WO2015168733A1 - Decoy molecules - Google Patents

Abstract

Disclosed are systems and methods for modulating gene expression. More particularly, the present invention discloses the use of decoy RNA molecules that are non-homologous to a RNA expression product of a gene of interest for reducing RNA silencing and thereby enhancing expression of that gene. The invention also discloses expression systems from which such decoy RNA molecules are producible as well as transgenic cells, tissues and organisms that contain such expression systems.

Description

TITLE OF THE INVENTION

"DECOY MOLECULES"

[0001] This application claims priority to Australian Provisional Application No. 2014901625 entitled "Decoy Molecules" filed 5 May 2014, the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates to systems and methods for modulating gene expression. More particularly, the present invention relates to the use of decoy RNA molecules that are non-homologous to a RNA expression product of a gene of interest for reducing RNA silencing and thereby enhancing expression of that gene. The invention also relates to expression systems from which such decoy RNA molecules are producible and to transgenic cells, tissues and organisms that contain such expression systems.

BACKGROUND OF THE INVENTION

[0003] Mechanisms that suppress the expression of specific cellular genes, viruses or mobile genetic elements (such as transposons and retroelements) are critical for normal cellular function in a variety of eukaryotes. A number of related processes, discovered independently in plants (Waterhouse et a\. 1998; Matzke et a/., 2001), animals (Fire et al., 1998) and fungi (Cogoni, 2001), including transcriptional gene silencing (TGS) and post transcriptional gene silencing (PTGS), result in the RNA- mediated suppression of gene expression (also known as "RNA silencing"). Each of these processes is triggered by molecules containing double stranded RNA (dsRNA) structure, such as transcripts containing inverted repeats or double stranded RNA intermediates formed during RNA virus replication. Non-dsRNAs, also referred to as aberrant RNAs, may also function as initiators of RNA silencing. Such aberrant RNAs may be converted into dsRNAs by silencing-associated RNA-dependent RNA polymerases (RDRs), which have been identified in plants, fungi and Caenorhabditis elegans (Tuschl, ChemBiochem, 2001).

[0004] Plants utilize small interfering RNA (siRNA)-mediated PTGS as a mediated defense strategy to combat molecular parasites (e.g., viruses), which can cause genome instability and manipulate cellular machinery for their proliferation, and to degrade aberrant transcripts generated from endogenous genes and transgenes. siRNA- mediated PTGS is triggered by double-stranded RNA (dsRNA) and results in a sequence- specific shut down of the expression of genes containing homologous or highly similar sequences to the trigger, as a result of mRNA cleavage or translational repression (Figure 1). The basic process involves cleavage of long dsRNA into 21-22 nucleotides (nt) long siRNAs that guide recognition and degradation of homologous mRNAs (Blevins et a/., 2006, Deleris et al. , 2006; Fusaro et al. 2006). The siRNAs are generated by the processing of dsRNA triggers by RNaselll-like enzymes called Dicer-like in plants.

Triggers of siRNA-mediated PTGS can be dsRNA derived from intermediates of replicating RNA viruses, intra-molecular fold-back structures within viral genomes, exogenously introduced synthetic RNA transcripts of engineered inverted genes (hairpins), or products from undesirable transcription of native or transgenes by neighboring promoters.

Alternatively, dsRNA may be synthesized by the action of one of six RNA-dependent RNA polymerases (RDRs) (RDRl-6) on aberrant single stranded RNAs (ssRNAs) or polymerase IV transcripts. dsRNA triggers of siRNA-mediated PTGS are predominantly processed by DCL4 or its surrogate DCL2 . Production of siRNAs also requires dsRNA- binding proteins (DRBs) such as DRB4, which enables the synthesis of DCL4-dependent siRNA from RNA and DNA viruses (Haas et al., 2008, Qu et al., 2008; Curtin et al. 2008). siRNAs are recruited by Argonaute (AGO) proteins (predominantly AGOl and AG02) and a selected siRNA strand is incorporated into an AGO-containing, RNA-induced silencing complex (RISC), which guides sequence specific inactivation of target RNA (Harvey et a/. , 2011, Jaubert et al., 2011, Morel et al., 2002, Qu et al., 2008, Scholthof et al. , 2011, Takeda et al., 2008, Wang et al. , 2011, Zhang et al., 2006).

[0005] Plants have evolved means to amplify the silencing response to dsRNA triggers through the production of secondary siRNAs. These are produced through the activity of cellular RNA-dependent RNA polymerases (RDRs), which convert single- stranded RNA (ssRNA) into new dsRNA substrates for dicing (Figure 1) (Dunoyer et a/., 2005, Himber et al. , 2003, Voinnet, 2008). Both primary and secondary siRNAs also have the potential to move to neighboring cells through plasmodesmata to spread the silencing signal through the plant. This aspect of RNA silencing represents the systemic arm of the siRNA mediated PTGS response. As a means of viral protection, the

transmission of the mobile siRNAs ahead of the infection front is essential, as it primes antiviral silencing in cells that are yet to be infected. As such, replication or movement of the virus into those cells is delayed or prevented (Havelda et a/. , 2003, Voinnet et a/., 2000, Voinnet et a/. , 1998). However, as a counter defense strategy most plant viruses produce suppressor proteins which can target different elements of the PTGS machinery in order to combat RNA silencing.

[0006] siRNA-mediated PTGS poses a significant constraint on the expression of transgenes in plant biotechnology, particularly on the production of recombinant proteins, whereby reduced transgene expressing results in reduced yields of target proteins. Plant virus-encoded suppressors of RNA silencing have proved useful tools for counteracting this phenomenon, however, their wide applicability in transgenic plants is limited because their expression often causes harmful developmental effects to the plant, and the proteins themselves are toxic in some species (Brigneti et a/. , 1998, Kasschau et a/., 2003, Nairn et a/., 2012, Voinnet et a/., 2003). Two novel approaches to avoid this problem were recently described by Alvarez et al. (2008) and Saxena et al. (2011). In the first study, transgenic tomatoes containing a silenced vaccine candidate gene were super-transformed with the TBSV pl9 silencing suppressor gene under the control of ethanol inducible promoter. Upon addition of ethanol, PTGS was reversed and these lines accumulated the antigenic protein to levels up to 3-times higher in the fruit than non- silenced elite tomato lines. In contrast, Saxena et a/. (2011) mutated the TBSV pl9 silencing suppressor such that it retained the ability to sequester siRNA but did not cause a harmful phenotype when over-expressed in N. benthamiana. Transgenic plants co- expressing green fluorescent protein (GFP) and the pl9/R43W mutant showed elevated accumulation of GFP compared with plants without the suppressor. Further, transgenic expression of P19/R43W caused little to no morphological defects and plants produced normal-looking flowers and fertile seed. Despite the success of this mutant, its effectiveness in suppressing PTGS was about half that of the native pl9 and one may assume constitutive expression of this mutant form would likely compromise the plants viral defenses. For these reasons the use of viral silencing suppressors, to reduce PTGS of transgenes, is generally limited to transient expression via Agrobacterium infiltration.

[0007] Enhanced transgene expression has also been achieved by hairpin RNA (hpRNA) directed suppression of a component of the RNA silencing pathway, RDR6 (Yoon et a/., 2012). Such hpRNA directed silencing, in which an inverted repeat sequence with homology to a target mRNA is expressed to provide triggering dsRNA, is demonstrated to direct efficient suppression of homologous targets (Waterhouse 1998, Smith 2000).

[0008] Although there has been progress in developing technologies for suppressing RNA-mediated gene silencing, there is still a need for alternative strategies of enhancing expression of a gene of interest by modulating RNA silencing of that gene.

SUMMARY OF THE INVENTION

[0009] The present invention is predicated in part on the determination that the expression level of a transgene may be enhanced by permeating the silencing pathway with RNA molecules that lack homology to the transgene. In particular, several dsRNAs were co-expressed with transgenes that lack homology to the dsRNAs in Nicotiana benthamiana and the effect of co-expressed non-specific dsRNAs was analyzed and compared with the impact of the silencing suppressors P19, PI and PO. The present inventors found unexpectedly that co-expression of non-specific dsRNA effectively limits the host silencing response as evidenced by significant increases in the expression of the transgenes. Moreover, the transgene overexpression facilitated by the non-specific dsRNA was i) synergistic with the expression of a VSR known to reduce AGO levels and ii) antagonistic with a VSR known to sequester dsRNAs. These results suggest that the observed inhibition of RNA silencing targeted to the overexpressed transgene is due to permeation of the silencing machinery with non-specific dsRNAs. Notably, the present inventors established that co-expression of a transgene with non-specific dsRNA achieves consistent, high levels of transgene expression without the use of viral protein

suppressors and as such represents a unique tool for overexpressing a gene of interest in host cells in which RNA-mediated gene silencing occurs, as described hereafter.

[0010] Accordingly, one aspect of the present invention provides expression systems for expressing a target nucleic acid sequence in a host cell, suitably with reduced RNA silencing of the target nucleic acid sequence. These expression systems generally comprise, consist or consist essentially of a first expression system component (e.g., comprising one or more expression cassettes or constructs) and a second expression system component (e.g., comprising one or more expression cassettes or constructs), wherein the target nucleic acid sequence is expressible from the first expression system component, and wherein a modulator nucleic acid sequence is expressible from the second expression system component. Expression of the modulator nucleic acid sequence in the host cell produces a double stranded decoy RNA molecule that is a substrate of the RNA silencing pathway and that is non-homologous with a RNA expression product of the target nucleic acid sequence, wherein the decoy RNA molecule competes with other substrates of the RNA silencing pathway to thereby induce silencing of the modulator nucleic acid sequence with reduced silencing of the target nucleic acid sequence. Suitably, the reduced silencing enhances expression of the target nucleic acid sequence. The double stranded decoy RNA molecule, suitably its double stranded or duplex region, can be at least 17 nucleotides and as much as 3000 nucleotides in length (and all integer nucleotide lengths in between). The decoy RNA molecule can be selected from long dsRNA (e.g., a precursor dsRNA that is suitably a substrate for DICER or a DICER-like protein), siRNA and shRNA. In some embodiments, the decoy RNA molecule comprises a duplex region formed by base pairing of complementa ry RNA sequences, and a single stranded region that forms a loop connecting the complementary RNA

sequences.

[0011] Suitably, the decoy RNA molecule has no more than about 80%, 75%,

70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% sequence identity to the RNA expression product of the target nucleic acid sequence, suitably over the entire sequence of the RNA expression prod uct or over a subsequence (e.g., a subsequence of at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length) of the RNA expression product. In some embodiments, one strand of a double stranded or duplex region of the decoy RNA molecule has no more than about 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40% sequence identity over the entire sequence of the RNA expression product or over a subsequence (e.g., a subsequence of at least 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides in length) of the RNA expression product. In illustrative examples of this type, one strand of a double stranded or dupl ex region of the decoy RNA molecule has a sequence consisting of no more than 13, 12, 11 , 10, 9 nucleotides that are identical to a subsequence of the RNA expression product, wherein the subsequence is 17 nucleotides in length . In other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a sequence consisting of no more than 13, 12, 11, 10, 9 nucleotides that are identical to a

subsequence of the RNA expression product, wherein the subsequence is 18 nucleotides in length . In other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a sequence consisting of no more than 14, 13, 12, 11, 10 nucleotides that are identical to a subsequence of the RNA expression product, wherein the subsequence is 19 nucleotides in length . In other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a sequence consisting of no more than 15, 14, 13, 12, 11 nucleotides that are identical to a subsequence of the RNA expression product, wherein the subsequence is 20 nucleotides in length . In still other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a sequence consisting of no more than 15, 14, 13, 12, 11 nucleotides that are identical to a subsequence of the RNA expression product, wherein the subsequence is 21 nucleotides in length . In yet other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a sequence consisting of no more than 16, 15, 14, 13, 12 nucleotides that are identical to a subsequence of the RNA expression product, wherein the subsequence is 22 nucleotides in length . In other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a sequence consisting of no more than 17, 16, 15, 14, 13 nucleotides that are identical to a subsequence of the RNA expression product, wherein the subsequence is 23 nucleotides in length . In still other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a sequence consisting of no more than 18, 17, 16, 15, 14, nucleotides that are identical to a subsequence of the RNA expression product, wherein the subsequence is 24 nucleotides in length . In still other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a sequence consisting of no more than 19, 18, 17, 16, 15, nucleotides that are identical to a subsequence of the RNA expression product, wherein the subsequence is 25 nucleotides in length .

