factors such as light, temperature, chemicals, hormones).

[0206] A range of naturally-occurring promoters is known to be operable in plants and have been used to drive the expression of heterologous (both foreign and endogenous) genes in plants: for example, the constitutive 35S cauliflower mosaic virus (CaMV) promoter, the ripening- enhanced tomato polygalacturonase promoter (Bird et al., 1988), the E8 promoter (Diekman & Fischer, 1988) and the fruit specific 2A1 promoter (Pear et al., 1989) and many others, e.g., U2 and U5 snRNA promoters from maize, the promoter from alcohol dehydrogenase, the Z4 promoter from a gene encoding the Z4 22 kD zein protein, the Z10 promoter from a gene encoding a 10 kD zein protein, a Z27 promoter from a gene encoding a 27 kD zein protein, the A20 promoter from the gene encoding a 19 kD-zein protein, inducible promoters, such as the light inducible promoter derived from the pea rbcS gene and the actin promoter from rice, e.g. , the actin 2 promoter (WO 00/70067); seed specific promoters, such as the phaseolin promoter from beans, may also be used. The nucleotide sequences of this invention can also be expressed under the regulation of promoters that are chemically regulated. This enables the nucleic acid sequence or encoded polypeptide to be synthesized only when the crop plants are treated with the inducing chemicals. Chemical induction of gene expression is detailed in EP 0 332 104 (to Ciba-Geigy) and U.S. Pat. No. 5,614,395. A preferred promoter for chemical induction is the tobacco PR-la promoter.

[0207] Examples of some constitutive promoters which have been described include the rice actin 1 (Wang et al. , 1992; U.S. Pat. No. 5,641,876), CaMV 35S (Odell et al. , 1985), CaMV 19S (Lawton et al., 1987), nos, Adh, sucrose synthase; and the ubiquitin promoters.

[0208] Examples of tissue specific promoters which have been described include the lectin (Vodkin, 1983; Lindstrom et al., 1990) corn alcohol dehydrogenase 1 (Vogel et al. , 1989; Dennis et al., 1984), corn light harvesting complex (Simpson, 1986; Bansal et al., 1992), corn heat shock protein (Odell et al., 1985), pea small subunit RuBP carboxylase (Poulsen et al., 1986), Ti plasmid mannopine synthase (Langridge et al. , 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (vanTunen et al. , 1988), bean glycine rich protein 1 (Keller et al. , 1989), truncated CaMV 35S (Odell et al., 1985), potato patatin (Wenzler et al., 1989), root cell (Yamamoto et al., 1990), maize zein (Reina et al. , 1990; Kriz et al., 1987; Wandelt et al., 1989; Langridge et al., 1983; Reina et al., 1990), globulin-1 (Belanger et al., 1991), . alpha. - tubulin, cab (Sullivan et al. , 1989), PEPCase (Hudspeth & Grula, 1989), R gene complex-associated promoters (Chandler et al., 1989), histone, and chalcone synthase promoters (Franken et al., 1991). Tissue specific enhancers are described in Fromm et al. (1989).

[0209] Inducible promoters that have been described include the ABA- and turgor- inducible promoters, the promoter of the auxin-binding protein gene (Schwob et al., 1993), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston et al., 1988), the MPI proteinase inhibitor promoter (Cordero et al., 1994), and the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., 1995; Quigley et al., 1989; Martinez et al., 1989).

[0210] Several other tissue-specific regulated genes and/or promoters have been reported in plants. These include genes encoding the seed storage proteins (such as napin, cruciferin, β-conglycinin, and phaseolin) zein or oil body proteins (such as oleosin), or genes involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase. And fatty acid desaturases (fad 2-1)), and other genes expressed during embryo development (such as Bce4, see, for example, EP 255378 and Kridl et a/., 1991). Particularly useful for seed-specific expression is the pea vicilin promoter (Czako et al., 1992). (See also U.S. Pat. No. 5,625,136, herein incorporated by reference). Other useful promoters for expression in mature leaves are those that are switched on at the onset of senescence, such as the SAG promoter from Arabidopsis (Gan et al., 1995).

[0211] A class of fruit-specific promoters expressed at or during antithesis through fruit development, at least until the beginning of ripening, is discussed in U.S. Pat. No. 4,943,674. cDNA clones that are preferentially expressed in cotton fiber have been isolated (John et a/., 1992). cDNA clones from tomato displaying differential expression during fruit development have been isolated and characterized (Mansson et al. , 1985, Slater et al., 1985). The promoter for polygalacturonase gene is active in fruit ripening. The polygalacturonase gene is described in U.S. Pat. No. 4,535,060, U.S. Pat. No. 4,769,061, U.S. Pat. No. 4,801,590, and U.S. Pat. No. 5, 107,065, which disclosures are incorporated herein by reference.

[0212] Other examples of tissue-specific promoters include those that direct expression in leaf cells following damage to the leaf (for example, from chewing insects), in tubers (for example, patatin gene promoter), and in fiber cells (an example of a developmentally-regulated fiber cell protein is E6 (John et a/., 1992). The E6 gene is most active in fiber, although low levels of transcripts are found in leaf, ovule and flower.

[0213] Examples of other plant promoters reported in the literature include the chloroplast glutamine synthetase GS2 promoter from pea, the chloroplast fructose-1,6- biphosphatase (FBPase) promoter from wheat, the nuclear photosynthetic ST-LS1 promoter from potato, the serine/threonine kinase (PAL) promoter and the glucoamylase (CHS) promoter from Arabidopsis thaliana. Also reported to be active in photosynthetically active tissues are the ribulose-l,5-bisphosphate carboxylase (RbcS) promoter from eastern larch {Larix laricina), the promoter for the cab gene, cab6, from pine, the promoter for the Cab-1 gene from wheat, the promoter for the CAB-1 gene from spinach, the promoter for the cablR gene from rice, the pyruvate, orthophosphate dikinase (PPDK) promoter from corn, the promoter for the tobacco Lhcbl *2 gene, the Arabidopsis thaliana SUC2 sucrose-H+ symporter and the promoter for the thylakoid membrane proteins from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS). Other promoters for the chlorophyll a/b-binding proteins may also be utilised in the invention, such as the promoters for the LhcB gene and PsbP gene from white mustard.

[0214] The tissue-specificity of some "tissue-specific" promoters may not be absolute and may be tested by one skilled in the art using the diphtheria toxin sequence. One can also achieve tissue-specific expression with "leaky" expression by a combination of different tissue- specific promoters (Beals et al., 1997). Other tissue-specific promoters can be isolated by one skilled in the art (see U.S. Pat. No. 5,589,379). Several inducible promoters ("gene switches") have been reported. Many are described in the review by Gatz (1996) and Gatz (1997). These include tetracycline repressor system, Lac repressor system, copper-inducible systems, salicylate-inducible systems (such as the PRla system), glucocorticoid (Aoyama et a/., 1997) and ecdysome-inducible systems. Also included are the benzene sulfonamide (U.S. Pat. No. 5,364,780) and alcohol (WO 97/06269 and WO 97/06268) inducible systems and glutathione S-transferase promoters. Other studies have focused on genes inducibly regulated in response to environmental stress or stimuli such as increased salinity. Drought, pathogen and wounding. (Gra ham et al., 1985; Graham et a/., 1985, Smith et a/., 1986). Accumulation of metallocarboxypeptidase-inhibitor protein has been reported in leaves of wounded potato plants (Graham et a/., 1981). Other plant genes have been reported to be induced methyl jasmonate, elicitors, heat-shock, anaerobic stress, or herbicide safeners.

[0215] In some embodiments, the promoter is selected from a gamma zein promoter, an oleosin olel6 promoter, a globulinl promoter, an actin I promoter, an actin cl promoter, a sucrose synthetase promoter, an INOPS promoter, an EXM5 promoter, a globulinl promoter, a b- 32, ADPG-pyrophosphorylase promoter, an Ltpl promoter, an Ltp2 promoter, an oleosin olel7 promoter, an oleosin olel8 promoter, an actin 2 promoter, a pollen-specific protein promoter, a pollen-specific pectate lyase promoter, an anther-specific protein promoter, an anther-specific gene RTS2 promoter, a pollen-specific gene promoter, a tapeturn-specific gene promoter, tapetum- specific gene RAB24 promoter, a anthranilate synthase alpha subunit promoter, an alpha zein promoter, an anthranilate synthase beta subunit promoter, a dihydrodipicolinate synthase promoter, a Thil promoter, an alcohol dehydrogenase promoter, a cab binding protein promoter, an H3C4 promoter, a RUBISCO SS starch branching enzyme promoter, an ACCase promoter, an actin3 promoter, an actin7 promoter, a regulatory protein GF14-12 promoter, a ribosomal protein L9 promoter, a cellulose biosynthetic enzyme promoter, an S-adenosyl-L-homocysteine hydrolase promoter, a superoxide dismutase promoter, a C-kinase receptor promoter, a phosphoglycerate mutase promoter, a root-specific RCc3 mRNA promoter, a glucose-6 phosphate isomerase promoter, a pyrophosphate-fructose 6-phosphatelphosphotransferase promoter, an ubiquitin promoter, a beta-ketoacyl-ACP synthase promoter, a 33 kDa photosystem 11 promoter, an oxygen evolving protein promoter, a 69 kDa vacuolar ATPase subunit promoter, a metallothionein-like protein promoter, a glyceraldehyde-3-phosphate dehydrogenase promoter, an ABA- and ripening- inducible-like protein promoter, a phenylalanine ammonia lyase promoter, an adenosine triphosphatase S-adenosyl-L-homocysteine hydrolase promoter, an a-tubulin promoter, a cab promoter, a PEPCase promoter, an R gene promoter, a lectin promoter, a light harvesting complex promoter, a heat shock protein promoter, a chalcone synthase promoter, a zein promoter, a globulin-l promoter, an ABA promoter, an auxin-binding protein promoter, a UDP glucose flavonoid glycosyl-transferase gene promoter, an NTI promoter, an actin promoter, an opaque 2 promoter, a b70 promoter, an oleosin promoter, a CaMV 35S promoter, a CaMV 19S promoter, a histone promoter, a turgor-inducible promoter, a pea small subunit uBP carboxylase promoter, a Ti plasmid mannopine synthase promoter, Ti plasmid nopaline synthase promoter, a petunia chalcone isomerase promoter, a bean glycine rich protein I promoter, a CaMV 35S transcript promoter, a potato patatin promoter, or a S-E9 small subunit RuBP carboxylase promoter.

[0216] In some embodiments, the promoter is an alcohol dehydrogenase promoter {e.g., derived from Aspergillus nidulans such as AlcAP).

3.5 Other Regulatory Elements

[0217] In addition to promoters, a variety of 5' and 3' transcriptional regulatory sequences are also available for use in expressing a toxicant or virus impairment nucleic acid sequence of the invention.

3.6 Transcription terminators

[0218] The toxicant or virus impairment nucleic acid sequences of the present invention will typically be operably linked to a 3' non-translated sequence that functions in cells to terminate transcription and/or to cause addition of a polyadenylated nucleotide sequence to the 3' end of the RNA sequence transcribed from the relevant toxicant or virus impairment nucleic acid sequences. Thus, a 3' non-translated sequence refers to that portion of a gene comprising a nucleic acid segment that contains a transcriptional termination signal and/or a polyadenylation signal and any other regulatory signals (e.g., translational termination signals) capable of effecting mRNA processing or gene expression. The polyadenylation signal is characterised by modulating the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. Polyadenylation signals are commonly recognised by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon. The 3' non-translated regulatory sequence desirably includes from about 50 to 1,000 nucleotide base pairs and contains transcriptional and translational termination sequences.