[0012] Suitably, the decoy RNA molecule has no more than about 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% sequence identity to one or more (e.g., 2, 3, 4, 5, 6, 7,8 ,9, 10 or more) endogenous RNA expression products in the host cell, suitably over the entire sequence of individual endogenous RNA expression products or over a subsequence (e.g. , a subsequence of at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length) of individual endogenous RNA expression products. In some embodiments, one strand of a double stranded or duplex region of the decoy RNA molecule has no more than 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40% sequence identity over the entire sequence of an individual endogenous RNA expression product or over a subsequence (e.g. , a subsequence of at least 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides in length) of an individual endogenous RNA expression product. In illustrative examples of this type, one strand of a double stranded or duplex region of the decoy RNA molecule has a sequence consisting of no more than 13, 12, 11, 10, 9 nucleotides that are identical to a subsequence of an individual endogenous RNA expression product, wherein the subsequence is 17 nucleotides in length . In other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a seq uence consisting of no more than 13, 12, 11, 10, 9 nucleotides that are identical to a subsequence of an individual endogenous RNA expression product, wherein the subsequence is 18 nucleotides in length . In other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a sequence consisting of no more than 14, 13, 12, 11, 10 nucleotides that a re identical to a subsequence of an individual endogenous RNA expression product, wherei n the subsequence is 19 nucleotides in length. In other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a sequence consisting of no more than 15, 14, 13, 12, 11 nucleotides that are identical to a subsequence of an individual endogenous RNA expression product, wherein the subsequence is 20 nucleotides in length . In still other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a sequence consisting of no more than 15, 14, 13, 12, 11 nucleotides that are identical to a subsequence of an individual endogenous RNA expression product, wherein the subsequence is 21 nucleotides in length . In yet other illustrative examples, one strand of a double stranded or du plex region of the decoy RNA molecule has a sequence consisting of no more than 16, 15, 14, 13, 12 nucleotides that are identical to a subsequence of an individual endogenous RNA expression product, wherein the subsequence is 22 nucleotides in length . In other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a seq uence consisting of no more than 17, 16, 15, 14, 13 nucleotides that are identical to a subsequence of an individual endogenous RNA expression product, wherein the subsequence is 23 nucleotides in length . In still other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a sequence consisting of no more than 18, 17, 16, 15, 14, nucleotides that are identical to a subsequence of an individual endogenous RNA expression product, wherein the subsequence is 24 nucleotides in length. In still other illustrative examples, one strand of a double stranded or duplex region of the decoy RNA molecule has a sequence consisting of no more than 19, 18, 17, 16, 15, nucleotides that are identical to a subsequence of an individual endogenous RNA expression product, wherein the subsequence is 25 nucleotides in length .

[0013] Suitably, the decoy RNA molecule is unable to hybridize under high, medium or low stringency conditions to the RNA expression product of the target nucleic acid sequence and/or to endogenous RNA expression products of the host cell .

[0014] In specific embodiments, one or both of the first expression system component and the second expression system component is stably introduced in the genome of the host cell . The host cell can be any eukaryotic cell in which RNA-mediated gene silencing occurs, including animal (e.g., mammalian) and plant host cells.

[0015] The target or modulator nucleic acid sequence can be, or correspond to, a heterologous nucleic acid sequence or an endogenous nucleic acid sequence. In some embodiments, the target nucleic acid sequence is, or corresponds to, an endogenous nucleic acid sequence a nd the modulator nucleic acid sequence is, or corresponds to, a heterologous nucleic acid sequence. In other embodiments, the target nucleic acid sequence and modulator nucleic acid sequence both are, or correspond to, heterologous nucleic acid sequences. In still other embodiments, the ta rget nucleic acid sequence is, or corresponds to, a heterologous nucleic acid sequence and the modulator nucleic acid sequence is, or corresponds to, an endogenous nucleic acid sequence. Suitably, one or both of the target nucleic acid sequence and the modulator nucleic acid sequence is conditionally expressible. In other embodiments, one or both of the target nucleic acid sequence and the modulator nucleic acid sequence is constitutively expressible. In still other embodiments, the target nucleic acid seq uence is constitutively expressible and the modulator nucleic acid sequence is conditionally expressible. In some embodiments, the target nucleic acid sequence and the modulator nucleic acid sequence are both conditionally expressible. In other embodiments, the target nucleic acid sequence is conditionally expressible and the modulator nucleic acid sequence is constitutively expressible. In further embodiments, the target nucleic acid sequence is conditionally expressible and the modulator nucleic acid sequence is constitutively and optionally conditionally expressible.

[0016] 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 target or modulator 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 (also referred to herein as a "contiguous nucleic acid entity" or "contiguous gene") or a plurality of non-contiguous sequences (also referred to herein as a "non-contiguous nucleic acid entity", "non- contiguous gene" or "split gene") that can conditionally form a contiguous sequence.

[0017] In preferred embodiments, one or both of the first expression system component and the second expression system component comprises an inactive replicon that comprises replicase c/^'s-acting elements, which facilitate, in the presence of a replicase, circularization and release from the inactive replicon 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 an effector nucleic acid sequence of the invention (i.e., a target or modulator nucleic acid sequence) is expressible.

[0018] In representative examples of this type, one or both of the first expression system component and the second expression system component 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, 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 an effector nucleic acid sequence of the invention (i.e., a target or modulator nucleic acid sequence) is expressible. In illustrative examples of this type, the proreplicon comprises an effector nucleic acid sequence, which is in the form of a contiguous sequence and which is operably connected to at least one transcriptional control sequence (e.g., a promoter, transcription terminator, c/^'s-acting sequence, etc.). In specific embodiments, the contiguous sequence is operably linked to a constitutive promoter for constitutively expressing the contiguous sequence. In non-limiting examples of this type, expression of the effector 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 effector nucleic acid sequence. In other embodiments, the contiguous sequence is operably linked to a regulated promoter for conditionally expressing the contiguous sequence. In representative examples of this type, expression of the rep gene and the effector 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 effector nucleic acid sequence.

[0019] In other representative examples, the proreplicon comprises an effector 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 effector nucleic acid sequence and a downstream member of the non-contiguous sequences corresponds to a 5' portion of the effector 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.). 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 effector 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 effector 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 effector 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 effector nucleic acid sequence. In non-limiting examples of this type, the effector nucleic acid sequence is a modulator nucleic acid sequence that encodes a double stranded decoy RNA molecule comprising a duplex region formed by base pairing 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.

[0020] In preferred embodiments, the effector 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 sepa rated from a n upstream or downstream Rep recognition element by a non-coding sequence (e.g., an intron) . Suitably, in these examples, the 3' portion of the effector nucleic acid sequence is sepa rated from an upstream Rep recognition element by a 3' portion of an intron and the 5' portion of the effector 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 rea rrangement of the construct to form a contiguous effector nucleic acid sequence, which comprises in operable linkage, from 5' to 3', the 5' portion of the effector 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 effector nucleic acid sequence. Suitably, a promoter (e.g., regulated or constitutive) is operably connected upstream of the 5' portion of the effector nucleic acid sequence to form an expression cassette.

[0021] In non-limiting examples, 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 target nucleic acid sequence, a 5' portion of the target nucleic acid sequence, a nd the second Rep recognition element. A promoter is suitably operably linked to the 5' portion of the target nucleic acid sequence and a transcription terminator is preferably but not essentially (optionally) operably linked to the 3' portion of the target 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 target nucleic acid sequence and the second Rep recognition element become operably connected to form a contiguous target nucleic acid sequence comprising, from 5' to 3', the 5' portion of the target nucleic acid sequence, the second Rep recognition element and the 3' portion of the target nucleic acid sequence.

Autonomous episomal replication of the target replicon results in amplification of the target replicon with expression of the contiguous target nucleic acid sequence. [0022] In non-limiting examples, the second expression system component comprises a proreplicon (a "modulator 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 "modulator 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 modulator nucleic acid sequence, a 5' portion of the modulator nucleic acid sequence, and the second Rep recognition element. Suitably, a promoter is operably linked to the 5' portion of the modulator nucleic acid sequence and a transcription terminator is preferably but not essentially (optionally) operably linked to the 3' portion of the modulator nucleic acid sequence. In these examples, a Rep protein interacts with the Rep recognition element(s) in the modulator proreplicon to facilitate circularization, release and autonomous episomal replication of the modulator replicon. Circularization of the modulator replicon results in rearrangement of the construct such that the 3' and 5' portions of the modulator nucleic acid sequence and the second Rep recognition element become operably connected to form a contiguous modulator nucleic acid sequence and comprising, from 5' to 3', the 5' portion of the modulator nucleic acid sequence, the second Rep recognition element and the 3' portion of the modulator nucleic acid sequence. Autonomous episomal replication of the modulator replicon results in amplification of the modulator replicon with expression of the contiguous modulator nucleic acid sequence.

[0023] In specific embodiments, the first expression system component comprises a proreplicon for expressing the target nucleic acid sequence and the second expression system component comprises a proreplicon for expressing modulator nucleic acid sequence. In other embodiments, the first expression system component comprises a proreplicon for expressing the target nucleic acid sequence and the second expression system component is in the form of a biphasic expression system component for expressing modulator nucleic acid sequence.

[0024] 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 host cell. Suitably, the rep gene is selected from among geminivirus (e.g., Mastrevirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Topocuvirus, Turncurtovirus), nanovirus (e.g., Nanovirus, Babuvirus), circovirus (e.g., Circovirus), and bacterial rep genes.

[0025] In certain embodiments 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/^'rivs-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.

[0026] In a related aspect, the present invention provides host cells that contain an expression system as broadly described above and elsewhere herein. In some embodiments, the first and/or second expression system components is/are stably introduced in the genome of the host cell. In some embodiments, the host cells are plant host cells, including monocotyledonous or dicotyledonous host cells. In other

embodiments, the host cells are non-plant eukaryotic cells, including yeast, fungus and animal cells (e.g., mammalian cells such as primate cells including human cells), in which RNA silencing occurs.

[0027] Certain aspects of the present invention are directed to the expression of target nucleic acid sequences that are endogenous to the host cell and that are suitably susceptible to RNA silencing and the modulator nucleic acid sequence is introduced into the host cell in order to inhibit the silencing to enhance expression of the target nucleic acid sequence. In these instances, it is not necessary to provide to the host cell a heterologous expression cassette from which the target nucleic acid sequence is expressible, as the target nucleic acid sequence is part of an endogenous expression cassette in the genome of the host cell . Accordingly, in another aspect, the present invention provides a host cell that comprises an endogenous expression system component from which a target nucleic acid sequence is expressible, and a heterologous expression system component from which a modulator nucleic acid sequence is expressible, wherein expression of the modulator nucleic acid sequence in the host cell produces a double stranded decoy RNA molecule that is a substrate of the RNA silencing pathway and that is non-homologous with a RNA expression product of the target nucleic acid sequence, wherein the decoy RNA molecule competes with other substrates of the RNA silencing pathway to thereby induce silencing of the modulator nucleic acid sequence with reduced silencing of the endogenous target nucleic acid sequence. Suitably, the reduced silencing enhances expression of the endogenous target nucleic acid sequence. In specific embodiments, the decoy RNA molecule has no more than about 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% sequence identity to the RNA expression product of the target nucleic acid sequence, suitably over the entire sequence of the RNA expression product or over a subsequence (e.g., a subsequence of at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length) of the RNA expression product, as broadly described above. In some embodiments, the decoy RNA molecule has no more than about 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% sequence identity to endogenous RNA expression products in the host cell , suitably over the entire sequence of an individual endogenous RNA expression product or over a subsequence (e.g., a subsequence of at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length) of an individual endogenous RNA expression product, as broadly described above. In some embodiments, the decoy RNA molecule is unable to hybridize under high, medium or low stringency conditions to the RNA expression product of the target nucleic acid sequence and/or to endogenous RNA expression products of the host cell .

[0028] In specific embodiments, the decoy RNA molecule targets an

endogenous RNA expression product with consequential silencing of an endogenous nucleic acid sequence (e.g., an endogenous gene) . Suitably, in these embodiments, the decoy RNA molecule has at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity over the entire sequence of the RNA expression product or over a subsequence (e.g., a subsequence of at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length) of the endogenous RNA expression product. In representative examples of this type, the decoy RNA molecule is unable to hybridize under medium or high stringency cond itions, as defined for example herein, to any sequence of nucleotides located within the RNA expression product of the target nucleic acid sequence but is able to hybridize to a sequence of nucleotides located within an endogenous RNA expression product of the host cell under the same conditions. The endogenous RNA expression product is preferably an expression product of a gene involved in the RNA silencing pathway. In preferred embodiments, the decoy RNA molecule is expressed from a replicon-based system, as described for example above and elsewhere herein.

[0029] In another aspect, the present invention provides transgenic organisms and parts thereof including organs and tissues, which comprise a host cell as broadly described above and elsewhere herein . In some embodiments, the transgenic organisms are transgenic plants, including monocotyledonous or dicotyledonous tra nsgenic plants. In other embodiments, the transgenic organisms are non-plant eukaryotic organisms, including yeast, fungus and animals (e.g., mammals such as primate including human), in which RNA silencing occurs. [0030] In another aspect, the present invention provides methods for enhancing expression of a target nucleic acid sequence in a host cell . These methods generally comprise, consist or consist essentially of co-expressing the target nucleic acid sequence and a modulator nucleic acid sequence in the host cell , wherein expression of the modulator nucleic acid sequence produces a double stranded decoy RNA molecule that is a substrate of the RNA silencing pathway and that is non -homologous with a RNA expression product of the target nucleic acid sequence, wherein the decoy RNA molecule competes with other substrates of the RNA silencing pathway to thereby induce si lencing of the modulator nucleic acid sequence with reduced silencing of the target nucleic acid sequence. Suitably, the methods further comprise introducing into the host cell at least one expression system component from which the target nucleic acid seq uence and/or the modulator nucleic acid sequence is/are expressible. In some embodiments, the target nucleic acid sequence a nd/or the modulator nucleic acid sequence is/are stably introduced in the genome of the host cell . In some embodiments, the host cel ls are plant host cells, including monocotyledonous or dicotyledonous host cells. In other

embodiments, the host cells are non-plant eukaryotic cells, including yeast, fungus and animal cells (e.g., mammalian cells such as primate cells including human cells), in which RNA silencing occurs.

[0031] In some embodiments, the methods further comprise exposing the host cell to one or more stimuli that stimulate or enhance expression of the target nucleic acid sequence, the modulator sequence or both the target nucleic acid sequence and the modulator sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a schematic representation illustrating decoy dsRNA- mediated post transcriptional gene silencing (PTGS) in plants. siRNA mediated PTGS is triggered by double-stranded RNA (dsRNA) which may be presented in the cell as dsRNA intermediates of replicating RNA viruses, intramolecular fold -back structures within viral genomes, exogenously introduced synthetic RNA transcript of engineered inverted genes (hairpins) or unintended transcription of nuclear or transgenes by neighboring promoters. Alternatively, dsRNA may be synthesized by the action of one of the six RNA-dependent RNA polymerases (RDRs) (RDR1-6) on aberrant ssRNAs or polymerase IV transcripts. The dsRNA triggers a re processed into siRNAs of approximately 21 nucleotides (nt) long by RNaselll-like enzymes called Dicer-like proteins (DCL4 and DCL2) . siRNAs are loaded into Argonaute (AGO)-containing RNA-induced silencing complexes (RISCs) to guide translational inhibition and/or slicing of homologous long RNAs (post transcriptional gene silencing ; PTGS) . Cleaved RNAs are also used by cellular RDRs and their cofactors to amplify the RNA silencing response through production of more dsRNA substrates for DCL processing . Both primary and secondary siRNAs also have the potential to move to neighboring cells through plasmodesmata to spread the silencing response through the plant.

[0033] Figure 2 is a schematic representation showing an illustrative construct for inducing production of a decoy RNA molecule. The elements of the construct include a promoter, a coding sequence (AlcR) for an alcohol receptor, a transcription terminator (T), an ethanol inducible promoter (AlcAP), a modulator nucleic acid sequence comprising two complementary portions (i.e. , 5' Decoy and 3' Decoy) spaced apart by an intervening nucleic acid spacer, and a transcription terminator (T), which encode the decoy RNA molecule.

[0034] Figure 3 is a schematic representation showing a non-limiting example of a construct for constitutively expressing a decoy RNA molecule. The elements of the construct include a constitutive promoter, a modulator nucleic acid sequence comprising two complementary portions (i.e. , 5' Decoy and 3' Decoy) spaced apart by an intervening nucleic acid spacer, and a transcription terminator (T) , which encode the decoy RNA molecule.

[0035] Figure 4 is a schematic representation showing an exemplary INPACT construct for conditionally expressing a decoy RNA molecule. The elements of this construct include a proreplicon that comprises first and second Rep recognition elements (Rep rec 1, Rep rec 2), wh ich mediate circularization and release of a replicon from the proreplicon in the presence of a Rep protein, a non-contiguous or split modulator nucleic acid sequence comprising two complementary portions (i.e. , 5' Decoy and 3' Decoy), a transcription terminator (T), a short intergenic region (SIR) and a promoter. When the repiicon circularizes, elements of the construct rearrange to provide a contiguous modulator nucleic acid entity comprising from 5' to 3', the 5' Decoy, the second Rep recognition element (Rep rec 2) and the 3' Decoy, which encode the decoy RNA molecule.

[0036] Figure 5 is a schematic representation showing a representative construct for inducing production of Rep protein. The elements of this construct include a promoter, a coding sequence (AlcR) for an alcohol receptor, a transcription terminator (T), an ethanol inducible promoter (AlcAP), a coding sequence for two rep gene expression products (Rep and RepA), and a transcription terminator (T).

[0037] Figure 6 is a schematic representation depicting a non-limiting construct, based on prorepiicon constructs described for example by 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), for expressing a decoy RNA molecule. The elements of this construct include a prorepiicon comprising first and second Rep recognition elements (Rep rec 1, Rep rec 2), which mediate circuiarization and release of a repiicon from the prorepiicon in the presence of a Rep protein, and an expression cassette for expressing a modulator nucleic acid sequence, which is in the form of a contiguous nucleic acid entity and which encodes a decoy RNA molecule. The expression cassette includes, from 5' to 3', a promoter, a modulator nucleic acid sequence comprising a first complementary portion (5' Decoy), an intervening nucleic acid spacer, a second complementary portion (3' Decoy), and a transcription terminator (T).

[0038] Figure 7 is a schematic representation showing a representative construct for inducing expression of a gene of interest (GOI) . The elements of this construct include a promoter, a coding sequence (AlcR) for an alcohol receptor, a transcription terminator (T), an ethanol inducible promoter (AlcAP), a GOI, and a transcription terminator (T).

[0039] Figure 8 is a schematic representation showing a representative construct for constitutively expressing a gene of i nterest (GOI). The elements of this construct comprises an expression cassette that includes a promoter, a GOI and a transcription terminator (T).

[0040] Figure 9 is a schematic representation showing an exemplary INPACT construct for conditionally expressing a gene of interest (GOI). The elements of this construct include a prorepiicon that comprises first and second Rep recognition elements (Rep rec 1, Rep rec 2), which mediate circuiarization and release of a repiicon from the prorepiicon in the presence of a Rep protein, a non-contiguous or split GOI comprising, from 5' to 3', a 3' portion of an intron (Intron 3'), a 3' portion of the GOI (3' GOI), a transcription terminator (T), a short intergenic region (SIR), a promoter, a 5' portion of the GOI (5' GOI), a 5' portion of the intron (Intron 5') and the second Rep recognition element (Rep rec 2). When the replicon circularizes, elements of the construct rearrange to provide, from 5' to 3', the promoter; a contiguous GOI entity comprising the 5' portion of the GOI (5' GOI), the 5' portion of the intron (Intron 5'), the second Rep recognition element (Rep rec 2), the 3' portion of the intron (Intron 3') and the 3' portion of the GOI (3' GOI); the transcription terminator (T), and the short intergenic region (SIR) intermediate the transcription terminator (T) and the promoter.

[0041] Figure 10 is a schematic representation depicting a non-limiting example of an expression system for enhancing expression of a gene of interest (GOI). The expression system comprises a first proreplicon for conditionally expressing a decoy RNA molecule and a second proreplicon for conditionally expressing the GOI. Elements of these constructs are the same as those illustrated in Figures 4 and 9.

[0042] Figure 11 is a schematic representation depicting a further non-limiting example of an expression system for enhancing expression of a gene of interest (GOI). The construct system comprises a first Yadav proreplicon construct for conditionally expressing decoy RNA molecule, and a second INPACT proreplicon construct for conditionally expressing the GOI. Elements of these constructs are the same as those illustrated in Figures 6 and 9.

[0043] Figure 12 is a schematic representation of illustrating the production of decoy RNA using an embodiment of the INPACT expression system. The elements of this system include: 35SP, CaMV 35S promoter; AlcR, alcohol receptor gene; nosT, nopaline synthase terminator; AlcAP, AlcA promoter; Rep/RepA, Replicase/Replicase A

[0044] Figure 13 is a schematic representation depicting high level expression of a decoy RNA that reduces PTGS of a transgene using the INPACT expression system.

[0045] Figure 14 is a schematic representation illustrating high level expression of a decoy RNA that reduces PTGS of an endogenous gene using the INPACT expression system.

[0046] Figure 15 is schematic representation of constructs used in the

Experimental section. Construct names are indicated on the left. The diagrams show each expression cassette between the right and left borders of the T-DNA (RB and LB respectively). The genes of interest (uidA encoding the GUS, GFP, TBSV P19, SMBV PI, and PEMVPO) are under the control of either the CaMV35S or the SSU promoters and either the CaMV35S, SSU or Nos terminators. For the p35S-CPMV-GUS, the uidA gene is between the 5' and 3' enhancer elements of the CPMV. The constructs encoding the hairpins have been designed with a sense (s) and antisense (as) fragments of the GFP, DCL1 and the chalcone synthase (CHS) coding sequence. TBSV, Tomato Bushy Stunt Virus; SBMV, Southern Bean Mosaic Virus; PEMV, Pea Enation Mosaic Virus; CaMV, Cauliflower Mosaic Virus; SSU, Rubisco Small SubUnit; Nos, Nopaline synthase.

[0047] Figure 16 is a photographic representation showing the effect of co- expression of silencing suppressor proteins and non-specific hairpin constructs on reporter proteins transient expression in N. benthamiana leaves. (A) Leaf of N.

benthamiana plant agro-infiltrated with the p35S-GFP GFP inducer vector (A-l) plus either P19 (A-2), P0^PE (A-3), PI (A-6) or hairpin expressing vectors p35S-DCLlhp and pSSU-CHShp (A-4 and 5 respectively). Leaf was photographed at 8 dpi under UV light. (B) Leaf of N. benthamiana plants infiltrated with the p35S-GUS or (C and D) the p35S- CPMVGUS, GUS inducer vectors, plus either P19 (B, C, D-2), hairpin expression vectors p35S-GFPhp targeting the GFP (B, D-3), or the chalcone synthase pSSU-CHShp (C-3), or a mix of both P19 and the corresponding hairpin expressing vector (B, C ,D-4). Leaf was photographed at 8 dpi after GUS staining assay.

[0048] Figure 17 is a graphical representation showing quantitative analysis

(enzyme-linked immunosorbent assay) of GUS accumulation in infiltrated N.

benthamiana leaves.

[0049] Figure 18 is a graphical representation showing Northern blot analysis of siRNAs in infiltrated N. benthamiana leaves. (1) Control ; (2) PO; (3) PI; (4) empty vector; (5) P19; (6) GFhp; (7) CHShp. At 8 DPI total RNAs from N. benthamiana leaves infiltrated with Agrobacterium suspension, carrying either the p35S-GUS (A) or p35S- CPMV-GUS (B) mixed with different either P19, GFPhp and CHShp, were blotted and probed for the presence of GUS and GFP siRNAs. Hybridization with U6 RNA is used as loading control.

[0050] Figure 19 is a graphical representation showing fluorometric

quantification of GUS activity performed in N. benthamiana leaves infiltrated with Agrobacterium suspension carrying either the p35S-GUS (A) or p35S-CPMV-GUS (B and C) mixed with different potential silencing suppressor proteins (SSP). P19, PI , P0^PE and GFPhp are referred here as SSPs. GUS activity is expressed in pg 4-methylumblliferone min^"1 mg^"1 protein, and a graph drawn of the average rate of GUS activity per collection of transgenics per construct. The quantification of GUS activity for each test was replicated three times. Statistical analysis was performed using least significant difference and homogeneity of variance test by SPSS 16.0, and one way ANOVA test was used for the statically analysis. Means with different lower-case or upper-case letters were statistically different at P < 0.05 among segments and between tissues,

respectively. Error bars on the graph represent SE with three replicates. [0051] Figure 20 is a graphical representation showing transient expression via Agrobacterium infiltration of GUS and a decoy hpRNA specific for GFP that lacks homology to GUS and other endogenous genes of Nicotiana tabacum.

[0052] Figure 21 is a graphical representation showing stable expression GUS and a decoy hpRNA specific for GFP that lacks homology to GUS and other endogenous genes of Nicotiana tabacum.

[0053] Figure 22 is a graphical representation showing fluorometric

quantification of GUS activity in stably Agrobacterium transformed plants of N. tabacum containing INPACT-GUS cassette + alcohol induced decoy hpRNA (Alc-MPhp, also referred to herein as Alc-hpl) or constitutively expressed decoy hpRNA (35S-MPhp, also referred to herein as 35S-hpl). Both hpRNA molecules are specific for the Movement Protein (MP) of Tobacco yellow leaf curl virus (TYLCV) and lack homology to GUS or other endogenous genes of N. tabacum.

[0054] Figure 23 is a graphical representation showing fluorometric

quantification of GUS activity performed in N. tabacum via Agrobacterium infiltration of GUS and an INPACT cassette containing a decoy hpRNA specific for GFP (35S-GFPhp) or MP (35S-MPhp) that lack homology to GUS or other endogenous genes of N. tabacum.

[0055] Figure 24 is a graphical representation showing fluorometric

quantification of GUS activity performed in N. benthamiana via Agrobacterium infiltration of a 35S>GUS expression cassette and an INPACT cassette containing a decoy hpRNA specific for GFP (GFPhp) or MP (MPhp) that lacks homology to GUS and other

endogenous genes of N. benthamiana.