[0219] Exemplary 3' non-translated sequences that are operable in plants include the CaMV35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 terminator, the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato, although other 3' elements known to those of skill in the art can also be employed. Alternatively, one also could use a gamma coixin, oleosin 3 or other terminator from the genus Coix. Exemplary 3' elements include those from the nopaline synthase gene of Agrobacterium tumefaciens (Bevan ef a/., 1983), the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato.

3.7 Untranslated leader sequences

[0220] As the nucleic acid sequence between the transcription initiation site and the start of the coding sequence, i.e., the untranslated leader sequence, can influence gene expression, one may also wish to employ a particular leader sequence. Suitable leader sequences are contemplated to include those which comprise sequences predicted to direct optimum expression of the attached gene, i.e., to include a suitable consensus leader sequence which may increase or maintain mRNA stability and prevent inappropriate initiation of translation. The choice of such sequences will be known to those of skill in the art in light of the present disclosure.

Sequences that are derived from genes that are highly expressed in plants will be most preferred.

3.8 Introns

[0221] Additional sequences that have been found to enhance gene expression in transgenic plants include intron sequences (e.g., from Adhl , bronzel , actinl , actin 2 (WO

00/760067), or the sucrose synthase intron) and viral leader sequences (e.g., from TMV, MCMV and AMV). For example, a number of non-translated leader sequences derived from viruses are known to enhance expression. Specifically, leader sequences from Tobacco Mosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. , Gallie et al. , 1987; Skuzeski et al., 1990). Other leaders known in the art include but are not limited to: Picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5 noncoding region) (Elroy-Stein et al., 1989); Potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) ; MDMV leader (Maize Dwarf Mosaic Virus) ; Human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejak et al., 1991); Untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling et al. , 1987; Tobacco mosaic virus leader (TMV), (Gallie et al., 1989; and Maize Chlorotic Mottle Virus leader (MCMV) (Lommel et al., 1991)). See also, Della-Cioppa et al., 1987.

[0222] Introns for use in the present invention contain the required 3' and 5' splice sites to facilitate splicing at the intron/exon junction and subsequent removal of the intron sequence during transcription. Introns that are recognized and spliced by plant cellular machinery are well known in the art and any such intron of functional fragment can be used in the methods and transgenic plants of the present invention. Exemplary introns for use in the present methods include those from plants, such as the intron from potato light-inducible tissue specific ST-LS1 gene, as well as synthetic plant introns (see e.g. Goodall et al., (1990) Plant Mol Biol. 14(5) : 727- 33). In the constructs of the present invention that comprise a split genes and 3' and 5' regions of an intron, the 3' and 5' regions of an intron can be 3' and 5' regions of a single intron or can be a 3' region of one intron and a 5' region of another intron, providing the 3' and 5' regions contain the necessary splice sites for splicing. Regulatory elements such as Adh intron 1 (Callis et al., 1987), sucrose synthase intron (Vasil et al., 1989) or TMV omega element (Gallie, et al., 1989), may further be included where desired.

3.9 Enhancers

[0223] Examples of enhancers include elements from the CaMV 35S promoter, octopine synthase genes (Ellis et al., 1987), the rice actin I gene, the maize alcohol dehydrogenase gene (Callis et al., 1987), the maize shrunken I gene (Vasil ei al., 1989), TMV Omega element (Gallie et al., 1989) and promoters from non-plant eukaryotes (e.g. yeast; Ma et a/., 1988).

3.10 Other sequences

[0224] Other sequences can be included within or adjacent to the expression cassettes or constructs described herein to promote any one or more of integration of the constructs into the plant genome, selection or screening of transgenic plant cells and/or transgenic plants.

[0225] The expression cassettes or constructs can also be introduced into a vector, such as a plasmid. They can be introduced into the same vector or different vectors. Furthermore, a vector can include two or more of a first component or expression cassette, and/or two or more of a second component or expression cassette, so that the vector comprises two or more copies of the toxicant nucleic acid sequence and/or two or more copies of the virus impairment nucleic acid sequence. Similarly, in embodiments where a third construct is utilised (e.g., to express a rep gene), a vector can include two or more copies of the third component. Plasmid vectors include additional DNA sequences that provide for easy selection, amplification, and transformation of the expression construct in prokaryotic and eukaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors. Additional nucleic acid sequences include origins of replication to provide for autonomous replication of the vector, selectable marker genes, desirably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert nucleic acid sequences or genes.

[0226] Desirably, the vector contains one or more elements that permit stable integration of the construct into the plant cell genome or autonomous replication of the vector in the cell independent of the genome of the cell. In particular embodiments, the vector contains one or more elements so that the construct is stably integrated into the plant cell genome when the vector is introduced into a plant cell. In some examples, the vector contains additional nucleic acid sequences for directing integration by homologous recombination into the genome of the plant cell, which facilitate integration of the construct into the plant cell genome at a precise location in the chromosome. To increase the likelihood of integration at a precise location, the integrational elements should desirably contain a sufficient number of nucleic acids, such as 100 to 1,500 nts, usually 400 to 1,500 nts and more usually 800 to 1,500 nts, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the plant cell. Furthermore, the integrational elements may be non-coding or coding nucleic acid sequences.

[0227] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the plant cell in question.

[0228] To facilitate identification of transformants, a selectable or screenable marker gene is included adjacent to or within the constructs of the present invention. The actual choice of a marker is not crucial as long as it is functional in combination with the plant cell of choice. The marker gene and toxicant nucleic acid sequence (and optionally a rep gene) do not have to be linked, since co-transformation of unlinked genes is also an efficient process in transfection or transformation, especially transformation of plants (see e.g., U.S. Pat. No. 4,399,216).

[0229] Included within the terms selectable or screenable marker genes are genes that encode a "secretabie marker" whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers that encode a secretabie antigen that can be identified by antibody interaction, or secretabie enzymes that can be detected by their catalytic activity. Secretabie proteins include, but are not restricted to, proteins that are inserted or trapped in the cell wall (e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S); small, diffusible proteins detectable, e.g. by ELISA; and small active enzymes detectable in extracellular solution (e.g., -amylase, β-lactamase, phosphinothricin acetyltransferase).

[0230] Exemplary selectable markers for selection of plant transformants include, but are not limited to, a hyg gene which encodes hygromycin B resistance; a neomycin phosphotransferase (neo) gene conferring resistance to kanamycin, paromomycin, G418 and the like; a glutathione-S-transferase gene from rat liver conferring resistance to glutathione derived herbicides; a glutamine synthetase gene conferring, upon expression, resistance to glutamine synthetase inhibitors such as phosphinothricin; an acetyl transferase gene from Streptomyces viridochromogenes conferring resistance to the selective agent phosphinothricin; a gene encoding a 5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance to N- phosphonomethylglycine; a bar gene conferring resistance against bialaphos; a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil; a dihydrofolate reductase (DHF ) gene conferring resistance to methotrexate; a mutant acetolactate synthase gene (ALS), which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals; a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan; or a dalapon dehalogenase gene that confers resistance to the herbicide 2,2-dichloropropionic acid.

[0231] Exemplary screenable markers include, but are not limited to, a uidA gene encoding a β-glucuronidase (GUS) enzyme for which various chromogenic substrates are known; a β-galactosidase gene encoding an enzyme for which chromogenic substrates are known; an aequorin gene which may be employed in calcium-sensitive bioluminescence detection; a green fluorescent protein gene; a luciferase (/i/c) gene, which allows for bioluminescence detection; a β- lactamase gene, which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); an R-locus gene, encoding a product that regulates the production of anthocyanin pigments (red colour) in plant tissues; an a-amylase gene; a tyrosinase gene, which encodes an enzyme capable of oxidizing tyrosine to dopa and dopaquinone which in turn condenses to form the easily detectable compound melanin; or a xylE gene, which encodes a catechol dioxygenase that can convert chromogenic catechols.

4. Methods of producing transgenic plants

[0232] In certain embodiments, the plant is selected from monocotyledons, dicotyledons, and gymnosperms. The plant may be an ornamental plant or crop plant. Illustrative examples of plant cells from ornamental plants include, but are not limited to, plant cells from Malus spp, Crataegus spp, Rosa spp., Betula spp, Sorbus spp, Olea spp, Nerium spp, Salix spp and Populus spp. Illustrative examples of plant cells from crop plants include plant cells from plant species that are cultivated in order to produce a harvestable product such as, but not limited to, Abelmoschus esculentus (okra), Acacia spp., Agave fourcroydes (henequen), Agave sisalana (sisal), Albizia spp., Allium fistulosum (bunching onion), Allium sativum (garlic), Allium spp.

(onions), Alpinia galanga (greater galanga), Amaranthus caudatus, Amaranthus spp., Anacardium spp. (cashew), Ananas comosus (pineapple), Anethum graveolens (dill), Annona cherimola (cherimoya), Apios americana (American potatobean), Arachis hypogaea (peanut), Arctium spp. (burdock), Artemisia spp. (wormwood), Aspalathus linearis (redbush tea), Athertonia diversifolia, Atriplex nummularia (old man saltbush), Averrhoa carambola (starfruit), Azadirachta indica (neem), Backhousia spp., Bambusa spp. (bamboo), Beta vulgaris (sugar beet), Boehmeria nivea (ramie), bok choy, Boronia megastigma (sweet boronia), Brassica carinata (Abyssinian mustard), Brassica juncea (Indian mustard), Brassica napus (rapeseed), Brassica oleracea (cabbage, broccoli), Brassica oleracea var Albogabra (gai lum), Brassica parachinensis (choi sum), Brassica pekensis (Wong bok or Chinese cabbage), Brassica spp., Burcella obovata, Cajanus cajan (pigeon pea), Camellia sinensis (tea), Cannabis sativa (non-drug hemp), Capsicum spp., Carica spp.