TABLE 1

BRIEF DESCRIPTION OF THE SEQUENCES

SEQUENCE ID

SEQUENCE NUMBER

SEQ ID NO: 1 Banana b unchy top virus (DNA-1 Rep)

SEQ ID NO: 2 Tobacco /ellow dwarf virus (Rep/RepA)

SEQ ID NO: 3 Maize str eak virus (Rep/RepA)

SEQ ID NO: 4 Tomato 1 eaf curl virus (A2 Rep)

SEQ ID NO: 5 Bean gok den mosaic virus (AC2 Rep)

SEQ ID NO: 6 Beet curl Y top virus (C2)

SEQ ID NO: 7 Tomato p seudo-curly top virus (C2)

SEQ ID NO: 8 Geminivi 'uses IR consensus nonanucleotide

SEQ ID NO: 9 Geminivi 'uses IR alternate nonanucleotide

SEQ ID NO: 10 Begomov irus DNA-β IR consensus nonanucleotide

SEQ ID NO: 11 Begomov irus DNA-a IR consensus nonanucleotide

SEQ ID NO: 12 Nanoviru s IR consensus nonanucleotide

SEQ ID NO: 13 Nanoviru s IR alternate nonanucleotide

SEQ ID NO: 14 TGMV co< at protein promoter

SEQ ID NO: 15 wheat dv\ /arf virus LIR

SEQ ID NO: 16 Tobacco /ellow dwarf virus LIR

SEQ ID NO: 17 Tobacco /ellow dwarf virus SIR

SEQ ID NO: 18 Maize str eak virus LIR

SEQ ID NO: 19 Maize str eak virus SIR

SEQ ID NO: 20 Tomato y ellow leaf curl virus

SEQ ID NO: 21 Bean gok den mosaic virus DNA-A IR

SEQ ID NO: 22 Bean gok den mosaic virus DNA-B IR

SEQ ID NO: 23 Beet curl y top virus

SEQ ID NO: 24 Banana b unchy top virus DNA-1 IR

SEQ ID NO: 25 Banana b unchy top virus DNA-2 IR

SEQ ID NO: 26 Banana b unchy top virus DNA-3 IR

SEQ ID NO: 27 Banana b unchy top virus DNA-4 IR

SEQ ID NO: 28 Banana b unchy top virus DNA-5 IR

SEQ ID NO: 29 Banana b unchy top virus DNA-6 IR

SEQ ID NO: 30 Ageratun 1 leaf curl virus DNA-β IR

SEQ ID NO: 31 Ageratun 1 yellow vein virus DNA-β IR

SEQ ID NO: 32 Chilli leaf curl virus DNA-β IR

SEQ ID NO: 33 Cotton le af curl virus DNA-β IR

SEQ ID NO: 34 EupatoriL i m yellow vein virus DNA-β IRrgerg

SEQ ID NO: 35 Mimosa y 'ellow leaf curl virus DNA-β IR

SEQ ID NO: 36 Malachra yellow vein mosaic virus DNA-β IR

SEQ ID NO: 37 Pepper y< Bllow leaf curl virus DNA-β IR SEQUENCE ID

SEQUENCE NUMBER

SEQ ID NO: 38 Tobacco curly shoot virus DNA-β IR

SEQ ID NO: 39 Tomato leaf curl virus (Thailand) DNA-β IR

SEQ ID NO: 40 Tomato yellow leaf curl virus DNA-β IR

SEQ ID NO: 41 pT181 origin

SEQ ID NO: 42 pT181 minimal origin

SEQ ID NO: 43 Modified Tobacco yellow dwarf virus (TYDV) LIR

SEQ ID NO: 44 Barnase gene

SEQ ID NO: 45 TMV replicase

SEQ ID NO: 46 TMV replicase 50 kDa fragment

SEQ ID NO: 47 INPACT-GUS vector

GFPhp (also referred to herein as "hp2") ; sequence

SEQ ID NO: 48

corresponding to the sense arm of the hairpin is provided

CHShp; sequence corresponding to the sense arm of the hairpin

SEQ ID NO: 49

is provided

DCLlhp; sequence corresponding to the sense arm of the hairpin

SEQ ID NO: 50

is provided

MPhp (also referred to herein as "hpl") ; sequence corresponding

SEQ ID NO: 51

to the sense arm of the hairpin is provided

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[0056] 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.

[0057] 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. Thus, for example, the term "c/^'s-acting sequence" also includes a plurality of c/^'s-acting sequences.

[0058] 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).

[0059] 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.

[0060] 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 i nvention. 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.

[0061] 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.

[0062] 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 .

[0063] "Autonomous" or "c/^'s" replication refers to replication of a replicon that contains all c/^'s- and trans-acting sequences (such as the replication gene (rep)) required for replication.

[0064] "Cells", "host cells", "transformed host cells", "regenerate host 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.

[0065] The terms "c/^'s-acting element", "c/^'s-acting seq uence" 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.

[0066] "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.

[0067] 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. [0068] 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 host 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] "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.

[0074] "Constitutive promoter" refers to an unregulated promoter that di rects 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.

[0075] 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 any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear 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 linked. Constructs of the present invention will generally include the necessary elements to direct expression of a nucleic acid sequence of interest that is also contained in the construct, such as, for example, a target nucleic acid sequence or a modulator nucleic acid sequence. Such elements may include control elements such as a promoter that is operably linked to (so as to direct transcription of) the nucleic acid sequence of interest, and often includes a

polyadenylation sequence as well. Within certain embodiments of the invention, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and/or elements to facilitate stable integration of the construct into the genome of a host cell . Two or more constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nucleic acid molecules, such as two or more separate vectors. An "expression construct" generally includes at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in an organism or part thereof including a host cell. For the practice of the present invention, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular Cloning : A Laboratory Manual, 3^rd edition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0076] As used herein, the term "contiguous" in the context of a nucleic acid sequence means that the sequence is a single sequence, uninterrupted by any

intervening sequence or sequences.

[0077] The term "contiguous nucleic acid entity" defines an entity (e.g., a gene) comprised of a linear series or complete sequence of nucleotides, suitably within a larger polynucleotide sequence, which defines the nucleic acid entity (e.g., a modulator nucleic acid sequence, a target nucleic acid sequence etc.). A "non-contiguous nucleic acid entity" is an entity that is comprised of a series of nucleotides within a polynucleotide sequence, which is non-linear in alignment, that is that the nucleotides are spaced or grouped in a non-continuous manner along the length of a polynucleotide sequence. A non-contiguous nucleic acid entity (also referred to herein as a "split gene") can be a discontinuous nucleic acid entity wherein the nucleotides are grouped into 2 linear sequences (e.g., each comprising a different open reading frame (ORF)) arranged along the length of the polynucleotide, which together define the entire sequence of the nucleic acid entity (e.g., a modulator nucleic acid sequence, a target nucleic acid sequence etc.). Alternatively, the non-contiguous nucleic acid entity can be a discontinuous 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) arranged along the length of the polynucleotide.

Illustrative non-contiguous nucleic acid entities include those in which a 5' portion of a contiguous nucleic acid entity is located on a nucleic acid molecule downstream 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 modulator nucleic acid entity, a non-contiguous target 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.

[0078] 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 host 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.

[0079] 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).

[0080] 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, wh ich 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 seq uence") 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 ta rget sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches a re most tolerated in the terminal regions a nd, if present, a re 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.

[0081] 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.

[0082] 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 a n expression system component of the invention is introduced .

[0083] As used herein, the term "episome" or "replicon" refers to a DNA or RNA virus or a vector that undergoes episomal replication in host cells (e.g., 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 ACl and ALl genes in ACMV and TGMV geminiviruses, respectively). It may or may not contain a nucleic acid sequence of interest for expression in the host cell.

[0084] "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.

[0085] 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.

[0086] As used herein, the term "expression cassette" refers to a polynucleotide sequence capable of effecting expression of a gene of interest (e.g. , a target nucleic acid sequence, a modulator nucleic acid sequence etc.) in a host 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 host cell or may be part of a construct.

[0087] 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 host cell in which they reside or are proposed to reside, provided that at least one them (e.g. , used to express the modulator nucleic acid sequence) of the expression system is heterologous with respect to the host 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 host 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 host cell (e.g., a plant cell) in a regulated manner. Both constructs may be stably introduced into the host 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.

[0088] 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.

[0089] 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 i nclude siRNA, shRNA, miRNA (including pri-miRNA and pre-miRNA), nucleic acid aptamers (including RNA aptamers and DNA aptamers), ribozymes (including deoxyribozymes), riboswitches, U l adaptors, molecular beacons, and transcriptional factor-binding regions.

[0090] 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.

[0091] "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.

[0092] 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).

[0093] 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).

[0094] 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 M 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", "nonhomologous 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 decoy 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.

[0095] The term "host" refers to any organism, or cell thereof, whether eukaryotic or prokaryotic into which a construct of the invention can be introduced, particularly, hosts in which RNA silencing occurs. In particular embodiments, the term "host" refers to eukaryotes, including unicellular eukaryotes such as yeast and fungi as well as multicellular eukaryotes such as: plants, illustrative examples of which include angiosperms, gymnosperms, monocotyledons (monocots) and dicotyledons (dicots), and animals non-limiting examples of which include invertebrate animals (e.g., insects, cnidarians, echinoderms, nematodes, etc.); eukaryotic parasites (e.g., malarial parasites, such as Plasmodium falciparum, helminths, etc.); vertebrate animals (e.g., fish, amphibian, reptile, bird, mammal); and mammals (e.g., rodents, primates such as humans and non-human primates). Thus, the term "host cell" suitably encompasses cells of such eukaryotes as well as cell lines derived from such euka ryotes.

[0096] 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 "cis-acting sequence" and "c/^'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.

[0097] 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 vira l 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 non-functional 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.

[0098] "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.

[0099] 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 betasatellites (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.

[0100] "Introducing" in the context of a host cell including an animal cell, animal part, and/or animal organ, plant cell, plant part and/or plant organ means contacting a nucleic acid molecule with the animal cell, animal part, and/or animal organ, or 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 animal cell, animal part, and/or animal organ, or 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 host 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] The term "operably connected" or "operably linked" as used herein refers to a juxtaposition 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 c/^'s-acting sequence to a promoter encompasses positioning and/or orientation of the c/^'s-acting sequence relative to the promoter so that the c/^'s-acting sequence regulates {e.g. , inhibits, abrogates, stimulates or enhances) promoter activity. Alternatively, "operably connecting" noncontiguous nucleic acid sequences of a non-contiguous 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 target or modulator 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 the coding sequence of the contiguous nucleic acid entity.

[0107] 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 amena ble 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, onio n, asparagus, lily, coconut, and the like.

[0108] 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.

[0109] As used herein, the term "plant part" includes but is not limited to embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ea rs, 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.

[0110] The term "plant organ" refers to plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant.

[0111] "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 pa rts 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.

[0112] The term "promoter" refers to a nucleotide sequence, usually upstream (5') to a transcribable sequence, which controls the expression of the transcribabl e 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.

[0113] "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.

[0114] 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., target or modulator 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.

[0115] The term "rearrangement" refers to the rearrangement of noncontiguous nucleic acid sequences such that they become operably connected with one another to form a contiguous nucleic acid entity (e.g., a contiguous target or modulator nucleic acid sequence). This term encompasses one or more changes in the order of spaced subsequences of a modulator or target 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.

[0116] 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 ecdysome-inducible systems. [0117] "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.

[0118] 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 organism and those that are heterologous to the host organism (such as introduced into the host organism by recombi nant 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 natu re. 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.

[0119] A "rep coding sequence" as used herein refers to a sequence of nucleic acids from which a single transcript encoding a Rep protein can be produced .

[0120] 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 organism 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.

[0121] 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 c/^'s 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.

[0122] 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 is the 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. [0123] 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.

[0124] 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.

[0125] 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.

[0126] 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 ?es.25 : 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et a/., "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15.

[0127] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of a RNA molecule formed during the transcription of a RNA from a gene or nucleic acid sequence of interest, including RNA (e.g., mRNA) that is a product of RNA processing of a primary transcription product.

[0128] "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.

[0129] 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 (ACl or ALl in ACMV or TGMV geminiviruses, respectively), which can function in replication without being on the replicon.

[0130] "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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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" organism, such as a transgenic plant, transgenic microorganism, or transgenic animal, is an organism into which a transgene has been delivered or introduced and the transgene can be expressed in the transgenic organism to produce a product, the presence of which can impart an effect and/or a phenotype in the organism.

[0135] As used herein, the term "transient expression" refers to expression in cells in which a transgene is introduced into a host 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

[0136] 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.

[0137] 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.

[0138] 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 host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host 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 host 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 host 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. [0139] 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.

[0140] 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.

[0141] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

2. Abbreviations

[0142] The following abbreviations are used throughout the application :

3. Construct system for expression of target nucleic acid sequences

[0143] The present invention is directed to expression systems for expressing a target nucleic acid sequence in a host cell, suitably with reduced RNA silencing of the target nucleic acid sequence. The expression systems generally comprise at least two expression system components, in which a first expression system component expresses the target nucleic acid sequence and a second expression system component expresses a modulator nucleic acid sequence that encodes a double stranded decoy RNA molecule that is a substrate of the RNA silencing pathway and that is non-homologous with a RNA expression product (i.e. , transcript) of the target nucleic acid sequence. In operation, the decoy RNA molecule competes with other substrates of the RNA silencing pathway to thereby induce silencing of the modulator nucleic acid sequence with reduced silencing of the target nucleic acid sequence. Without wishing to be bound by any one theory or mode of operation, it is proposed that permeation of the silencing pathway with RNA molecules that lack homology to the target nucleic acid sequence reduces the host silencing response to other nucleic acid sequences including the target nucleic acid sequence. The reduced silencing effectively leads to enhanced expression of the target nucleic acid sequence. In some embodiments, both of the first and second expression system components are heterologous with respect to the host cell. In other

embodiments, the first expression system component is endogenous and the second expression system component is heterologous with respect to the host cell.

3.1 Decoy RNA molecules and encoding modulator nucleic acid sequences

[0144] In accordance with the present invention, a double stranded decoy RNA molecule is capable of entering the RNA silencing pathway to silence the expression of a modulator nucleic acid that encodes the decoy RNA. The double stranded decoy RNA, which is also referred to herein as "dsRNA", 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 dsRNA may include two nucleotide polymers that have one or more region(s) of complementarity to each other.

[0145] 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.

[0146] 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 panhand le structure.

[0147] 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.

[0148] In other embodiments, as e.g., 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.

[0149] Suitably, the dsRNA molecule is selected from long dsRNA (e.g., a precursor dsRNA that is suitably a substrate for DICER or a DICER-like protein), 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.