(papaya), Carthamus tinctorius (safflower), Carum carvi (caraway), Cassinia spp., Castanospermum australe (blackbean), Casuarina cunninghamiana (beefwood), Ceratonia siliqua (carob), Chamaemelum nobile (chamomile), Chamelaucium spp. (Geraldton wax), Chenopodium quinoa (quinoa), Chrysanthemum (Tanacetum), cinerariifolium (pyrethrum), Cicer arietinum (chickpea), Cichorium intybus (chicory), Clematis spp., Clianthus formosus (Sturt's desert pea), Cocos nucifera (coconut), Coffea spp. (coffee), Colocasia esculenta (taro), Coriandrum sativum (coriander), Crambe abyssinica (crambe), Crocus sativus (saffron), Cucurbita foetidissima (buffalo gourd), Cucurbita spp. (gourd), Cyamopsis tetragonoloba (guar), Cymbopogon spp. (lemongrass), Cytisus proliferus (tagasaste), Daucus carota (carrot), Desmanthus spp., Dioscorea esculenta (Asiatic yam), Dioscorea spp. (yams), Diospyros spp. (persimmon), Doronicum sp., Echinacea spp., Eleocharis dulcis (water chestnut), Eleusine coracana (finger millet), Emanthus arundinaceus, Eragrostis tef (tef), Erianthus arundinaceus, Eriobotrya japonica (loquat), Eucalyptus spp., Eucalyptus spp. (gil mallee), Euclea spp., Eugenia malaccensis (jumba), Euphorbia spp., Euphoria longana (longan), Eutrema wasabi (wasabi), Fagopyrum esculentum (buckwheat), Festuca arundinacea (tall fescue), Ficus spp. (fig), Flacourtia inermis, Flindersia grayliana (Queensland maple), Foeniculum olearia, Foeniculum vulgare (fennel), Garcinia mangostana (mangosteen), Glycine latifolia, Glycine max (soybean). Glycine max (vegetable soybean), Glycyrrhiza glabra (licorice), Gossypium spp. (cottons), Grevillea spp., Grindelia spp., Guizotia abyssinica (niger), Harpagophyllum sp., Helianthus annuus (high oleic sunflowers), Helianthus annuus (monosun sunflowers), Helianthus tuberosus (Jerusalem artichoke), Hibiscus cannabinus (kenaf), Hordeum bulbosum, Hordeum spp. (waxy barley), Hordeum vulgare (barley), Hordeum vulgare subsp.

spontaneum, Humulus lupulus (hops), Hydrastis canadensis (golden seal), Hymenachne spp., Hyssopus officinalis (hyssop), Indigofera spp., Inga edulis (ice cream bean), Inocarpus tugiter, Ipomoea batatas (sweet potato), Ipomoea sp. (kang kong), Lablab purpureus (white lablab), Lactuca spp. (lettuce), Lathyrus spp. (vetch), Lavandula spp. (lavender), Lens spp. (lentil), Lesquerella spp. (bladderpod), Leucaena spp., Lilium spp., Limnanthes spp. (meadowfoam), Linum usitatissimum (flax), Linum usitatissimum (linseed), Linum usitatissimum (Linola.TM.), Litchi chinensis (lychee), Lotus corniculatus (birdsfoot trefoil), Lotus pedunculatus, Lotus sp., Luffa spp., Lunaria annua (honesty), Lupinus mutabilis (pearl lupin), Lupinus spp. (lupin), Macadamia spp., Mangifera indica (mango), Manihot esculenta (cassava), Medicago spp. (lucerne), Medicago spp., Melaleuca spp. (tea tree), Melaleuca uncinata (broombush), Mentha tasmannia, Mentha spicata (spearmint), Mentha X piperita (peppermint), Momordica charantia (bitter melon), Musa spp. (banana), Myrciaria cauliflora (jaboticaba), Myrothamnus flabelli folia, Nephelium lappaceum (rambutan), Nerine spp., Ocimum basilicum (basil), Oenanthe javanica (water dropwort),

Oenothera biennis (evening primrose), Olea europaea (olive), Olearia sp., Origanum spp.

(marjoram, oregano), Oryza spp. (rice), Oxalis tuberosa (oca), Ozothamnus spp. (rice flower), Pachyrrhizus ahipa (yam bean), Panax spp. (ginseng), Panicum miliaceum (common millet), Papaver spp. (poppy;, Parthenium argentatum (guayule), Passiflora sp., Paulownia tomemtosa (princess tree), Pelargonium graveolens (rose geranium), Pelargonium sp., Pennisetum

americanum (bulrush or pearl millet), Persoonia spp., Petroselinum crispum (parsley), Phacelia tanacetifolia (tansy), Phalaris canadensis (canary grass), Phalaris sp., Phaseolus coccineus (scarlet runner bean), Phaseolus lunatus (lima bean), Phaseolus spp., Phaseolus vulgaris (culinary bean),

Phaseolus vulgaris (navy bean), Phaseolus vulgaris (red kidney bean), Pisum sativum (field pea),

Plantago ovata (psyllium), Polygonum minus, Polygonum odoratum, Prunus mume (Japanese apricot), Psidium guajava (guava), Psophocarpus tetragonolobus (winged bean), Pyrus spp.

(nashi), Raphanus satulus (long white radish or Daikon), Rhagodia spp. (saltbush), Ribes nigrum (black currant), Ricinus communis (castor bean), Rosmarinus officinalis (rosemary), Rungia klossii (rungia), Saccharum officinarum (sugar cane), Salvia officinalis (sage), Salvia sclarea (clary sage), Salvia sp., Sandersonia sp., Santalum acuminatum (sweet quandong), Santalum spp.

(sandalwood), Sderocarya caffra (marula), Scutellaria galericulata (scullcap), Secale cereale (rye), Sesamum indicum (sesame), Setaria italica (foxtail millet), Simmondsia spp. (jojoba), Solanum spp., Sorghum almum (sorghum), Stachys betonica (wood betony), Stenanthemum scortechenii, Strychnos cocculoides (monkey orange), Stylosanthes spp. (stylo), Syzygium spp., Tasmannia lanceolata (mountain pepper), Terminalia karnbachii, Theobroma cacao (cocoa), Thymus vulgaris (thyme), Toona australis (red cedar), Trifoliium spp. (clovers), Trifolium alexandrinum (berseem clover), Trifolium resupinatum (persian clover), Triticum spp., Triticum tauschii, Tylosema esculentum (morama bean), Valeriana sp. (valerian), Vernonia spp., Vetiver zizanioides (vetiver grass), Vicia benghalensis (purple vetch), Vicia faba (faba bean), Vicia narbonensis (narbon bean), Vicia sativa, Vicia spp., Vigna aconitifolia (mothbean), Vigna angularis (adzuki bean), Vigna mungo (black gram), Vigna radiata (mung bean), Vigna spp., Vigna unguiculata (cowpea), Vitis spp. (grapes), Voandzeia subterranea (bambarra groundnut), Triticosecale (triticale), Zea mays (bicolour sweetcorn), Zea mays (maize), Zea mays (sweet corn), Zea mays subsp. mexicana (teosinte), Zieria spp., Zingiber officinale (ginger), Zizania spp. (wild rice), Ziziphus jujuba (common jujube). In particular embodiments, the first and second constructs are introduced into Gossypium spp. (cottons), Nicotiana tabacum (tobacco), Ananas comosus (pineapple), Saccharum spp (sugar cane), Musa spp (banana), Lycopersicon esculentum (tomato) and Solanum tuberosum (potato) cell, Manihot spp. (cassava), Zea mays (maize), Triticum spp. (wheat), bean, Capsicum spp. (pepper), Lactuca sativa (lettuce), Carica papaya (papaya), Beta vulgaris (beet), Brassica oleracea convar. capitata (cabbage), Ipomoea batatas (sweet potato) or Fabaceae family

(legumes) plant cells.

[0233] Constructs corresponding to the expression system of the invention may be introduced directly into a desired host or into one or more of its parts (e.g. , cell or tissue types root, leaf, flower, stalk or meristem). Alternatively, the construct may be introduced into a progenitor of the organism and the progenitor is then grown or cultured for a time an under conditions sufficient to produce the organism of interest, whereby the synthetic construct is contained in one or more cell types of that organism. Suitable progenitor cells include bat are not limited to, stem cells such as embryonic stem cell, pluripotent immune cells, meristematic cells and embryonic callus. In certain embodiments, the synthetic construct is introduced into the organism of interest using a particular route of administration (e.g., for plants, administration to flowers, meristem, roots, leaves or stalk). Practitioners in the art will recognise that the route of administration will differ depending on the choice of organism of interest and the sought virus to which resistance is to be conferred. Desirably, the synthetic constructs are introduced into the same of the organism of interest (e.g., autologous cells), or into a cell that is compatible with the organism of interest (e.g., syngeneic or allogeneic cells) and the genetically-modified cell so produced is introduced into the organism of interest at a selected site or into a part of that organism.

[0234] Constructs corresponding to the subject expression system may be introduced into an organism of interest or part thereof using any suitable method, and the kind of method employed will direr depending on the intended cell type, part and/or organism of interest. For example, four general classes of methods for delivering nucleic acid molecules into cells have been described : (1) chemical methods such as calcium phosphate precipitation, polyethylene glycol (PEG)-mediate precipitation and lipofection; (2) physical methods such as microinjection, electroporation, acceleration methods and vacuum infiltration; (3) vector based methods such as bacterial and viral vector- mediated transformation; and (4) receptor-mediated. Transformation techniques that fall within these and other classes are well known to workers in the art, and new techniques are continually becoming known. The particular choice of a transformation technology will be determined by its efficiency to transform certain host species as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce a synthetic construct of the invention into cells is not essential to or a limitation of the invention provided it achieves an acceptable level of nucleic acid transfer. Thus, the constructs can be introduced into tissues or cells by any number of routes, including viral infection, phage infection, microinjection, electroporation, or fusion of vesicles, lipofection, infection by Agrobacterium tumefaciens or A. rhizogenes, or protoplast fusion. Jet injection may also be used for intra- muscular administration (as described for example by Furth et al., 1992 Anal Biochem 205: 265-

368). The synthetic constructs may be coated onto microprojectiles, and delivered into a host plant cell or into tissue by a particle bombardment device, or "gene gun" (see, for example, Tang et al., 1992, Nature 356: 152-154). Alternatively, the constructs can be fed directly to, or injected into, the host organism or it may be introduced into the cell (i.e., intracellular^) or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, etc. Methods for oral introduction include direct mixing of the constructs with food or the organism. In certain embodiments, a hydrodynamic nucleic acid administration protocol in employed (e.g., see Chang et al., 2001, J. Virol. 75: 3469-3473; Liu et al., 1999, Gene Ther. 6: 1258-1266; Wolff et al., 1990, Science 247: 1465-1468; Zhang et al., 1999, Hum Gene. Ther. 10: 1735-1737; and Zhang et al., 1999, Gene Ther. 7: 1344-1349). Other methods of nucleic acid delivery include, but are not limited to, liposome mediated transfer, naked DNA delivery (direct injection) and receptor- mediated transfer (ligand-DNA complex).

[0235] Specific embodiments of the present invention relate to the introduction of expression system components into plant hosts. Guidance in the practical implementation of transformation systems for plant improvement is widely available to those skilled in the art (see, e.g. , Birch (1997) An. Rev. Plant Physiol. Plant Mol. Biol. 48 : 297-326). Non-limiting examples of methods for the transformation of plants include transformation via bacterial mediated nucleic acid delivery (e.g., via Agrobacteria), viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker mediated nucleic acid delivery, liposome mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium-phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEF- mediated nucleic acid uptake, as well as any other electrical, chemical, nucleic acid into the plant cell, including any combination thereof. General guides to various plant transformation methods known in the art include Miki et al., ("Procedures for Introducing Foreign DNA into Plants") in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R., and Thompson J.E. Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska {Cell. Mol. Biol. Lett. 7:849-858 (2002)). [0236] Thus, in some particular embodiments, the introducing into a plant host is via bacterial-mediated transformation, particle bombardment transformation, calcium-phosphate- mediated transformation

[0237] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

EXAMPLES

EXAMPLE 1

ENGINEERING RESISTANCE TO TOBACCO YELLOW DWARF MASTREVIRUS USING INPACT EXPRESSION PLATFORM

[0238] An INPACT cassette was assembled based on the replication machinery of TYDV and capable of expressing the lethal ribonuclease, barnase. This construct was called pINPACT- TYDV-barnase and contains an hph expression cassette in the T-DNA that confers resistance to the antibiotic hygromycin in plants. Importantly, no barnase expression occurs when the INPACT cassette is integrated in the host genome.

[0239] Upon exposure to TYDV Rep mediates replicative release of the INPACT cassette from the host genome. Reconstitution of the barnase expression cassette. Circular INPACT episome are amplified extra-chromosomally, and barnase ribonuclease is produced from processed mRNA resulting in cell death.

[0240] Vector pi NPACT-TYDV- barnase was mobilised into Agrobacterium tumefacies

(strain LBA4404) and used to transform tobacco (Nicotiana tabacum cv. Samsun) by the leaf disk method. At least 15 independent transgenic events were established and of these, two lines (#1-3 and #1-25) were identified as having some resistance to TYDV infection, based on small-scale artificial infectivity trials after ten weeks (results not shown). Using Southern hybridization, line #1-3 was shown to contain a single copy of the INPACT cassette whereas line #1-25 contained two integrated copies (see, Figure 1).

[0241] For large-scale TYDV resistance trials, twenty copies of INPACT tobacco lines #1-3 and 1-25 were prepared from nodal cuttings and acclimatised in the glasshouse. As controls, twenty wild-type (Wt) tobacco plants were included. All plants were challenged with TYDV by syringe infiltration of recombinant Agrobacteria (strain Agll) containing an infectious TYDV l.lmer clone. Following virus challenge, the top leaf from each plant was sampled every month and total DNA extracted using the CTAB protocol. Samples were screened for TYDV using PCR and primers designed to amplify the TYDV movement protein gene.

[0242] As demonstrated in Figure 2, the results show that 12 weeks post-inoculation 16 of 20 (80%) wild-type tobacco plants were infected with TYDV. In comparison, only 3 of 20 plants (15%) representing line #1-3 and 2 of 20 plants (10%) representing line #1-25 were positive for the virus. This result confirms these transgenic lines have some level of resistance/tolerance to TYDV compared to the non-transgenic controls. Only plants testing PCR positive for the virus showed typical symptoms associated with TYDV infection (see, Figure 4).

Materials and Methods

Vector Construction [0243] Construction of vector pINPACT-TYDV-barnase (formerly pINPACT-cytoB) has previously been described (Dugdale et al., In plant activation: an inducible, hyperexpression platform for recombinant protein production in plants, 2013, Plant Cell., 25: 2429-43). Specifically, the full nucleic acid sequence for the pI PACT-TYDV-barnase is as follows:

aattcctcaagtaccgtacgatgaaactctattcttaactagtgagtgagccagtgagccaggtttatgtggcatttatattggtcgg tggaccgttcggatcttgacgcgtggggcacactcacggattttaatattacccgtgagtgctctcttggccccacgcgagcccttta gggcgagcgaaagtgcgccgtagtttcctttagtatttaattcgattatttaatctaactttttattttttattttgttttcttgcagggag ggcaagctgcccggcaagagcggccgcacctggcgcgaggccgacatcaactacaccagcggcttccgcaacagcgaccgcat cctgta ca g ca g eg a ctg g ctg a tcta ca a g a ccaccg a cca eta cca g a ccttca cca a g a tccg ctg a g a gctccg ttca a a c atttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgta ataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaa acaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggctcgagaaaaatgtcgttat tttgatttcatattaatgagcttcagtgtagagaaattcaaatcttattaataaaaacccggaatacaaaaacacacgaaaacgaa aaaaagacaccttacaatcattacacactatataccctcctatgaggagaggcacgttcaggcgcgcctcatggagtcaaagatt caaatagaggacctaacagaactcgccgtaaagactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaa atcttcgtcaacatggtggagcacgacacacttgtctactccaaaaatatcaaagatacagtctcagaagaccaaagggcaattg agacttttcaacaaagggtaatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagatagtggaaa aggaaggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagtggtcccaaag atggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctc cactgacgtaagggatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagagaac acgggggactcttgaccatggcccaggtcatcaacaccttcgacggcgtcgccgactacctgcagacctaccacaagctgcccga caactacatcaccaagagcgaggcccaggccttgggctgggtcgccagcaagggcaacctggccgacgtcgcccccggcaaga gcatcggcggcgacatcttcagcaacaggtaagatttttattttttatttaattaactcaagtaccgtacgatgaaactctattcttaa ctagtgagtgagccagtgagcccggtttatgtggcatttatattggtcggtggaccgttcggatcttgacgcgtggggcacactcac ggattttaatattacccgtgagtgctctcttggccccacgcgagccctttagggcgagcgaaagtgcgccgtagtttcctttagtaa gcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatcccccttt cgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgctagagcag cttgagcttggatcagattgtcgtttcccgccttcagtttaaactatcagtgtttgacaggatatattggcgggtaaacctaagagaa aagagcgtttattagaataacggatatttaaaagggcgtgaaaaggtttatccgttcgtccatttgtatgtgcatgccaaccacagg gttcccctcgggatcaaagtactttgatccaacccctccgctgctatagtgcagtcggcttctgacgttcagtgcagccgtcttctga aaacgacatgtcgcacaagtcctaagttacgcgacaggctgccgccctgcccttttcctggcgttttcttgtcgcgtgttttagtcgca taaagtagaatacttgcgactagaaccggagacattacgccatgaacaagagcgccgccgctggcctgctgggctatgcccgcg tcagcaccgacgaccaggacttgaccaaccaacgggccgaactgcacgcggccggctgcaccaagctgttttccgagaagatca ccggcaccaggcgcgaccgcccggagctggccaggatgcttgaccacctacgccctggcgacgttgtgacagtgaccaggctag accgcctggcccgcagcacccgcgacctactggacattgccgagcgcatccaggaggccggcgcgggcctgcgtagcctggca gagccgtgggccgacaccaccacgccggccggccgcatggtgttgaccgtgttcgccggcattgccgagttcgagcgttccctaa tcatcgaccgcacccggagcgggcgcgaggccgccaaggcccgaggcgtgaagtttggcccccgccctaccctcaccccggcac agatcgcgcacgcccgcgagctgatcgaccaggaaggccgcaccgtgaaagaggcggctgcactgcttggcgtgcatcgctcg accctgtaccgcgcacttgagcgcagcgaggaagtgacgcccaccgaggccaggcggcgcggtgccttccgtgaggacgcatt gaccgaggccgacgccctggcggccgccgagaatgaacgccaagaggaacaagcatgaaaccgcaccaggacggccagga cgaaccgtttttcattaccgaagagatcgaggcggagatgatcgcggccgggtacgtgttcgagccgcccgcgcacgtctcaacc gtgcggctgcatgaaatcctggccggtttgtctgatgccaagctggcggcctggccggccagcttggccgctgaagaaaccgagc gccgccgtctaaaaaggtgatgtgtatttgagtaaaacagcttgcgtcatgcggtcgctgcgtatatgatgcgatgagtaaataaa caaatacgcaaggggaacgcatgaaggttatcgctgtacttaaccagaaaggcgggtcaggcaagacgaccatcgcaacccat ctagcccgcgccctgcaactcgccggggccgatgttctgttagtcgattccgatccccagggcagtgcccgcgattgggcggccgt gcgggaagatcaaccgctaaccgttgtcggcatcgaccgcccgacgattgaccgcgacgtgaaggccatcggccggcgcgactt cgtagtgatcgacggagcgccccaggcggcggacttggctgtgtccgcgatcaaggcagccgacttcgtgctgattccggtgcag ccaagcccttacgacatatgggccaccgccgacctggtggagctggttaagcagcgcattgaggtcacggatggaaggctacaa gcggcctttgtcgtgtcgcgggcgatcaaaggcacgcgcatcggcggtgaggttgccgaggcgctggccgggtacgagctgccc attcttgagtcccgtatcacgcagcgcgtgagctacccaggcactgccgccgccggcacaaccgttcttgaatcagaacccgagg gcgacgctgcccgcgaggtccaggcgctggccgctgaaattaaatcaaaactcatttgagttaatgaggtaaagagaaaatgag caaaagcacaaacacgctaagtgccggccgtccgagcgcacgcagcagcaaggctgcaacgttggccagcctggcagacacg ccagccatgaagcgggtcaactttcagttgccggcggaggatcacaccaagctgaagatgtacgcggtacgccaaggcaagac cattaccgagctgctatctgaatacatcgcgcagctaccagagtaaatgagcaaatgaataaatgagtagatgaattttagcggc taaaggaggcggcatggaaaatcaagaacaaccaggcaccgacgccgtggaatgccccatgtgtggaggaacgggcggttgg ccaggcgtaagcggctgggttgtctgccggccctgcaatggcactggaacccccaagcccgaggaatcggcgtgacggtcgca aaccatccggcccggtacaaatcggcgcggcgctgggtgatgacctggtggagaagttgaaggccgcgcaggccgcccagcg gcaacgcatcgaggcagaagcacgccccggtgaatcgtggcaagcggccgctgatcgaatccgcaaagaatcccggcaaccg ccggcagccggtgcgccgtcgattaggaagccgcccaagggcgacgagcaaccagattttttcgttccgatgctctatgacgtgg gcacccgcgatagtcgcagcatcatggacgtggccgttttccgtctgtcgaagcgtgaccgacgagctggcgaggtgatccgcta cgagcttccagacgggcacgtagaggtttccgcagggccggccggcatggccagtgtgtgggattacgacctggtactgatggc ggtttcccatctaaccgaatccatgaaccgataccgggaagggaagggagacaagcccggccgcgtgttccgtccacacgttgc ggacgtactcaagttctgccggcgagccgatggcggaaagcagaaagacgacctggtagaaacctgcattcggttaaacacca cgcacgttgccatgcagcgtacgaagaaggccaagaacggccgcctggtgacggtatccgagggtgaagccttgattagccgct acaagatcgtaaagagcgaaaccgggcggccggagtacatcgagatcgagctagctgattggatgtaccgcgagatcacaga aggcaagaacccggacgtgctgacggttcaccccgattactttttgatcgatcccggcatcggccgttttctctaccgcctggcacg ccgcgccgcaggcaaggcagaagccagatggttgttcaagacgatctacgaacgcagtggcagcgccggagagttcaagaag ttctgtttcaccgtgcgcaagctgatcgggtcaaatgacctgccggagtacgatttgaaggaggaggcggggcaggctggcccg atcctagtcatgcgctaccgcaacctgatcgagggcgaagcatccgccggttcctaatgtacggagcagatgctagggcaaattg ccctagcaggggaaaaaggtcgaaaaggtctctttcctgtggatagcacgtacattgggaacccaaagccgtacattgggaacc ggaacccgtacattgggaacccaaagccgtacattgggaaccggtcacacatgtaagtgactgatataaaagagaaaaaaggc g a tttttccg ccta a a a ctcttta a a a ctta tta a a a ctctta a aa cccg cctg g cctgtg cata a ctgtctg g cca g eg ca ca g ccg aagagctgcaaaaagcgcctacccttcggtcgctgcgctccctacgccccgccgcttcgcgtcggcctatcgcggccgctggccgc tcaaaaatggctggcctacggccaggcaatctaccagggcgcggacaagccgcgccgtcgccactcgaccgccggcgcccaca tcaaggcaccctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctg taagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagt cacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaa ataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcg gctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgt gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagc atcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccc tcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcac gctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcct tatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcag agcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgc gctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggttttttt gtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaa cgaaaactcacgttaagggattttggtcatgcattctaggtactaaaacaattcatccagtaaaatataatattttattttctcccaat caggcttgatccccagtaagtcaaaaaatagctcgacatactgttcttccccgatatcctccctgatcgaccggacgcagaaggca atgtcataccacttgtccgccctgccgcttctcccaagatcaataaagccacttactttgccatctttcacaaagatgttgctgtctccc aggtcgccgtgggaaaagacaagttcctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctttaaatggagtg tcttcttcccagttttcgcaatccacatcggccagatcgttattcagtaagtaatccaattcggctaagcggctgtctaagctattcgt atagggacaatccgatatgtcgatggagtgaaagagcctgatgcactccgcatacagctcgataatcttttcagggctttgttcatc ttcatactcttccgagcaaaggacgccatcggcctcactcatgagcagattgctccagccatcatgccgttcaaagtgcaggacctt tggaacaggcagctttccttccagccatagcatcatgtccttttcccgttccacatcataggtggtccctttataccggctgtccgtcat ttttaaatataggttttcattttctcccaccagcttatataccttagcaggagacattccttccgtatcttttacgcagcggtatttttcga tcagttttttcaattccggtgatattctcattttagccatttattatttccttcctcttttctacagtatttaaagataccccaagaagctaa ttataacaagacgaactccaattcactgttccttgcattctaaaaccttaaataccagaaaacagctttttcaaagttgttttcaaagt tggcgtataacatagtatcgacggagccgattttgaaaccgcggtgatcacaggcagcaacgctctgtcatcgttacaatcaacat gctaccctccgcgagatcatccgtgtttcaaacccggcagcttagttgccgttcttccgaatagcatcggtaacatgagcaaagtct gccgccttacaacggctctcccgctgacgccgtcccggactgatgggctgcctgtatcgagtggtgattttgtgccgagctgccggt cggggagctgttggctggctggtggcaggatatattgtggtgtaaacaaattgacgcttagacaacttaataacacattgcggac gtttttaatgtactgaattaacgccgaattaattcgggggatctggattttagtactggattttggttttaggaattagaaattttattg atagaagtattttacaaatacaaatacatactaagggtttcttatatgctcaacacatgagcgaaaccctataggaaccctaattcc cttatctgggaactactcacacattattatggagaaactcgagcttgtcgatcgacagatccggtcggcatctactctatttctttgcc ctcggacgagtgctggggcgtcggtttccactatcggcgagtacttctacacagccatcggtccagacggccgcgcttctgcgggc gatttgtgtacgcccgacagtcccggctccggatcggacgattgcgtcgcatcgaccctgcgcccaagctgcatcatcgaaattgc cgtcaaccaagctctgatagagttggtcaagaccaatgcggagcatatacgcccggagtcgtggcgatcctgcaagctccggat gcctccgctcgaagtagcgcgtctgctgctccatacaagccaaccacggcctccagaagaagatgttggcgacctcgtattggga atccccgaacatcgcctcgctccagtcaatgaccgctgttatgcggccattgtccgtcaggacattgttggagccgaaatccgcgtg cacgaggtgccggacttcggggcagtcctcggcccaaagcatcagctcatcgagagcctgcgcgacggacgcactgacggtgtc gtccatcacagtttgccagtgatacacatggggatcagcaatcgcgcatatgaaatcacgccatgtagtgtattgaccgattccttg cggtccgaatgggccgaacccgctcgtctggctaagatcggccgcagcgatcgcatccatagcctccgcgaccggttgtagaaca gcgggcagttcggtttcaggcaggtcttgcaacgtgacaccctgtgcacggcgggagatgcaataggtcaggctctcgctaaact ccccaatgtcaagcacttccggaatcgggagcgcggccgatgcaaagtgccgataaacataacgatctttgtagaaaccatcgg cgcagctatttacccgcaggacatatccacgccctcctacatcgaagctgaaagcacgagattcttcgccctccgagagctgcatc aggtcggagacgctgtcgaacttttcgatcagaaacttctcgacagacgtcgcggtgagttcaggctttttcatatctcattgccccc cgggatctgcgaaagctcgagagagatagatttgtagagagagactggtgatttcagcgtgtcctctccaaatgaaatgaacttc cttatatagaggaaggtcttgcgaaggatagtgggattgtgcgtcatcccttacgtcagtggagatatcacatcaatccacttgcttt gaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatctt gaacgatagcctttcctttatcgcaatgatggcatttgtaggtgccaccttccttttctactgtccttttgatgaagtgacagatagctg ggcaatggaatccgaggaggtttcccgatattaccctttgttgaaaagtctcaatagccctttggtcttctgagactgtatctttgata ttcttggagtagacgagagtgtcgtgctccaccatgttatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttcc acgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttgaacgatagcctttcctttatcgcaatga tggcatttgtaggtgccaccttccttttctactgtccttttgatgaagtgacagatagctgggcaatggaatccgaggaggtttcccg atattaccctttgttgaaaagtctcaatagccctttggtcttctgagactgtatctttgatattcttggagtagacgagagtgtcgtgct ccaccatgttggcaagctgctctagccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgaca ggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacacttt atgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacg [SEQ