[0150] The dsRNA molecules of the present invention are suitably sufficiently distinct in sequence from the RNA expression product of a target nucleic acid sequence that is expressed or desired to be expressed 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 decoy RNA molecule and the expression product of the target nucleic acid polynucleotide seq uence 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.

[0151] 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 target 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.

[0152] 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 target 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 target 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.

[0153] In certain embodiments, the modulator nucleic acid sequence that encodes, and is the subject of RNA silencing by, the decoy RNA molecule is a nucleic acid sequence that is heterologous to the host cell (e.g., artificial or from a different genetic source or organism to which the host cell expressing the modulator nucleic acid sequence relates).

[0154] In other 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 a sequence of nucleotides of an endogenous nucleic acid sequence 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. Alternatively, or in addition, the dsRNA molecule, particularly its antisense or guide strand, is generally unable to hybridize (i.e., above background) under medium or high stringency conditions, as defined for example herein, to any sequence of nucleotides located within a RNA expression product of the target nucleic acid sequence but is able to hybridize to a sequence of nucleotides located within an endogenous RNA expression product of the host cell under the same conditions. Illustrative endogenous nucleic acid sequences for targeting by the dsRNA decoy molecules of the invention include any sequence whose expression is desired to be inhibited, and which suitably provides a beneficial effect (e.g., to expression of the target nucleic acid sequence in the host cell) . For example, dsRNA molecules can be designed for silencing expression of genes involved in the RNA silencing pathway, representative examples of which include Agola, Agolb, Ago2, Ago4a, Ago4b, Ago5, Ago6, Ago7, AgolO, Cmt3a, Cmt3b, Dell, Dcl2, Dcl3, Dcl4, Drbla, Drblb, Drb2a, Drb2b, Drb3a, Drb3b, Drb4, Drb5, Drdl , Drm3, Henl , Metl, Nrpdla, Nrpdlb, Nrpd2a, Rdrl, Rdr2,

Rdr5, Rdr6a, Rdr6b* and Sgs3. Without wishing to be bound by any one theory or mode of operation, it is proposed that silencing expression of any one or more genes involved in the RNA silencing pathway will lead to a reduction in gene silencing in the host cell , with consequential further reduction in silencing of the target nucleic acid sequence . In these embodiments, the dsRNA molecule is preferably expressed from a replicon-based system, as described for example herein.

[0155] 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 ta rget 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.

[0156] The modulator 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 modulator nucleic acid sequence, and also depending on the host cell in which this sequence is desired to be expressed . In some cases, expression in m ultiple 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 modulator nucleic acid sequence can also be controlled at the level of replication .

[0157] The modulator nucleic acid sequence may be in the form of a contiguous nucleic acid entity that encodes an intact or uninterrupted dsRNA molecule. Alternatively, the modulator 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 capa ble of rearranging (e.g., by replication or recombination) to form a contiguous nucleic acid entity that encodes an intact dsRNA molecule.

3.2 Target nucleic acid sequences

[0158] Target nucleic acid sequences a re suitably genes of interest that are reflective of the commercial markets and interests of those involved in the development of transgenic hosts and host cells and are generally dependent on the use or uses to which they are put. Exemplary hosts include all organisms in which RNA silencing occurs, illustrative examples of which include eukaryotic hosts, including unicellular eukaryotes such as yeast and fungi as well as multicellular eukaryotes such as: plants and animals.

3.2.1 Plant genes of interest

[0159] In specific embodiments, the host is a plant and the gene of interest suitably imparts, improves or modulates a desirable agronomic trait or characteristic illustrative examples of which include herbicide resistance or tolerance; insect resistance or tolerance; disease resistance or tolerance (viral, bacterial, fungal, nematode); stress tolerance and/or resistance, as exemplified by resistance or tolerance to drought, heat, chilling, freezing, excessive moisture, salt stress; oxidative stress; increased yields; food content and makeup; physical appearance; male sterility; drydown; standability;

prolificacy; starch properties; oil quantity and quality; and the like. One may desire to incorporate one or more genes conferring any such desirable trait or traits, such as, for example, a gene or genes encoding pathogen resistance. General categories of genes of interest for these embodiments include, for example, those genes involved in

information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include genes encoding proteins conferring resistance to abiotic stress, such as drought, temperature, salinity, and toxins such as pesticides and herbicides, or to biotic stress, such as attacks by fungi, viruses, bacteria, insects, and nematodes, and development of diseases associated with these organisms. Various changes in phenotype are of interest including modifying expression of a gene in a plant, altering a plant's pathogen or insect defense mechanism, increasing the plant's tolerance to herbicides, altering plant development to respond to environmental stress, and the like. The results can be achieved by providing expression of heterologous or increased expression of endogenous products in plants. Alternatively, the results can be achieved by providing for a reduction of expression of one or more endogenous products, particularly enzymes, transporters, or cofactors, or affecting nutrients uptake in the plant. These changes can result in a change in phenotype of the transformed plant. It is recognized that any gene of interest can be operably linked to the promoter sequences of the embodiments and expressed in a plant.

[0160] Non-limiting examples of genes of interest that provide beneficial agronomic traits to plants (e.g. , crop plants) include nucleic acid sequences that modulate herbicide resistance (U.S. Pat. No. 5,633,435; U.S. Pat. No. 5,463,175), increased yield (U.S. Pat. No. 5,716,837), insect control (U.S. Pat. No. 6,063,597; U.S. Pat. No. 6,063,756; U.S. Pat. No. 6,093,695; U.S. Pat. No. 5,942,664; U.S. Pat. No.

6,110,464), fungal disease resistance (U.S. Pat. No. 5,516,671 ; U.S. Pat. No. 5,773,696;

U.S. Pat. No. 6,121,436; and U.S. Pat. No. 6,316,407, and U.S. Pat. No. 6,506,962), virus resistance (U.S. Pat. No. 5,304,730 and U.S. Pat. No. 6,013,864), nematode resistance (U.S. Pat. No. 6,228,992), bacterial disease resistance (U.S. Pat. No.

5,516,671), starch production (U.S. Pat. No. 5,750,876 and U.S. Pat. No. 6,476,295), modified oils production (U.S. Pat. No. 6,444,876), high oil production (U.S. Pat. No. 5,608,149 and U.S. Pat. No. 6,476,295), modified fatty acid content (U.S. Pat. No.

6,537,750), high protein production (U.S. Pat. No. 6,380,466), fruit ripening (U.S. Pat. No. 5,512,466), enhanced animal and human nutrition (U.S. Pat. No. 5,985,605 and U.S. Pat. No. 6,171,640), biopolymers (U.S. Pat. No. 5,958,745 and US Patent Publication No. US20030028917), environmental stress resistance (U.S. Pat. No. 6,072,103),

pharmaceutical peptides (U.S. Pat. No. 6,080,560), improved processing traits (U.S. Pat. No. 6,476,295), improved digestibility (U.S. Pat. No. 6,531,648) low raffinose (U.S. Pat. No. 6,166,292), industrial enzyme production (U.S. Pat. No. 5,543,576), improved flavor (U.S. Pat. No. 6,011,199), nitrogen fixation (U.S. Pat. No. 5,229,114), hybrid seed production (U.S. Pat. No. 5,689,041), and biofuel production (U.S. Pat. No. 5,998,700). The patent documents disclosing these nucleic acid sequences are hereby incorporated by reference herein in their entirety.

[0161] The gene of interest may encode a marker that when expressed imparts a distinct phenotype to the plant host expressing the marker and thus allows such transformed plant host to be distinguished from those that do not have the marker. Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic, herbicide, or the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g., the R-locus trait, color, fluorescence, etc.).

[0162] The present invention also contemplates genes of interest for expressing RNA transcripts that function to affect plant phenotype yet are not translated into protein. Two non-limiting examples are antisense RNA and RNA with ribozyme activity. Both may serve possible functions in reducing or eliminating expression of native or introduced plant genes. For example, genes may be constructed or isolated, which when transcribed, produce antisense RNA that is complementary to all or part(s) of a targeted messenger RNA(s). The antisense RNA reduces production of the polypeptide product of the messenger RNA. The polypeptide product may be any protein encoded by the plant genome. The aforementioned genes will be referred to as antisense genes. An antisense gene may thus be introduced into a plant by transformation methods to produce a novel transgenic plant with reduced expression of a selected protein of interest. For example, the protein may be an enzyme that catalyzes a reaction in the plant. Reduction of the enzyme activity may reduce or eliminate products of the reaction which include any enzymatically synthesized compound in the plant such as fatty acids, amino acids, carbohydrates, nucleic acids and the like. Alternatively, the protein may be a storage protein, such as a zein, or a structural protein, the decreased expression of which may lead to changes in seed amino acid composition or plant morphological changes respectively. The possibilities cited above are provided only by way of example and do not represent the full range of applications. Alternatively, genes may also be constructed or isolated, which when transcribed produce RNA enzymes, or ribozymes, which can act as endoribonucleases and catalyze the cleavage of RNA molecules with selected sequences. The cleavage of selected messenger RNAs can result in the reduced production of their encoded polypeptide products.

3.2.2 Other genes of interest

[0163] Other genes of interest for expression in eukaryotic hosts, including yeast hosts and animal hosts (e.g. , mammalian hosts), include genes that code for therapeutic proteins such as but not limited to cytokines and receptors (such as interleukins 1-36 and interferons, as well as their receptors), growth factors and receptors (such as such as epidermal growth factor (EGF), acid fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor AA, AB, and BB(PDGF AA, AB and BB), insulin-like growth factor (IGF), transforming growth factor (TGF) and their receptors, human serum albumin, a-fetoprotein, antibodies (such as full length immunoglobulins comprising two light and two heavy chains, Fabs, scFvs (single chain variable fragment), camelid-type antibodies, antibody fragments, antibody fragment-fusions, antibody-receptor fusions, etc.), chemokines, hematopoietic growth factors (such as GM-CSF, G-CSF, etc.), coagulation factors, complement factors, steroid hormones and their receptors (such as glucocorticoid hormones, mineralocortical hormones, sexual steroid hormones, etc. and their receptors), matrix proteins (such as fibronectin, collagen, vitronectin, etc.), other bioactive peptides (such as

adrenocorticotropic hormone and fragments, angiotensin and related peptides, atrial natriuretic peptides, bradykinin and related peptides, chemotactic peptides, dynorphin and related peptides, endorphins and β-lipotropin fragments, enkephalin and related peptides, enzyme inhibitors, gastrointestinal peptides, growth hormone releasing peptides, luteinizing hormone releasing hormone and related peptides, melanocyte stimulating hormone and related peptides, neurotensin and related peptides, opioid peptides, oxytocin, vasopressin, vasotocin and related peptides, parathyroid hormone and fragments, protein kinase related peptides (including PKC), somatostatin and related peptides, substance P and related peptides, toxins, conditional toxins, antigens, tumor suppressor proteins, membrane proteins, vasoactive proteins and peptides, and anti-viral proteins. Alternatively, the genes of interest may encode industrial enzymes,

representative examples of which include lipases, proteases, cellulases, pectinases, amylases, esterases, oxidoreductases, transferases, lactases, isomerases, and

invertases. 3.3 Expression cassettes

[0164] The target and modulator nucleic acid sequences of the invention ("effector nucleic acid sequences") 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 effector nucleic acid sequences in a transgenic host.

3.3.1 Promoters

[0165] 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

[0166] 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 target 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 vice 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 effector nucleic acid sequences in the desired host cell.

[0167] These promoters include, but are not limited to, constitutive, inducible, temporally regulated, developmentally 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.

[0168] 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.

[0169] 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).

[0170] 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 a/., 1988), the E8 promoter (Diekman & Fischer, 1988) and the fruit specific 2A1 promoter (Pear et a/. , 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.

[0171] Examples of some constitutive promoters which have been described include the rice actin 1 (Wang et a/., 1992; U.S. Pat. No. 5,641,876), CaMV 35S (Odell et al., 1985), CaMV 19S (Lawton et a/., 1987), nos, Adh, sucrose synthase; and the ubiquitin promoters.

[0172] 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 a/. , 1984), corn light harvesting complex (Simpson, 1986; Bansal et al., 1992), corn heat shock protein (Odell et a/., 1985), pea small subunit RuBP carboxylase (Poulsen et a/. , 1986), Ti plasmid mannopine synthase (Langridge et a/., 1989), Ti plasmid nopaline synthase (Langridge et a/., 1989), petunia chalcone isomerase (vanTunen et a/. , 1988), bean glycine rich protein 1 (Keller et a/. , 1989), truncated CaMV 35S (Odell et a/. , 1985), potato patatin (Wenzler et a/., 1989), root cell (Yamamoto et al., 1990), maize zein (Reina et a/., 1990; Kriz et a/., 1987; Wandelt et a/., 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 a/., 1989), histone, and chalcone synthase promoters (Franken et a/. , 1991). Tissue specific enhancers are described in Fromm et a/. (1989).

[0173] 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 a/., 1988), the MPI proteinase inhibitor promoter (Cordero et a/. , 1994), and the

glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et a/., 1995; Quigley et a/., 1989; Martinez et a/. , 1989).

[0174] 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 a/., 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 a/., 1995).

[0175] A class of fruit-specific promoters expressed at or during antithesis through fruit development, at least until the beg inning 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 a/., 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.

[0176] 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.

[0177] 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.

[0178] 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 al. , 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 a nd 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. (Graham et al. , 1985; Graham et al. , 1985, Smith et al., 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.