ID NO: 173].

[0244] TYDV nucleic acid (GenBank accession number M81103.1) was isolated from infected plant material using the CTAB method. The TYDV genome was PCR amplified in two fragments; (i) partial genome from nucleotides 1872 to 268 using primers TY-F1 (5'- ggatcccctccctctacatctgcggacc-3') and TY-R1 (5'- aagctttttcaactttgggatgtcacatcatc-3') and (ii) complete genome from nucleotide 1870 using primers TY-F1 and TY-R2 (5'- ggatcctggttccagcccttctaggttcctgga-3'). Thermocycling conditions were as above. PCR products were ligated into pGEM-T. Easy vector, cloned and sequenced. Fragment 1 was ligated into pBIN-Plus vector backbone using Hindlll and BamHI restriction sites. Fragment 2 was then ligated in using the unique BamHI restriction site. The resulting construct contained a greater-than-genome-length version of the TYDV genome (l. lmer) and was called pBIN-TYDV-l. lmer.

Tobacco Transformation

[0245] pINPACT-TYDV-barnase vector was mobilised into Agrobacterium tumefaciens (strain LBA4404) and used to transform wild-type tobacco {Nicotiana tabacum cv. Samsun) by the leaf disk method. Plants transformed with pI PACT-TYDV-barnase were selected in media containing hygromycin. Tissue culture plants were soil acclimated and transferred to either a glasshouse or growth cabinets with a 16 hour photoperiod and constant temperature of 27°C. Plants were grown until the 10 to 12 leaf stage prior to virus challenge.

Virus Challenge

[0246] Vector pBIN-TYDV-l. lmer was mobilised into Agrobacterium tumefaciens (strain Agll or GV3101). Recombinant Agrobacteria were grown to an optical density at 600 nm (OD600) of 1.0 in LB broth and then prepared for infiltration in MMA media (10 mM MES (2-[N- morpholinojethanesulfonic acid), 10 mM magnesium chloride, 100 μΜ acetosyringone) using the method of Sainsbury et al. (2009). Bacteria were infiltrated into 4 zones on the underside of 2 leaves using a needleless 1.0 ml_ syringe. Also, approximately 100 μΙ_ of bacteria was directly injected into the crown of the plant with a needle and syringe.

Screening transgenic tobacco lines following virus challenge

[0247] Approximately 100 mg of leaf material was sampled from the top leaves of tobacco plants and immediately snap frozen in liquid nitrogen. Total DNA was isolated using the CTAB method. For detection of TYDV MP gene, primers MPs-F and MPs-R were used. For detection of TYDV Rep gene primers CP-F (5'-atggcgggccggtataagggtttgg-3') and CP-R (5'- ttattgattgccaactgatttgaaatac-3') was used. Thermocycling conditions were as follows: 95 °C for 5 min, followed by 25 cycles of 95 °C for 30 sec, 50 °C for 30 sec, 68 °C for 30 sec with a final extension step of 68 °C for 10 min. PCR products were electro phoresed through a 1.5% agarose gel and visualised using SYBR-Safe DNA gel stain.

EXAMPLE 2

PARTIAL RESISTANCE CONFERRED BY SIRNA STRATEGY ALONE

[0248] A siRNA expression cassette targeting the TYDV movement protein (MP) gene was assembled in pBIN-Plus vector. This cassette contained a 300 bp region of the TYDV MP in both sense and antisense orientations spaced by a small synthetic intron and placed under the transcriptional control of the CaMV 35S promoter and nos terminator. This construct was called pBIN-MP.hp and contained an nptll expression cassette in the T-DNA that confers resistance to the antibiotic kanamycin in plants.

[0249] Vector pBIN-MP.hp was mobilised into Agrobacterium tumefaciens (strain

LBA4404) and used to transform wildtype tobacco (Nicotiana tabacum cv. Samsun) and super- transform elite INPACT parent line #1-3 by the leaf disk method. At least ten independent transgenic events were established for each transformation.

[0250] Five copies of each transgenic line were established by nodal cuttings and acclimatised in a climate controlled growth cabinet. As controls, five wildtype tobacco plants were included. Plants were challenged with TYDV by syringe infiltration of recombinant Agrobacteria (strain GV3101) harbouring an infectious TYDV l. lmer clone. Two weeks later, Agroinfiltration of the TYDV infectious clone was repeated for each plant. Following the second virus challenge, the top leaf from each plant was sampled every three weeks and total DNA extracted using the CTAB protocol. Total DNA extracts were screened for TYDV using PCR and primers designed to amplify the TYDV coat protein gene. PCR results over a 6 week period are displayed below.

[0251] PCR results confirmed 80% of wild-type tobacco plants were infected with TYDV, 6 weeks post-inoculation. Infection rates in transgenic tobacco lines (1-10) transformed with pBIN- MP.hp ranged from 20-100% (see, Figure 4). This result suggests that expression of a RNA hairpin targeting the TYDV MP gene does not confer complete resistance to TYDV infection and spread. Those transgenic plants that tested positive for TYDV by PCR also displayed typical TYDV symptoms.