[0179] 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-1 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 RuBP 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. [0180] In some embodiments, the promoter is an alcohol dehydrogenase promoter (e.g., derived from Aspergillus nidulans such as AlcAP).

Animal promoters

[0181] Numerous promoters are known for driving constitutive or conditional expression in animal hosts including mammals, illustrative examples of which include viral promoters such as but not limited to the SV40 early (SV40e) promoter region, the promoter contained in the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters of the E1A or major late promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, baculovirus IE1 promoter, adenovirus promoters, and papillomavirus promoters, and promoters of animal (e.g., mammalian) genes, non-limiting examples of which include elongation factor 1 alpha (EF1) promoter, phosphoglycerate kinase (PGK) promoter, heat-shock promoters, ubiquitin (Ubc) promoter, albumin promoter, metallothionein promoters, the ubiquitous promoters (HPRT, vimentin, a-actin, tubulin and the like), the promoters of the intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (of the MDR, CFTR or factor VIII type, and the like), pathogenesis or disease related -promoters, and promoters that exhibit tissue specificity and have been utilized in transgenic animals, such as the elastase I gene control region which is active in pancreatic acinar cells; insulin gene control region active in pancreatic beta cells, immunoglobulin gene control region active in lymphoid cells, mouse mammary tumor virus control region active in testicular, breast, lymphoid and mast cells; albumin gene, Apo AI and Apo All control regions active in liver, alpha-fetoprotein gene control region active in liver, alpha 1-antitrypsin gene control region active in the liver, beta-globin gene control region active in myeloid cells, myelin basic protein gene control region active in oligodendrocyte cells in the brain, myosin light chain-2 gene control region active in skeletal muscle, and gonadotropic releasing hormone gene control region active in the hypothalamus, pyruvate kinase promoter, villin promoter, promoter of the fatty acid binding intestinal protein, promoter of the smooth muscle cell a-actin, and the like. In addition, these expression sequences may be modified by addition of enhancer or regulatory sequences and the like.

Yeast promoters

[0182] Representative examples of yeast promoters that are suitable for the present invention include CYC1, H1S3, GAL1, GAL10, ADH1, PGK, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOK1 (useful for expression in Pichia). Non-limiting constitutive promoters suitable for use in yeast include FBA1, TDH3 (GPD), ADH1, ILV5, and GPM 1, and illustrative inducible promoters suitable for use in yeast include GAL1, GAL10, OLE1, and CUP1. Other yeast promoters include hybrid promoters such as but not limited to UAS(PGKl)-FBAlp, UAS(PGKl)-EN02p, UAS(FBAl)-PDClp, UAS(PGKl)-PDClp, and UAS(PGK)-OLElp, which are described for example in U.S. Pat. Appl. Pub. No. 2014/0030783, the contents of which a re hereby incorporated herein by reference in their entirety.

3.3.2 Other Regulatory Elements

[0183] In addition to promoters, a variety of 5' and 3' transcriptional regulatory sequences is also available for use in expressing an effector nucleic acid sequence of the invention.

Transcription terminators

[0184] The effector 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 effector 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.

[0185] 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 et al. , 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. Non-limiting examples of transcription terminators useful in animal cells (e.g.,

mammalian cells), include those derived from viruses including SV40, as described in

Sambrook et al. , supra, as well as growth hormone transcriptional terminators (see, e.g., U.S. Pat. No. 5,122,458), and the like. Suitable transcriptional terminators for use in yeast include, but are not limited to FBAt, GPDt, GPMt, ERGl Ot, GALl t, CYCl, and ADHl transcription terminators.

Untranslated leader sequences

[0186] 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.

Intro ns

[0187] 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.

[0188] 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 a/., 1987), sucrose synthase intron (Vasil et a/., 1989) or TMV omega element (Gallie, et a/. , 1989), may further be included where desired.

Enhancers

[0189] Examples of enhancers include elements from the CaMV 35S promoter, octopine synthase genes (Ellis et a/., 1987), the rice actin I gene, the maize alcohol dehydrogenase gene (Callis et a/., 1987), the maize shrunken I gene (Vasil et a/., 1989), TMV Omega element (Gallie et a/., 1989) and promoters from non-plant eukaryotes (e.g. yeast; Ma et a/., 1988).

[0190] Enhancers that may be used for enhancing expression in animal hosts (e.g., mammalian hosts) include but are not limited to: an SV40 enhancer, a

cytomegalovirus (CMV) enhancer, an elongation factor 1 (EF1) enhancer, yeast enhancers, viral gene enhancers, and the like.

Site-specific recombinase activation

[0191] Regulated expression of a nucleic acid sequence of interest can also be regulated by other genetic strategies including recombinase-mediated gene activation in which a blocking nucleic acid sequence comprising transcription termination sequence bound by site-specific sequences ("recombinase recognition sites") is placed between a promoter and the effector nucleic acid sequence, thereby blocking the expression of the effector nucleic acid sequence from the promoter. The blocking nucleic acid sequence can be removed by expression of a coding sequence for a site-specific recombinase that mediates excision of the blocking sequence, thereby resulting in the expression of the effector nucleic acid sequence. In this case, the recombinase gene, the effector nucleic acid sequence, or both can be under the control of tissue-specific, developmental-specific or inducible promoters. Illustrative recombinases, which are site-specific, include Cre, modified Cre, Dre, Hp, FLP-wild type (wt), FLP-L, FLPe, Flpo or phiC31. Non-limiting examples of recombinase recognition sites include loxP, FRT, rax and attP/B.

Recombination may be effected by any art-known method, e.g., the method of

Doetschman et al. (1987, Nature 330 : 576-578); the method of Thomas et al. (1986, Cell 44:419-428); the Cre-loxP recombination system (Sternberg and Hamilton, 1981, J. Mol. Biol. 150 :467-486; Lakso et a/., 1992, Proc. Natl. Acad. Sci. USA 89 : 6232-6236); the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et a/. , 1991, Science 251 : 1351-1355; Lyznik et a/., 1996, Nucleic Acids Res. 24(19) : 3784-3789); the Cre- loxP-tetracycline control switch (Gossen and Bujard, 1992, Proc. Natl. Acad. Sci. USA 89: 5547-51); and ligand-regulated recombinase system (Kellendonk et a/., 1999, J. Mol. Biol. 285 : 175-82). Desirably, the recombinase is highly active, e.g. , the Cre-loxP or the FLPe system, and has enhanced thermostability (Rodrguez et a/., 2000, Nature Genetics 25: 139-40).

[0192] In specific embodiments, site-specific recombination is used for reconstituting a functional rep gene in an ancillary construct that comprises the rep gene in a non-contiguous form. Reconstitution of the rep gene leads to production of a Rep protein in trans for the replication of an associated proreplicon.

tRNA suppressor genes

[0193] An alternate genetic strategy is the use of tRNA suppressor gene. For example, the regulated expression of a tRNA suppressor gene can conditionally control expression of an effector nucleic acid sequence containing an appropriate termination codon as described by Ulmasov et al. 1997. Again, either the tRNA suppressor gene, the effector nucleic acid sequence, or both can be under the control of tissue-specific, developmental-specific or inducible promoters.

Site-specific replicase activation

[0194] In specific embodiments, expression of an effector nucleic acid sequence of the invention is regulated using replicase-mediated gene activation. In these embodiments, the effector nucleic acid sequence, which may be in the form of a contiguous 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., viral sequences) flanking the effector nucleic acid sequence, which are required for replication, but is incapable of episomal replication in cells (e.g., plant cells) because it lacks a functional rep gene(s) essential for replication. Under appropriate stimulus, the transactivating rep gene expresses the replication protein (Rep) (e.g. , viral Rep) missing in the proreplicon and allows the release of a replicon from the proreplicon and its episomal replication in a cell autonomous manner. Typically the replication elements are derived from viruses, as described for example below. Non-liming examples of such binary expression systems are described by Dale et al. (U.S. Pat. No. 7,863,430), Dugdale 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 herein in their entirety.

[0195] Thus, replicon replication can be targeted to specific cells by controlling the expression of replication protein(s) to those cel ls. The proreplicon embodiments of the present invention are particularly advantageous for expressing effector nucleic acid sequences in plant hosts. Plants are generally sensitive to cellular toxicity and/or the detrimental effect of viral replication and/or replication protein(s) in early stages of plant growth and differentiation that involve cell division and differentiation. Thus, controlling the expression of the replication protein and repiicon replication entirely or largely to non-dividing, terminally-differentiated cells will reduce the detrimental effect of repiicon replication on plant growth and development. Examples of such terminally-differentiated cells include, but are not limited to, the storage cells of seed and root tissues and mature leaf cells. Furthermore, the proreplicon when introduced into a plant host serves as a master copy for replicons not only in different generations but also in the same generation if cell divisions occur after the onset of episomal replication. This strategy will also solve the problem of episomal instability through cell divisions, since episomes are unstable in the absence of selection. Furthermore, repiicon replication is expected to achieve high level expression of effector nucleic acid sequences through gene

amplification that is heritable when stably integrated into the host chromosome and cell autonomous.

[0196] Replicase genes are selected so that they recognize the Rep recognition elements required for release of a repiicon from the proreplicon and autonomous episomal replication of the repiicon. Exemplary rep genes include those from ssDNA plant viruses, such as Geminiviruses and Nanoviruses, as well as those from bacteria, including phytoplasmal rep genes. For example, a Mastrevirus rep gene encoding both Rep and RepA proteins can be included in a construct for expressing an effector nucleic acid sequence. In other examples, a Curtovirus, Topocuvirus or Begomovirus rep gene is included. In further examples, a Nanovirus rep gene encoding the master replication initiation protein (M-Rep) is included. Non-limiting examples of rep genes for use in the expression system described herein include those set forth in Table 2.

Table 2. Exemplary rep genes

[0197] 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 initiate rolling 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 Vadivukarasi et al. (2007) J Biosc 32: 17-29). Accordingly, those skilled in the art would understand which regions of the rep gene can be modified while still retaining the required activities, and 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 et al. (2008) J Gen Virol 89: 2636-2641).

Exemplary Rep recognition elements

[0198] 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.

[0199] 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 beta satellites (DNA-β satellites) or alphasatellites.

[0200] 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 toxicity gene 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.

[0201] 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 toxicity gene, such as between the terminator that is operably linked to the 3' portion of the rep gene or toxicity gene and the promoter that is operably linked to the 5' portion of the rep gene or the toxicity gene. 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 Mastreviruses. 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.

[0202] 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 3.

Table 3. Exemplary Mastrevirus LIRs and SIRs

[0203] 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 of the 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 Sina loa 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). Table 4 sets forth non-limiting examples of specific Begomovirus IRs that can be used in the expression system described herein.

Table 4. Exemplary Begomovirus IRs

[0204] In other embodiments, Topocuvirus IRs, including fragments or variants thereof that retain the necessary features for rolling circle replication, are utilized as Rep recognition elements. An exemplary Topocuvirus IR is the IR from Tomato pseudo-curly top virus (TPCTV).

[0205] In further embodiments, the Rep recognition elements used in the present invention are Curtovirus IRs, including fragments or variants thereof, such as Curtovirus CRs, that retain the necessary features for rolling circle replication. Exemplary Curtovirus IRs include, but are not limited to, those from Beet curly top Iran virus (BCTIV); Beet curly top virus (BCTV; including the Beet curly top virus-California/Logan, Sugarbeet curly leaf virus, Sugarbeet curly top virus, Sugarbeet virus 1, Tomato yellow virus and Western yellow blight virus); Beet mild curly top virus (BMCTV); Beet severe curly top virus (BSCTV); Horseradish curly top virus (HrCTV); Pepper curly top virus (PepCTV); and Spinach curly top virus (SpCTV). Non-limiting examples of specific

Curtovirus IRs that can be used in the expression system of the present invention are set forth in Table 5.

Table 5. Exemplary Curtovirus IRs

[0206] In other examples, the Rep recognition elements used in the expression system described herein are Nanovirus IRs, including fragments or variants thereof that retain the necessary features for rolling circle replication. For example, IRs from Banana bunchy top virus (BBTV), Faba bean necrotic stunt virus (FBNSV), Faba bean necrotic yellows virus (FBNYV), Milk vetch dwarf virus (MDV); Pea necrotic yellow dwarf virus (PNYDV), or Subterranean clover stunt virus (SCSV) can be used as the Rep recognition elements in the subject expression system. Table 6 sets forth exemplary Nanovirus IRs for use in the invention.

Table 6. Exemplary Nanovirus IRs

[0207] 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, Begomoviruses 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 (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 (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).

[0208] Table 7 sets forth exemplary DNA-β satellite IRs for use in the expression system described herein.

Table 7. Exemplary DNA-β satellite IRs

[0209] 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:41 and 42.

[0210] 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 7 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 (SEQ ID NO:43).

3.4 Other sequences

[0211] 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 host cells and/or transgenic hosts.

[0212] 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 construct or expression cassette, and/or two or more of a second construct or expression cassette, so that the vector comprises two or more copies of the a modulator nucleic acid sequence and/or two or more copies of a target 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 construct. 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. [0213] Desirably, the vector contains one or more elements that permit stable integration of the construct into the host 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 host cell genome when the vector is introduced into a host cell. In some examples, the vector contains additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell, which facilitate integration of the construct into the host 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 host cell. Furthermore, the integrational elements may be non-coding or coding nucleic acid sequences.

[0214] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.

[0215] 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 host cell of choice. The marker gene and effector 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).