Materials and Methods

pBIN.MP. hp Vector Construction

[0252] A hairpin cassette targeting a ~300 bp region of the TYDV movement protein (MP) gene (nts 269 to 574, GenBank Accession M81103.1) was assembled in pBIN-Plus vector backbone. Sense and antisense MP sequences were PCR amplified from cloned components using the following primer pairs MPas-F (5'-gcgatcgccatggaccggcccgccattagggtttccttc-3) and MPas-R (5'- agatctatgtatcccgccaaataccaagtgg-3'), MPs-F (5'-ggtaccatgtatcccgccaaataccaagtggtc-3') and MPs-R (5'-gagctctaccggcccgccattagggtttcc-3'), respectively. Thermocycling conditions were 95°C for 2 min followed by 20 cycles of 95°C for 30 s, 50°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 5 min. PCR products were ligated into pGEM-T.Easy vector, cloned and sequenced. Sense and antisense MP sequences were inserted between a CaMV 35S promoter and nos terminator and spaced using a synthetic intron (syntron; Dugdale et al., 2013) loop sequence. The MP antisense sequence was inserted using AsiSI and Bg III restriction sites and the downstream sense MP sequence inserted using Kpnl and Sad restriction sites. The resulting TYDV MP hairpin construct was called pBIN-MP.hp, and has the following nucleic acid sequence:

ccgggctggttgccctcgccgctgggctggcggccgtctatggccctgcaaacgcgccagaaacgccgtcgaagccgtgtgcga gacaccgcggccgccggcgttgtggatacctcgcggaaaacttggccctcactgacagatgaggggcggacgttgacacttgag gggccgactcacccggcgcggcgttgacagatgaggggcaggctcgatttcggccggcgacgtggagctggccagcctcgcaa atcggcgaaaacgcctgattttacgcgagtttcccacagatgatgtggacaagcctggggataagtgccctgcggtattgacactt gaggggcgcgactactgacagatgaggggcgcgatccttgacacttgaggggcagagtgctgacagatgaggggcgcaccta ttgacatttgaggggctgtccacaggcagaaaatccagcatttgcaagggtttccgcccgtttttcggccaccgctaacctgtctttt aacctgcttttaaaccaatatttataaaccttgtttttaaccagggctgcgccctgtgcgcgtgaccgcgcacgccgaaggggggtg cccccccttctcg a a ccctcccg g cccg eta a eg eg gg cctccca tcccccca g gg g ctg eg cccctcg g ccg eg a a egg cctca c cccaaaaatggcagcgctggcagtccttgccattgccgggatcggggcagtaacgggatgggcgatcagcccgagcgcgacgc ccggaagcattgacgtgccgcaggtgctggcatcgacattcagcgaccaggtgccgggcagtgagggcggcggcctgggtggc ggcctgcccttcacttcggccgtcggggcattcacggacttcatggcggggccggcaatttttaccttgggcattcttggcatagtg gtcgcgggtgccgtgctcgtgttcgggggtgcgataaacccagcgaaccatttgaggtgataggtaagattataccgaggtatga aaacgagaattggacctttacagaattactctatgaagcgccatatttaaaaagctaccaagacgaagaggatgaagaggatga ggaggcagattgccttgaatatattgacaatactgataagataatatatcttttatatagaagatatcgccgtatgtaaggatttca gggggcaaggcataggcagcgcgcttatcaatatatctatagaatgggcaaagcataaaaacttgcatggactaatgcttgaaa cccaggacaataaccttatagcttgtaaattctatcataattgggtaatgactccaacttattgatagtgttttatgttcagataatgc ccgatgactttgtcatgcagctccaccgattttgagaacgacagcgacttccgtcccagccgtgccaggtgctgcctcagattcagg ttatgccgctcaattcgctgcgtatatcgcttgctgattacgtgcagctttcccttcaggcgggattcatacagcggccagccatccgt catccatatcaccacgtcaaagggtgacagcaggctcataagacgccccagcgtcgccatagtgcgttcaccgaatacgtgcgca acaaccgtcttccggagactgtcatacgcgtaaaacagccagcgctggcgcgatttagccccgacatagccccactgttcgtccat ttccgcgcagacgatgacgtcactgcccggctgtatgcgcgaggttactatgcggtgtgaaataccgcacagatgcgtaaggag aaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctca ctcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcc aggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtc agaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctg ccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgta ggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtc caacccggtaagacacgacttatcgccactggcagcaggttaccgactgcggcctgagttttttaagtgacgtaaaatcgtgttga ggccaacgcccataatgcgggctgttgcccggcatccaacgccattcatggccatatcaatgattttctggtgcgtaccgggttga gaagcggtgtaagtgaactgcagttgccatgttttacggcagtgagagcagagatagcgctgatgtccggcggtgcttttgccgtt acgcaccaccccgtcagtagctgaacaggagggacagctgatagacacagaagccactggagcacctcaaaaacaccatcata cactaaatcagtaagttggcagcatcacccataattgtggtttcaaaatcggctccgtcgatactatgttatacgccaactttgaaaa caactttgaaaaagctgttttctggtatttaaggttttagaatgcaaggaacagtgaattggagttcgtcttgttataattagcttctt ggggtatctttaaatactgtagaaaagaggaaggaaataataaatggctaaaatgagaatatcaccggaattgaaaaaactgat cgaaaaataccgctgcgtaaaagatacggaaggaatgtctcctgctaaggtatataagctggtgggagaaaatgaaaacctata tttaaaaatgacggacagccggtataaagggaccacctatgatgtggaacgggaaaaggacatgatgctatggctggaaggaa agctgcctgttccaaaggtcctgcactttgaacggcatgatggctggagcaatctgctcatgagtgaggccgatggcgtcctttgct cggaagagtatgaagatgaacaaagccctgaaaagattatcgagctgtatgcggagtgcatcaggctctttcactccatcgacat atcggattgtccctatacgaatagcttagacagccgcttagccgaattggattacttactgaataacgatctggccgatgtggattg cgaaaactgggaagaagacactccatttaaagatccgcgcgagctgtatgattttttaaagacggaaaagcccgaagaggaact tgtcttttcccacggcgacctgggagacagcaacatctttgtgaaagatggcaaagtaagtggctttattgatcttgggagaagcg gcagggcggacaagtggtatgacattgccttctgcgtccggtcgatcagggaggatatcggggaagaacagtatgtcgagctat tttttgacttactggggatcaagcctgattgggagaaaataaaatattatattttactggatgaattgttttagtacctagatgtggcg caacgatgccggcgacaagcaggagcgcaccgacttcttccgcatcaagtgttttggctctcaggccgaggcccacggcaagtat ttgggcaaggggtcgctggtattcgtgcagggcaagattcggaataccaagtacgagaaggacggccagacggtctacgggac cgacttcattgccgataaggtggattatctggacaccaaggcaccaggcgggtcaaatcaggaataagggcacattgccccggc gtgagtcggggcaatcccgcaaggagggtgaatgaatcggacgtttgaccggaaggcatacaggcaagaactgatcgacgcg gggttttccgccgaggatgccgaaaccatcgcaagccgcaccgtcatgcgtgcgccccgcgaaaccttccagtccgtcggctcga tggtccagcaagctacggccaagatcgagcgcgacagcgtgcaactggctccccctgccctgcccgcgccatcggccgccgtgg agcgttcgcgtcgtctcgaacaggaggcggcaggtttggcgaagtcgatgaccatcgacacgcgaggaactatgacgaccaag aagcgaaaaaccgccggcgaggacctggcaaaacaggtcagcgaggccaagcaggccgcgttgctgaaacacacgaagcag cagatcaaggaaatgcagctttccttgttcgatattgcgccgtggccggacacgatgcgagcgatgccaaacgacacggcccgct ctgccctgttcaccacgcgcaacaagaaaatcccgcgcgaggcgctgcaaaacaaggtcattttccacgtcaacaaggacgtga agatcacctacaccggcgtcgagctgcgggccgacgatgacgaactggtgtggcagcaggtgttggagtacgcgaagcgcacc cctatcggcgagccgatcaccttcacgttctacgagctttgccaggacctgggctggtcgatcaatggccggtattacacgaaggc cgaggaatgcctgtcgcgcctacaggcgacggcgatgggcttcacgtccgaccgcgttgggcacctggaatcggtgtcgctgctg caccgcttccgcgtcctggaccgtggcaagaaaacgtcccgttgccaggtcctgatcgacgaggaaatcgtcgtgctgtttgctgg cgaccactacacgaaattcatatgggagaagtaccgcaagctgtcgccgacggcccgacggatgttcgactatttcagctcgcac cgggagccgtacccgctcaagctggaaaccttccgcctcatgtgcggatcggattccacccgcgtgaagaagtggcgcgagcag gtcggcgaagcctgcgaagagttgcgaggcagcggcctggtggaacacgcctgggtcaatgatgacctggtgcattgcaaacg ctagggccttgtggggtcagttccggctgggggttcagcagccagcgctttactggcatttcaggaacaagcgggcactgctcga cgcacttgcttcgctcagtatcgctcgggacgcacggcgcgctctacgaactgccgataaacagaggattaaaattgacaattgtg attaaggctcagattcgacggcttggagcggccgacgtgcaggatttccgcgagatccgattgtcggccctgaagaaagctccag agatgttcgggtccgtttacgagcacgaggagaaaaagcccatggaggcgttcgctgaacggttgcgagatgccgtggcattcg gcgcctacatcgacggcgagatcattgggctgtcggtcttcaaacaggaggacggccccaaggacgctcacaaggcgcatctgt ccggcgttttcgtggagcccgaacagcgaggccgaggggtcgccggtatgctgctgcgggcgttgccggcgggtttattgctcgt gatgatcgtccgacagattccaacgggaatctggtggatgcgcatcttcatcctcggcgcacttaatatttcgctattctggagcttg ttgtttatttcggtctaccgcctgccgggcggggtcgcggcgacggtaggcgctgtgcagccgctgatggtcgtgttcatctctgcc gctctgctaggtagcccgatacgattgatggcggtcctgggggctatttgcggaactgcgggcgtggcgctgttggtgttgacacc aaacgcagcgctagatcctgtcggcgtcgcagcgggcctggcgggggcggtttccatggcgttcggaaccgtgctgacccgcaa gtggcaacctcccgtgcctctgctcacctttaccgcctggcaactggcggccggaggacttctgctcgttccagtagctttagtgttt gatccgccaatcccgatgcctacaggaaccaatgttctcggcctggcgtggctcggcctgatcggagcgggtttaacctacttcctt tggttccgggggatctcgcgactcgaacctacagttgtttccttactgggctttctcagccccagatctggggtcgatcagccgggg atgcatcaggccgacagtcggaacttcgggtccccgacctgtaccattcggtgagcaatggataggggagttgatatcgtcaacg ttcacttctaaagaaatagcgccactcagcttcctcagcggctttatccagcgatttcctattatgtcggcatagttctcaagatcgac agcctgtcacggttaagcgagaaatgaataagaaggctgataattcggatctctgcgagggagatgatatttgatcacaggcag caacgctctgtcatcgttacaatcaacatgctaccctccgcgagatcatccgtgtttcaaacccggcagcttagttgccgttcttccg aatagcatcggtaacatgagcaaagtctgccgccttacaacggctctcccgctgacgccgtcccggactgatgggctgcctgtatc gagtggtgattttgtgccgagctgccggtcggggagctgttggctggctggtggcaggatatattgtggtgtaaacaaattgacgc ttagacaacttaataacacattgcggacgtttttaatgtactggggtggtttttcttttcaccagtgagacgggcaacagctgattgc ccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggtt ccgaaatcggcaaaatcccttataaatcaaaagaatagcccgagatagggttgagtgttgttccagtttggaacaagagtccact attaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccacaaactgaaggcgggaaacga caatctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcgggacaagccgttttacgtttggaact gacagaaccgcaacgttgaaggagccactcagccgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattatt gcgcgttcaaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgttccataaattcccctcg gtatccaattagagtctcatattcactctcaatccaaataatctgcaccggatctggatcgtttcgcatgattgaacaagatggattg cacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccg tgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggca gcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgc tattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcgg cggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaag ccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgca tgcccgacggcgatgatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattca tcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcga atgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctg agcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatg aaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccac gggatctctgcggaacaggcggtcgaaggtgccgatatcattacgacagcaacggccgacaagcacaacgccacgatcctgag cgacaatatgatcgggcccggcgtccacatcaacggcgtcggcggcgactgcccaggcaagaccgagatgcaccgcgatatctt gctgcgttcggatattttcgtggagttcccgccacagacccggatgatccccgatcgttcaaacatttggcaataaagtttcttaaga ttgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacg ttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactag gataaattatcgcgcgcggtgtcatctatgttactagatcgggcctcctgtcaatgctggcggcggctctggtggtggttctggtgg cggctctgagggtggtggctctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctctggttcc ggtgattttgattatgaaaagatggcaaacgctaataagggggctatgaccgaaaatgccgatgaaaacgcgctacagtctgac gctaaaggcaaacttgattctgtcgctactgattacggtgctgctatcgatggtttcattggtgacgtttccggccttgctaatggtaa tggtgctactggtgattttgctggctctaattcccaaatggctcaagtcggtgacggtgataattcacctttaatgaataatttccgtc aatatttaccttccctccctcaatcggttgaatgtcgcccttttgtctttggcccaatacgcaaaccgcctctccccgcgcgttggccga ttcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcat taggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagc tatgaccatgattacgccaagctggcgcgcctcatggagtcaaagattcaaatagaggacctaacagaactcgccgtaaagact ggcgaacagttcatacagagtctcttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacacacttgtct actccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcct cggattccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggctcctacaaatgccatcattgcgataa aggaaaggccatcgttgaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaag aagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactatcct tcgcaagacccttcctctatataaggaagttcatttcatttggagagaacacgggggactcttgagcgatcgccatggaccggccc gccattagggtttccttcaccaggtcttctgagaggggtattaccaaaccctatctcagttgtcgtcttctgcttctttgctttcaaaag aaggatcaaatccttcaagaataacgtatacgctaagtatacgatacctacagcaaaaaggataacaatcaaagcaacaacaac tttcgaaaagaagtgctccgatgactcacccgctttctgcggctgatattgctcaaaagtaccaacaggggtatctgagtaatttat accagatgggaccacttggtatttggcgggatacatagatctaggtaagatttttattttttatttaattaagaattggcgcgccattt aaatctaactttttattttttattttgttttcttgcaggtaccatgtatcccgccaaataccaagtggtcccatctggtataaattactcag atacccctgttggtacttttgagcaatatcagccgcagaaagcgggtgagtcatcggagcacttcttttcgaaagttgttgttgcttt gattgttatcctttttgctgtaggtatcgtatacttagcgtatacgttattcttgaaggatttgatccttcttttgaaagcaaagaagca gaagacgacaactgagatagggtttggtaatacccctctcagaagacctggtgaaggaaaccctaatggcgggccggtagagc tccgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgt taagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacg cgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggctcgaggaat tcttaattaacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcaca tccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgcccgct cctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttag tgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacaaactatcagtgtttgacaggatatattggcgggtaa acctaagagaaaagagcgtttattagaataatcggatatttaaaagggcgtgaaaaggtttatccgttcgtccatttgtatgtgcat gccaaccacagggttccccagatctggcgccggccagcgagacgagcaagattggccgccgcccgaaacgatccgacagcgc gcccagcacaggtgcgcaggcaaattgcaccaacgcatacagcgccagcagaatgccatagtgggcggtgacgtcgttcgagt gaaccagatcgcgcaggaggcccggcagcaccggcataatcaggccgatgccgacagcgtcgagcgcgacagtgctcagaat tacgatcaggggtatgttgggtttcacgtctggcctccggaccagcctccgctggtccgattgaacgcgcggattctttatcactgat aagttggtggacatattatgtttatcagtgataaagtgtcaagcatgacaaagttgcagccgaatacagtgatccgtgccgccctg gacctgttgaacgaggtcggcgtagacggtctgacgacacgcaaactggcggaacggttgggggttcagcagccggcgctttac tggcacttcaggaacaagcgggcgctgctcgacgcactggccgaagccatgctggcggagaatcatacgcattcggtgccgag agccgacgacgactggcgctcatttctgatcgggaatgcccgcagcttcaggcaggcgctgctcgcctaccgcgatggcgcgcgc atccatgccggcacgcgaccgggcgcaccgcagatggaaacggccgacgcgcagcttcgcttcctctgcgaggcgggtttttcg gccggggacgccgtcaatgcgctgatgacaatcagctacttcactgttggggccgtgcttgaggagcaggccggcgacagcgat gccggcgagcgcggcggcaccgttgaacaggctccgctctcgccgctgttgcgggccgcgatagacgccttcgacgaagccggt ccggacgcagcgttcgagcagggactcgcggtgattgtcgatggattggcgaaaaggaggctcgttgtcaggaacgttgaagg accgagaaagggtgacgattgatcaggaccgctgccggagcgcaacccactcactacagcagagccatgtagacaacatcccc tccccctttccaccgcgtcagacgcccgtagcagcccgctacgggctttttcatgccctgccctagcgtccaagcctcacggccgcg ctcggcctctctggcggccttctggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtat cagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagca aaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacg ctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttc cgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgcttttccgctgcataaccctgcttcggggtcat tatagcgattttttcggtatatccatcctttttcgcacgatatacaggattttgccaaagggttcgtgtagactttccttggtgtatccaa cggcgtcagccgggcaggataggtgaagtaggcccacccgcgagcgggtgttccttcttcactgtcccttattcgcacctggcggt gctcaacgggaatcctgctctgcgaggctggccggctaccgccggcgtaacagatgagggcaagcggatggctgatgaaacca agccaaccaggaagggcagcccacctatcaaggtgtactgccttccagacgaacgaagagcgattgaggaaaaggcggcggc ggccggcatgagcctgtcggcctacctgctggccgtcggccagggctacaaaatcacgggcgtcgtggactatgagcacgtccg cgagctggcccgcatcaatggcgacctgggccgcctgggcggcctgctgaaactctggctcaccgacgacccgcgcacggcgcg gttcggtgatgccacgatcctcgccctgctggcgaagatcgaagagaagcaggacgagcttggcaaggtcatgatgggcgtggt ccgcccgagggcagagccatgacttttttagccgctaaaacggccggggggtgcgcgtgattgccaagcacgtccccatgcgctc catcaagaagagcgacttcgcggagctggtgaagtacatcaccgacgagcaaggcaagaccgagcgcctttgcgacgctca