[0216] Included within the terms selectable or screenable marker genes are genes that encode a "secretable marker" whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers that encode a secretable antigen that can be identified by antibody interaction, or secretable enzymes that can be detected by their catalytic activity. Secretable 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., a-amylase, β-lactamase,

phosphinothricin acetyltransferase) .

[0217] 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 encod ing 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 (DHFR) 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.

[0218] 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 (luc) gene, which allows for bioluminescence detection; a β-lactamase gene, which encodes an enzyme for which various chromogenic substrates are known (e.g., PAD AC, a chromogenic cephalosporin); an R-locus gene, encoding a product that regulates the production of anthocyanin pigments (red colour) in plant tissues; an - 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.

3.5 Exemplary constructs

[0219] In accordance with the present invention, the subject expression system comprises a component for expressing a target nucleic acid sequence and a component for expressing a modulator nucleic acid sequence that codes for the decoy RNA molecule. Individual components may comprise one or more constructs or one or more expression cassettes to achieve expression of the relevant effector nucleic acid sequence. The present inventors have developed illustrative expression system components to achieve this purpose.

[0220] For example a schematic representation depicting an illustrative construct for inducing production of a decoy RNA molecule is shown in Figure 2. This construct comprises a first expression cassette that includes a modulator nucleic acid sequence that encodes a decoy RNA molecule and that is operably connected to an ethanol inducible promoter (AlcAP) and a transcription terminator (T) (e.g., nopaline synthase terminator). The modulator nucleic acid sequence comprises two

complementary portions (i.e., 5' Decoy and 3' Decoy) spaced apart by an intervening nucleic acid spacer. The construct further comprises a second expression cassette comprising a coding sequence (AlcR) for an alcohol receptor operably linked to a promoter (e.g., a constitutive promoter such as the CaMV 35S promoter) and to a transcription terminator (T) (e.g., nopaline synthase terminator). In the presence of ethanol, the alcohol receptor expressed from AlcR of the second expression cassette binds to c/^'s-acting elements in AlcAP to drive expression of the modulator nucleic acid sequence of the first expression cassette and production of a double stranded decoy RNA molecule that comprises (1) a duplex region formed by base pairing of complementary RNA sequences corresponding to the complementary portions (i.e., 5' Decoy and 3' Decoy) and (2) a single stranded region corresponding to the intervening nucleic acid spacer, which forms a loop connecting the complementary RNA sequences.

[0221] Figure 3 shows a non-limiting example of a construct for constitutively expressing a decoy RNA molecule. This construct comprises an expression cassette comprising a modulator nucleic acid sequence that codes for a decoy RNA molecule and that is operably connected to a constitutive promoter (e.g., CaMV 35S promoter) and to a transcription terminator (T) (e.g., nopaline synthase terminator). The modulator nucleic acid sequence comprises two complementary portions (i.e., 5' Decoy and 3' Decoy) spaced apart by an intervening nucleic acid spacer. Expression of the modulator nucleic acid sequence from the constitutive promoter leads to constitutive production of a double stranded decoy RNA molecule that comprises (1) a duplex region formed by base pairing of complementary RNA sequences corresponding to the complementary portions (i.e., 5' Decoy and 3' Decoy) and (2) a single stranded region corresponding to the intervening nucleic acid spacer, which forms a loop connecting the complementary RNA sequences.

[0222] Figure 4 illustrates an exemplary expression system for conditionally expressing a decoy RNA molecule. The expression system is based on a binary INPACT proreplicon-based expression system (Dugdale et al., 2013), which is useful for tightly regulating gene expression. This system comprises a proreplicon comprising first and second Rep recognition elements (Rep rec 1, Rep rec 2), which mediate ci rcularization and release of a replicon from the proreplicon in the presence of a Rep protein, and an expression cassette for expressing a modulator nucleic acid sequence, which is in the form of a non-contiguous nucleic acid entity or split gene and which encodes a decoy RNA. The expression cassette includes, from 5' to 3', a first complementary portion (3' Decoy) of a modulator nucleic acid sequence, a transcription terminator (T) (e.g., nopaline synthase terminator), a short intergenic region (SIR) (e.g. , a Mastrevirus SIR), a promoter (e.g., a regulated or constitutive promoter), a second complementary portion (5' Decoy) of the modulator nucleic acid sequence, and the second Rep recognition element (Rep rec 2). When the proreplicon is circularized in the presence of a Rep protein, the second Rep recognition element (Rep rec 2) forms an intervening portion of the modulator nucleic acid sequence, which separates the first and second

complementary portions (i.e., 3' Decoy and 5' Decoy, respectively). In operation, expression of a rep gene from an ancillary construct (e.g., an ethanol inducible construct) results in production of the Rep protein, which in turn interacts with the Rep recognition elements (Rep rec 1 and 2) of the proreplicon to facilitate circularization and release of a replicon from the proreplicon and rearrangement of the expression cassette to form a contiguous modulator nucleic acid entity, which comprises in operable linkage, from 5' to 3', the second complementary portion (5' Decoy), the second Rep recognition element (Rep rec 2), the first complementary portion (3' Decoy) and the SIR. Interaction of the Rep protein with the Rep recognition elements (Rep rec 1 and 2) also results in rolling circle replication of the replicon, to thereby amplify the replicon with expression of the contiguous modulator nucleic acid entity. This leads to production of a double stranded decoy RNA that comprises (1) a duplex region formed by base pairing of complementary RNA sequences corresponding to the first and second complementary portions (i.e., 3' Decoy and 5' Decoy) and (2) a single stranded region corresponding to the second Rep recognition element (Rep rec 2), which forms a loop connecting the complementary RNA sequences.

[0223] Figure 5 showing a representative construct for inducing production of Rep protein. This construct comprises a first expression cassette that includes a rep gene encoding two rep gene expression products (Rep and RepA), which is operably connected to an ethanol inducible promoter (AlcAP) and a transcription terminator (T) (e.g., nopaline synthase terminator). The construct further comprises a second expression cassette comprising a coding sequence (AlcR) for an alcohol receptor operably linked to a promoter (e.g., a constitutive promoter such as the CaMV 35S promoter) and to a transcription terminator (T) (e.g., nopaline synthase terminator). In the presence of ethanol, the alcohol receptor expressed from AlcR of the second expression cassette binds to c/^'s-acting elements in AlcAP to drive expression of the rep gene of the first expression cassette and production of Rep protein.

[0224] Figure 6 depicts another non-limiting example of a construct for expressing a decoy RNA molecule. This construct comprises a proreplicon comprising first and second Rep recognition elements (Rep rec 1, Rep rec 2), which mediate

circularization and release of a replicon from the proreplicon in the presence of a Rep protein, and an expression cassette for expressing a modulator nucleic acid sequence, which is in the form of a contiguous nucleic acid entity and which encodes a decoy RNA molecule. The expression cassette includes, from 5' to 3', a promoter (e.g., a regulated or constitutive promoter), a modulator nucleic acid sequence comprising a first complementary portion (5' Decoy), an intervening nucleic acid spacer, a second complementary portion (3' Decoy), and a transcription terminator (T) (e.g., nopaline synthase terminator). In embodiments in which the promoter is a constitutive promoter (e.g., the CaMV 35S promoter), the modulator nucleic acid sequence is constitutively expressed, leading to production of a double stranded decoy RNA molecule that comprises (1) a duplex region formed by base pairing of complementary RNA sequences corresponding to the first and second complementary portions (i.e. , 5' Decoy and 3' Decoy) and (2) a single stranded region corresponding to the second Rep recognition element (Rep rec 2), which forms a loop connecting the complementary RNA sequences. Expression of the modulator nucleic acid sequence and production of the decoy RNA molecule can be boosted by expression of a rep gene from an ancillary construct (e.g., an ethanol inducible construct), which results in production of a Rep protein that interacts with the Rep recognition elements (Rep rec 1 and 2) of the proreplicon to facilitate circularization, release and rolling circle replication of a replicon, thereby amplifying the replicon with boosted expression of the modulator nucleic acid sequence. In embodiments in which the promoter is regulated (e.g., ethanol inducible promoter), expression of the modulator nucleic acid sequence is suitably regulated so that the modulator nucleic acid sequence is expressed largely under the same conditions that induce the transcriptional activity of the promoter that is operably connected to the rep gene. In representative examples of this type, the promoters used for expression of the rep gene and the modulator nucleic acid sequence are inducible/activatable under the same conditions to concurrently stimulate or enhance expression of the rep gene and the modulator nucleic acid sequence.

[0225] Figure 7 shows a representative construct for inducing expression of a gene of interest (GOI). This construct comprises a first expression cassette that includes a GOI encoding a corresponding expression product (e.g., protein or functional RNA), which is operably connected to an ethanol inducible promoter (AlcAP) and a transcription terminator (T) (e.g. , nopaline synthase terminator). The construct further comprises a second expression cassette comprising a coding sequence (AlcR) for an alcohol receptor operably linked to a promoter (e.g., a constitutive promoter such as the CaMV 35S promoter) and to a transcription terminator (T) (e.g., nopaline synthase terminator). In the presence of ethanol, the alcohol receptor expressed from AlcR of the second expression cassette binds to c/^'s-acting elements in AlcAP to drive expression of the GOI and production of its encoded expression product. [0226] Another non-limiting construct for constitutively expressing a gene of interest (GOI) is shown in Figure 8. This construct comprises an expression cassette that includes a GOI encoding a corresponding expression product (e.g., protein or functional RNA), which is operably connected to a constitutive promoter (e.g. , CaMV 35S promoter) and a transcription terminator (T) (e.g., nopaline synthase terminator) .

[0227] Figure 9 illustrates an exemplary IN PACT expression system for conditionally expressing a gene of interest (GOI) . This system comprises a proreplicon comprising first and second Rep recognition elements (Rep rec 1, Rep rec 2), which mediate circularization and release of a replicon from the proreplicon in the presence of a Rep protein, and an expression cassette for expressing a GOI, which is in the form of a split gene and which encodes a corresponding expression product (e.g., protein or functional RNA) . The expression cassette includes, from 5' to 3', a 3' portion of an intron (Intron 3'), a 3' portion of the GOI (3' GOI), a transcription terminator (T) (e.g., nopaline synthase terminator), a short intergenic region (SIR) (e.g., a Mastrevirus SIR), a promoter (e.g., a regulated or constitutive promoter), a 5' portion of the GOI (5' GOI), a 5' portion of the intron (Intron 5') and the second Rep recognition element (Rep rec 2) . When the replicon circularizes in the presence of a Rep protein, the expression cassette is rea rranged to provide in operable connection, from 5' to 3': the promoter; a contiguous GOI entity comprising the 5' portion of the GOI (5' GOI), the 5' portion of the intron (Intron 5'), the second Rep recognition element (Rep rec 2), the 3' portion of the intron (Intron 3') and the 3' portion of the GOI (3' GOI) ; the transcription terminator (T), and the short intergenic region (SIR) intermediate the transcription terminator (T) and the promoter. Expression of the contiguous GOI entity results in an expression product encoded by the 5' and 3' portions of the GOI.

[0228] Another representative example of a n expression system for enhancing expression of a gene of interest (GOI) I shown in Figure 10. This system comprises a first proreplicon for conditionally expressing a decoy RNA molecule and a second proreplicon for conditionally expressing the target nucleic acid sequence. The first proreplicon comprises first and second Rep recognition elements (Rep rec 1, Rep rec 2), which mediate circularization and release of a first replicon from the first proreplicon in the presence of a Rep protein, and a first expression cassette for expressing a modulator nucleic acid sequence, which is in the form of a split gene and which encodes a decoy RNA molecule. The first expression cassette includes, from 5' to 3', a first complementary portion (3' Decoy) of a modulator nucleic acid sequence, a transcription terminator (T) (e.g., nopaline synthase terminator), a short intergenic region (SIR) (e.g., a Mastrevirus SIR), a constitutive promoter (e.g., the CaMV 35S promoter), a second complementary portion (5' Decoy) of the modulator nucleic acid sequence, and the second Rep recognition element (Rep rec 2) that forms an intervening portion of the modulator nucleic acid sequence, which separates the first copy of the first complementary portion (3' Decoy) and the second complementary portion (5' Decoy) when the proreplicon circularizes in the presence of the Rep protein. The second proreplicon comprises first and second Rep recognition elements (Rep rec 1, Rep rec 2), which mediate

circularization and release of a second replicon from the second proreplicon in the presence of the Rep protein, and a second expression cassette for expressing the GOI, wherein the GOI is in the form of a split gene. The second expression cassette includes, from 5' to 3', a 3' portion of an intron (Intron 3'), a 3' portion of the GOI (3' GOI), a transcription terminator (T) (e.g., nopaline synthase terminator), a SIR (e.g. , a

Mastrevirus SIR), a constitutive promoter (e.g., the CaMV 35S promoter), a 5' portion of the GOI (5' GOI), a 5' portion of the intron (Intron 5'), and the second Rep recognition element (Rep rec 2). Expression of a rep gene from an ancillary construct (e.g., an ethanol inducible construct) results in production of the Rep protein that interacts with the Rep recognition elements (Rep rec 1 and 2) of the first and second proreplicons to facilitate circularization, release and rolling circle replication of the first and second replicons. Circularization of the first replicon results in rearrangement of the first expression cassette to form a contiguous modulator nucleic acid entity, which comprises in operable linkage, from 5' to 3', the second complementary portion (5' Decoy), the second Rep recognition element (Rep rec 2), the first copy of the first complementary portion (3' Decoy) and the SIR. Interaction of the Rep protein with the Rep recognition elements (Rep rec 1 and 2) of the first proreplicon also results in rolling circle replication of the first replicon, thereby amplifying the first replicon with expression of the modulator nucleic acid sequence. This leads to production of a double stranded decoy RNA molecule that comprises (1) a duplex region formed by base pairing of complementary RNA sequences corresponding to the first and second complementary portions (i.e. , 3' Decoy and 5' Decoy) and (2) a single stranded region corresponding to the second Rep recognition element (Rep rec 2), which forms a loop connecting the complementary RNA sequences. Circularization of the second replicon results in rearrangement of the second expression cassette to form a contiguous GOI entity, which comprises, from 5' to 3', the 5' portion the GOI (5' GOI), the 5' portion of the intron (Intron 5'), the second Rep recognition element (Rep rec 2), the 3' portion of the intron (Intron 3'), the 3' portion of the GOI (3' GOI), the transcription terminator (T) and the SIR. Interaction of the Rep protein with the Rep recognition elements (Rep rec 1 and 2) of the second proreplicon also results in rolling circle replication of the second replicon, thereby amplifying the second replicon with expression of the contiguous GOI entity and production of its corresponding expression product (e.g., functional RNA or protein).