[SEQ ID N0: 174].

Tobacco Transformation with pBIN-MP.hp

[0253] pBIN-MP. hp was transformed into wild-type tobacco using the method described in Example 1. Plants transformed with pBIN-MP. hp were selected with kanamycin. Tissue culture plants were soil acclimated and transferred to either a glasshouse or growth cabinets with a 16 hour photoperiod and constant temperature of 27°C. Plants were grown until the 10 to 12 leaf stage prior to virus challenge.

EXAMPLE 3

GENERATION OF RESISTANT PLANT LINES

[0254] A siRNA expression cassette targeting the TYDV movement protein (MP) was assembled in pBIN-Plus. This cassette contained a 300 bp region of the TYDV MP in both sense and antisense orientations spaced by a small synthetic intron and placed under the transcriptional control of the CaMV 35S promoter and nos terminator. THs construct was called pBIN MP. hp and contained an nptll expression cassette in the T-DNA that confers resistance to the antibiotic kanamycin in plants. Vector pBIN-MP.jp was mobilised into Agrobacterium tumefaciens (strain LBA4404) and used to transform super-transform elite INPACT tobacco {Nicotiana tabacum cv. Samsun) parent line #1-3 by the leaf disk method. At least ten independent transgenic events were established for each transformation.

[0255] Again, five copies of teach transgenic cell line were established by nodal cuttings and acclimatised in a climate controlled growth cabinet. As controls, five wild-type tobacco plants were included. Plants were challenged with TYDV by syringe infiltration of recombinant

Agrobacteria (strain GV3101) harbouring an infectious TYDV l. lmer clone. Two weeks later Agroinfiltration of the TYDV infectious clone was repeated for each plant. Following the second virus challenge, the top leaf from each plant was sampled every three weeks and total DNA extracted using the CTAB protocol. Total DNA extracts were screened for TYDV using PCR and primers designed to amplify the TYDV coat protein gene. PC results over a 6-week period are displayed in Figure 5.

[0256] The PCR results shown in Figure 5 confirmed 100% of wild-type tobacco plants and 40% of the elite INPACT plants (#1-3) were infected with TYDV, 6 weeks post-inoculation. Infection rages in the tobacco lines (shown as 1 to 11 in Figure 5) representing elite INPACT plant lines (#1-3) super-transformed with pBIN-MP. hp ranged from 0-100%. Importantly, three plant lines (2, 3 and 5) tested negative for TYDV by PCR, 6 weeks post inoculation, suggesting a complete absence of the virus. These plants also remained symptom free. This result suggests that the expression of a RNA hairpin targeting the TYDV MP gene acts to enhance virus resistance conferred by an INPACT cassette alone.

Materials and Methods

Super-Transformation of elite INPACT cell lines

[0257] Agrobacterium tumefacies (strain LBA4404) harbouring pBIN-MP. hp was used to super-transform elite INPACT tobacco line (# 1-3) using the same methodology as described above.

EXAMPLE 4

GENERATION OF ADDITIONAL RESISTANT PLANT LINES

[0258] In order to generate plant lines that have resistance to other viruses (and to provide alternative plant lines resistant to TYDV), additional expression systems were designed in accordance with the methods described above. A list of expression systems generated include those listed in Table 8.

Table 8: Exemplary expression systems and viral targets

[0259] As described above, the genes of some virus groups (and the polypeptides that they encode), perform more than one virus function. For example, the banana bunch top babuvirus movement protein gene is also a silencing suppressor gene.

SUMMARY OF EXAMPLES

[0260] The inventors have shown an INPACT-based transgenic resistance strategy alone can provide significantly higher levels of resistance or tolerance to TYDV infection in tobacco plants but cannot provide complete resistance.

[0261] Two elite INPACT tobacco plant lines were identified that showed significantly lower TYDV infection rates compared to wild-type controls (i.e., 10-15% infections compared to 80% infection, respectively. A siRNA resistance strategy using a hairpin construct targeting the TYDV MP gene as a representative VVI, reduced infection rates in some plant lines but failed to provide complete resistance to the virus.

[0262] Combining both INPACT and siRNA resistance strategies produced several plant lines that were symptom free, six weeks post-inoculation. Considering the INPACT resistance strategy relies on Rep-mediated activation and expression of a lethal gene product to kill the cell in which the virus enters/ replicates, we hypothesize that siRNA targeting of an essential virus encoded gene, may act to delay the virus life-cycle or infection process (and thus inhibit the viability of cells infected with a virus). It is therefore the combination of virus viability impairment and rapid cell death, provided by both systems, that may provide the higher levels of resistance in plants that is observed when both INPACT and siRNA are used in combination.

[0263] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0264] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0265] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An expression system for conferring virus resistance to a plant, the expression system comprising a first expression system component (e.g., comprising at least one expression cassette or construct) and a second expression system component (e.g., comprising at least one expression cassette or construct); wherein an expression cassette of the first expression system component comprises a toxicant nucleic acid sequence encoding a toxicity protein operabiy connected to at least one transcriptional control sequence (e.g., a promoter, transcription terminator, c/s-acting sequence, etc.); and wherein a virus viability impairment (VVI) nucleic acid sequence is expressible from the second expression system component, and the expression of the VVI nucleic acid sequence in a plant cell produces a double stranded RNA molecule that induces silencing of a gene of the virus.

2. An expression system according to claim 1, wherein one or both of the toxicant nucleic acid sequence and the VVI nucleic acid sequence is conditionally expressible.

3. An expression system according to claims 1, wherein one or both of the toxicant nucleic acid sequence and the VVI nucleic acid sequence is constitutively expressible.

4. An expression system according to any one of claims 1 to 3, wherein both the toxicant nucleic acid sequence and the VVI nucleic acid sequence are conditionally expressible.

5. An expression system according to any one of claims 1 to 4, wherein the toxicant nucleic acid sequence is in the form of a contiguous sequence.

6. An expression system according to any one of claims 1 to 4, wherein one or both of the toxicant nucleic acid sequence and the VVI nucleic acid sequence is in the form of a plurality of non-contiguous sequences that can conditionally form a contiguous sequence.

7. An expression system according to claim 6, wherein one or both of the first expression cassette and the second expression cassette comprises an inactive repiicon that comprises replication c/s-acting elements, which facilitate, in the presence of a replication initiation protein, circuiarization and release from the inactive repiicon of a corresponding repiicon, and autonomous episomal replication (e.g., roiling circle replication) of the repiicon, wherein the repiicon comprises an expression cassette from which the toxicant nucleic acid sequence or the VVI nucleic acid sequence of the invention is expressible.

8. An expression system according to claim 7, wherein the toxicant nucleic acid sequence comprises a prorepiicon that lacks a functional rep gene for automous episomal replication (e.g., rolling circle replication) but comprises Rep recognition elements, which facilitate, in the presence of a Rep protein, circuiarization and release from the prorepiicon of a corresponding repiicon, and autonomous episomal replication (e.g., rolling circle replication) of the repiicon, wherein the repiicon comprises an expression cassette from which a toxicant nucleic acid sequence of the invention is expressible.

9. An expression system according to claim 8, wherein the inactive repiicon or prorepiicon comprises an toxicant nucleic acid sequence, which is in the form of a contiguous sequence and which is operabiy connected to at least one transcriptional control sequence (e.g., a promoter, transcription terminator, c/s-acting sequence, etc.).

10. An expression system according to claim 9, wherein the contiguous sequence is operabiy linked to a constitutive promoter for constitutively expressing the contiguous sequence.

11. An expression system according to claim 10, wherein the expression of the toxicant nucleic acid sequence is optionally boosted in the presence of a Rep protein.

12. An expression system according to claim 11, wherein the Rep protein is produced from a rep gene in an ancillary construct.

13. An expression system according to claim 12, wherein the Rep protein interacts with the Rep recognition elements of the proreplicon to facilitate circularization and release from the proreplicon of a corresponding replicon and autonomous episomal replication of the replicon, to thereby boost expression of the rep nucleic acid sequence.

14. An expression system according to claim 9, wherein the contiguous sequence is operably linked to a regulated promoter for conditionally expressing the contiguous sequence.

15. An expression system according to claim 14, wherein expression of a rep gene from an ancillary construct and the toxicant nucleic acid sequence occurs under control of regulated promoters whose transcriptional activity is stimulated or induced under the same conditions to thereby concurrently stimulate or induce expression of the rep gene and the toxicant nucleic acid sequence.

16. An expression system according to claim 9, wherein the proreplicon comprises an toxicant nucleic acid sequence, which is in the form of non-contiguous sequences {e.g. , a pair of discontinuous sequences), wherein an upstream member of the non-contiguous sequences corresponds to a 3' portion of the toxicant nucleic acid sequence and a downstream member of the non-contiguous sequences corresponds to a 5' portion of the toxicant nucleic acid sequence, wherein the 5' portion is operably connected to at least one transcriptional control sequence (e.g., a promoter, transcription terminator, c/s-acting sequence, etc.).

17. An expression system according to claim 16, wherein interaction of the Rep recognition elements of the proreplicon with a Rep protein, which is suitably produced from a rep gene in an ancillary expression cassette, facilitates circularization and release from the proreplicon of a corresponding replicon, and autonomous episomal replication (e.g., rolling circle replication) of the replicon comprising the expression cassette, wherein circularization of the replicon results in rearrangement of the expression cassette such that the non-contiguous sequences become operably connected with one another to form a contiguous toxicant nucleic acid sequence (i.e., a contiguous nucleic acid entity).

18. An expression system according to claim 17, wherein one of the Rep recognition sequences ("downstream Rep recognition sequence") is present in the expression cassette at a position downstream of the 5' portion of the toxicant nucleic acid sequence and/or the VVI nucleic acid sequence so that when circularization of the replicon occurs the downstream Rep recognition sequence is present in the circularized replicon at a location intermediate an upstream 5' portion and a downstream portion of the toxicant nucleic acid sequence and/or the VVI nucleic acid sequence.

19. An expression system according to claim 8, wherein the first expression system component comprises a proreplicon (a "target proreplicon") that includes an upstream first Rep recognition element and a downstream second Rep recognition element, which facilitate circularization, release and autonomous episomal replication {e.g., rolling circle replication) of a corresponding "target replicon" in the presence of a Rep protein, and a construct that comprises, from 5' to 3', a 3' portion of the toxicant nucleic acid sequence, a 5' portion of the toxicant nucleic acid sequence, and the second Rep recognition element.

20. An expression system according to claim 19, wherein a promoter is operably linked to the 5' portion of the toxicant nucleic acid sequence and a transcription terminator is optionally operably linked to the 3' portion of the toxicant nucleic acid sequence.

21. An expression system according to claim 20, wherein a Rep protein interacts with the Rep recognition element(s) in the target proreplicon to facilitate circularization, release and autonomous episomal replication of the target replicon, wherein circularization of the target replicon results in rearrangement of the construct such that the 3' and 5' portions of the toxicant nucleic acid sequence and the second Rep recognition element become operably connected to form a contiguous toxicant nucleic acid sequence comprising, from 5' to 3', the 5' portion of the toxicant nucleic acid sequence, the second Rep recognition element and the 3' portion of the toxicant nucleic acid sequence, and wherein autonomous episomal replication of the target replicon results in amplification of the target replicon with expression of the contiguous toxicant nucleic acid sequence.

22. An expression system according to claim 21, wherein the contiguous sequence is operably linked to a constitutive promoter for constitutively expressing the contiguous sequence.

23. An expression system according to claim 22, wherein expression of the toxicant nucleic acid sequence is optionally boosted in the presence of a Rep protein.

24. An expression system according to claim 23, wherein the Rep protein is produced from a rep gene in an ancillary construct.

25. An expression system according to claim 24, wherein the Rep protein interacts with the Rep recognition elements of the proreplicon to facilitate circularization and release from the proreplicon of a corresponding replicon and autonomous episomal replication of the replicon, to thereby boost expression of the toxicant nucleic acid sequence.

26. An expression system according to claim 25, wherein the proreplicon comprises a toxicant nucleic acid sequence, which is in the form of non-contiguous sequences (e.g. , a pair of discontinuous sequences), wherein an upstream member of the non-contiguous sequences corresponds to a 3' portion of the toxicant nucleic acid sequence and a downstream member of the non-contiguous sequences corresponds to a 5' portion of the toxicant nucleic acid sequence, wherein the 5' portion is operably connected to at least one transcriptional control sequence (e.g., a promoter, transcription terminator, c/^'s-acting sequence, etc.).

27. An expression system according to claim 26, wherein interaction of the Rep recognition elements of the proreplicon with a Rep protein, which is suitably produced from a rep gene in an ancillary expression cassette, facilitates circularization and release from the proreplicon of a corresponding replicon, and autonomous episomal replication (e.g., rolling circle replication) of the replicon comprising the expression cassette of the first component, wherein circularization of the replicon results in rearrangement of the expression cassette such that the non-contiguous sequences become operably connected with one another to form a contiguous toxicant nucleic acid sequence {i.e., a contiguous nucleic acid entity).

28. An expression system according to claim 27, wherein one of the Rep recognition sequences ("downstream Rep recognition sequence") is present in the expression cassette at a position downstream of the 5' portion of the toxicant nucleic acid sequence so that when circularization of the replicon occurs the downstream Rep recognition sequence is present in the circularized replicon at a location intermediate an upstream 5' portion and a downstream 3' portion of the toxicant nucleic acid sequence.

29. An expression system according to any one of claims 1-28, wherein the VVI nucleic acid is at least 17 nucleotides and as many as 3000 nucleotides in length (and all integer nucleotide lengths in between).

30. An expression system according to claim 29, wherein the virus impairment nucleic acid sequence encodes a double stranded RNA molecule comprising a duplex region formed by hybridization of complementary RNA sequences encoded respectively by the 5' and 3' portions, and a single stranded region that forms a loop connecting the complementary RNA sequences, which loop is encoded in whole or in part by the downstream Rep recognition sequence.

31. An expression system according to any one of claims 29 and 30, wherein the VVI nucleic acid sequence comprises a non-coding sequence (e.g., an intron) that separates individual sequences (e.g., sequences that encode protein or a functional RNA molecule) of the VVI nucleic acid sequence nucleic acid sequence.

32. An expression system according to claim 30, wherein the VVI nucleic acid sequence is in the form of non-contiguous sequences, wherein an individual non-contiguous sequence is separated from an upstream or downstream Rep recognition element by a non-coding sequence (e.g., an intron).

33. An expression system according to any one of claims 1 to 32, wherein the VVI nucleic acid sequence is selected from long dsRNA, siRNA, and shRNA.

34. An expression system according to any one of claims 1 to 33, wherein the VVI nucleic acid sequence comprises a duplex region formed by hybridization of complementary RNA sequences, and a single stranded region that forms a loop connecting the complementary RNA sequences.

35. An expression system according to any one of claims 1 to 34, wherein the toxicity protein is a ribosome inhibiting protein or hypersensitive response-elicitor polypeptide.

36. An expression system according to claim 35, wherein the ribosome inhibiting protein is a barnase.

37. An expression system according to any one of claims 1 to 36, wherein the VVI nucleic acid sequence silences a gene that is important for virus spread.

38. An expression system according to any one of claims 1 to 36, wherein the VVI nucleic acid silences a gene that is important for virus replication.

39. An expression vector according to any one of claims 1 to 38, wherein the VVI nucleic acid silences a virus gene selected from the group comprising or consisting of a movement protein gene, silencing suppressor gene, coat protein gene, nuclear shuttle protein gene, transactivator gene, cell cycle control protein gene, and replication initiation (associated) protein gene.

40. An expression system according to any one of claims 1 to 39, wherein the virus is a Geminivirus or a Nanovirus.

41. The expression system according to claim 40, wherein the Geminivirus is selected from among a Mastrevirus, Begomovirus, Curtovirus and Topocuvirus.

42. The expression system according to any one of claims 1 to 41, wherein the expression system confers resistance to multiple viruses.

43. A method for producing a transgenic plant that is resistant to infection by a ssDNA virus, the method comprising transforming a plant cell with an expression construct system as defined in any one of claims 1-42.

44. A plant comprising a cell that comprises the expression construct system according to any one of claims 1 to 43.

45. A plant according to claim 44, wherein the plant is a monocotyledonous plant.

46. A plant according to claim 44, wherein the plant is a dicotyledonous plant.