[0229] Yet another non-limiting example of an expression system for enhancing expression of a gene of interest (GOI) is illustrated in Figure 11. This system comprises a first construct for conditionally expressing decoy RNA molecule, and a second construct for conditionally expressing the target nucleic acid sequence. The first construct comprises a proreplicon ("modulator proreplicon") comprising first and second Rep recognition elements (Rep rec 1, Rep rec 2), which mediate circularization and release of a modulator replicon from the modulator proreplicon in the presence of a Rep protein, and an expression cassette ("modulator expression cassette") for expressing a modulator nucleic acid sequence, which is in the form of a contiguous nucleic acid entity and which encodes a decoy RNA molecule. The modulator expression cassette includes, from 5' to 3', a promoter (e.g. , a regulated or constitutive promoter), a modulator nucleic acid sequence comprising a first complementary portion (5' Decoy), an intervening nucleic acid spacer, a second complementary portion (3' Decoy), and a transcription terminator (T) (e.g., nopaline synthase terminator). The second construct, which is in the form of a proreplicon ("target proreplicon"), comprises first and second Rep recognition elements (Rep rec 1, Rep rec 2), which mediate circularization and release of a target replicon from the target proreplicon in the presence of the Rep protein, and an expression cassette ("target expression cassette") for expressing the GOI, which is in the form of a split gene. The target expression cassette includes, from 5' to 3', a 3' portion of an intron (Intron 3'), a 3' portion of the GOI (3' GOI), a transcription terminator (T) (e.g., nopaline synthase terminator), a short intergenic region (SIR) (e.g., a Mastrevirus SIR), a constitutive promoter (e.g., the CaMV 35S promoter), a 5' portion of the GOI (5' GOI), a 5' portion of the intron (Intron 5'), and the second Rep recognition element (Rep rec 2). In embodiments in which the promoter of the modulator proreplicon is a constitutive promoter (e.g., the CaMV 35S promoter), the modulator nucleic acid sequence is constitutively expressed with constitutive production of a double stranded decoy RNA molecule that comprises (1) a duplex region formed by base pairing of complementary RNA sequences corresponding to the first and second complementary portions (i.e., 5' Decoy and 3' Decoy) and (2) a single stranded region corresponding to the second Rep recognition element (Rep rec 2), which forms a loop connecting the complementary RNA sequences. Expression of the modulator nucleic acid sequence and production of the decoy RNA molecule can be boosted by expression of a rep gene from an ancillary construct (e.g., an ethanol inducible construct), which results in production of a Rep protein that interacts with the Rep recognition elements (Rep rec 1 and 2) of the proreplicon to facilitate circularization, release and rolling circle replication of the modulator replicon. This results in amplification of the modulator replicon with boosted expression of the modulator nucleic acid sequence. In embodiments in which the promoter is regulated (e.g., ethanol inducible promoter), expression of the modulator nucleic acid sequence is suitably regulated so that the modulator nucleic acid sequence is expressed largely under the same conditions that induce the transcriptional activity of the regulated promoter that drives expression of the rep gene. The Rep protein produced through expression of the rep gene also interacts with the Rep recognition elements (Rep rec 1 and 2) of the target proreplicon to facilitate circularization, release and rolling circle replication of the target replicon, which results in rearrangement of the target expression cassette to form a contiguous GOI entity comprising in operable connection, from 5' to 3', the 5' portion of the GOI (5' GOI), the 5' portion of the intron (Intron 5'), the second Rep recognition element (Rep rec 2), the 3' portion of the intron (Intron 3'), the 3' portion of the GOI (3' GOI), the transcription terminator (T) (e.g. , nopaline synthase terminator) and the SIR. Interaction of the Rep protein with the Rep recognition elements (Rep rec 1 and 2) of the target proreplicon also results in rolling circle replication of the target replicon, to thereby amplify the target replicon with expression of the Contiguous GOI entity and production of its corresponding expression product (e.g., functional RNA or protein).

4. Methods of producing transgenic hosts

[0230] The present invention contemplates introducing the subject expression system in any host in which silencing occurs. Representative hosts will include eukaryotic organisms such as, but not limited to, fungi such as yeast (e.g., yeast strains for fermentation including strains of the genus Sacchromyces spp. e.g., Sacchromyces cerevisiae (baker's yeast), Saccharomyces distaticus, Saccharomyces uvarum; the genus Kluyveromyces, e.g., species Kluyveromyces marxianus, Kluyveromyces fragilis; the genus Candida, e.g., Candida pseudotropicalis, and Candida brassicae, Pichia stipitis (a relative of Candida shehatae, the genus Clavispora, e.g., species Clavispora lusitaniae and Clavispora opuntiae the genus Pachysolen, e.g., species Pachysolen tannophilus, the genus Bretannomyces, e.g., species Bretannomyces clausenii (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol : Production and

Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212)) and filamentous fungi, including species of Aspergillus, Trichoderma, and Neurospora; animal hosts including vertebrate animals illustrative examples of which include fish (e.g., salmon, trout, tulapia, tuna, carp, flounder, halibut, swordfish, cod and zebrafish), birds (e.g., chickens, ducks, quail, pheasants and turkeys, and other jungle foul or game birds) and mammals (e.g., dogs, cats, horses, cows, buffalo, deer, sheep, rabbits, rodents such as mice, rats, hamsters and guinea pigs, goats, pigs, primates, marine mammals including dolphins and whales, as well as cell lines, such as human or other mammalian cell lines of any tissue or stem cell type (e.g., COS, NIH 3T3 CHO, BHK, 293, or HeLa cells), and stem cells, including pluripotent and non-pluripotent and embryonic stem cells, and non-human zygotes), as well as invertebrate animals illustrative examples of which include nematodes (representative genera of which include those that infect animals such as but not limited to Ancylostoma, Ascaridia, Ascaris, Bunostomum, Caenorhabditis, Capillaria, Chabertia, Cooperia, Dictyocaulus, Haernonchus, Heterakis, Nematodirus, Oesophagostomum, Ostertagia, Oxyuris, Parascaris, Strongylus,

Toxascaris, Trichuris, Trichostrongylus, Tflichonema, Toxocara, Uncinaria, and those that infect plants such as but not limited to Bursaphalenchus, Criconerriella, Diiylenchus, Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus, Melodoigyne,

Nacobbus, Paratylenchus, Pratylenchus, Radopholus, Rotelynchus, Tylenchus, and Xiphinerna) and other worms, drosophila, and other insects (such as from the families Apidae, Curculionidae, Scarabaeidae, Tephritidae, Tortricidae, amongst others, representative orders of which include Coleoptera, Diptera, Lepidoptera , and Homoptera.

[0231] In certain embodiments, the host is a plant which is suitably selected from monocotyledons, dicotyledons and gymnosperms. The plant may be an ornamental plant or crop plant. Illustrative examples of host cells from ornamental plants include, but are not limited to, host cells from Malus spp, Crataegus spp, Rosa spp., Betula spp, Sorbus spp, Olea spp, Nerium spp, Salix spp and Populus spp. Illustrative examples of host cells from crop plants include host 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 uncinate (broombush), Mentha tasmannia, Mentha spicata (spearmint), Mentha X piperita

(peppermint), Momordica charantia (bitter melon), Musa spp. (banana), Myrciaria cauliflora (jaboticaba), Myrothamnus flabellifolia, 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. (poppyj, 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 canariensis (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), Sclerocarya 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 lanceolate (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) host cells.

[0232] 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 (e.g ., a muscle, skin, brain, lung, kidney, pancreas, a reproductive organ such as testes, ovaries and breast, eye, liver, heart, vascular cell, root, leaf, flower, stalk or meristem) or into an organ of the organism. Alternatively, the construct may be introduced into a progenitor of the organism and the progenitor is then grown or cultured for a time and 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, but 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 mammals, by the oral, parenteral (e.g., intravenous, intramuscular, intraperitoneal, intaventricular, intrarticular), mucosal (e.g., intranasal, intrapulmonary, oral, buccal, sublingual, rectal, intravaginal), dermal (topical, subcutaneous, transdermal); for plants, administration to flowers, meristem, root, 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-after phenotype.

Desirably, the synthetic constructs are introduced into the same or corresponding site. In other embodiments, the synthetic construct is introduced into a cell 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.

[0233] 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 differ 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 host 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 : 365- 368). The synthetic constructs may be coated onto microprojectiles, and delivered into a host 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 of the organism. In certain embodiments, a hydrodynamic nucleic acid administration protocol is 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).

[0234] 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 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, PEG-mediated nucleic acid uptake, as well as any other electrical, chemical, physical (mechanical) and/or biological mechanism that results in the introduction of 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, 1 E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7: 849-858 (2002)).

[0235] Thus, in some particular embodiments, the introducing into a plant host is via bacterial-mediated transformation, particle bombardment transformation, calcium- phosphate-mediated transformation, cyclodextrin-mediated transformation,

electroporation, liposome-mediated transformation, nanoparticle-mediated

transformation, polymer-mediated transformation, virus-mediated nucleic acid delivery, whisker-mediated nucleic acid delivery, microinjection, sonication, infiltration, polyethylene glycol-mediated transformation, any other electrical, chemical, physical and/or biological mechanism that results in the introduction of nucleic acid into the plant host.

[0236] Agrobacterium-mediated transformation is a commonly used method for transforming plants, in particular, dicot plants, because of its high efficiency of transformation and because of its broad utility with many different species.

Agrobacterium- mediated transformation typically involves transfer of the binary vector carrying the foreign DNA of interest to an appropriate Agrobacterium strain that may depend on the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (Uknes et ai. (1993) Plant Cell 5: 159-169). The transfer of the recombinant binary vector to Agrobacterium can be accomplished by a triparental mating procedure using Escherichia coli carrying the recombinant binary vector, a helper E. coli strain that carries a plasmid that is able to mobilize the

recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by nucleic acid

transformation (Hofgen & Willmitzer (1988) Nucleic Acids Res. 16:9877). Transformation of a plant by recombinant Agrobacterium usually involves co-cultivation of the

Agrobacterium with explants from the plant and follows methods well known in the art. Transformed tissue is regenerated on selection medium carrying an antibiotic or herbicide resistance marker between the binary plasmid T-DNA borders.

[0237] Another method for transforming plant hosts involves propelling inert or biologically active particles at plant tissues and cells. See, e.g. , U.S. Patent Nos.

4,945,050; 5,036,006 and 5,100,792. Generally, this method involves propelling inert or biologically active particles at the plant cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the nucleic acid of interest. Alternatively, a cell or cells can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., a dried yeast cell, a dried bacterium or a bacteriophage, each containing one or more nucleic acids sought to be introduced) also can be propelled into plant tissue. Thus, in particular embodiments of the present invention, a plant cell can be transformed by any method known in the art and as described herein and intact plants can be regenerated from these transformed cells using any of a variety of known techniques. Plant regeneration from plant cells, plant tissue culture and/or cultured protoplasts is described, for example, in Evans et al. (Handbook of Plant Cell Cultures, Vol. 1, MacMilan Publishing Co. New York (1983)); and Vasil I. R. (ed.) (Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984), and Vol. II (1986)). Methods of selecting for transformed, transgenic plants, plant cells and/or plant tissue culture are routine in the art and can be employed in the methods of the invention provided herein. Likewise, the genetic properties engineered into the transgenic seeds and plants, plant parts, and/or plant cells of the present invention described above can be passed on by sexual reproduction or vegetative growth and therefore can be maintained and propagated in progeny plants. Generally, maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as harvesting, sowing or tilling. A nucleotide sequence therefore can be introduced into the plant, plant part and/or plant cell in any number of ways that are well known in the art. The methods of the invention do not depend on a particular method for introducing one or more nucleotide sequences into a plant, only that they gain access to the interior of at least one cell of the plant. Where more than one nucleotide sequence is to be introduced, the respective nucleotide sequences can be assembled as part of a single nucleic acid construct/molecule, or as separate nucleic acid constructs/molecules, and can be located on the same or different nucleic acid

constructs/molecules. Accordingly, the nucleotide sequences can be introduced into the cell of interest in a single transformation event, in separate transformation events, or, for example, in plants, as part of a breeding protocol. In some embodiments of this invention, the introduced nucleic acid molecule may be maintained in the plant cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosome(s). Alternatively, the introduced construct may be present on an extra-chromosomal non-replicating vector and be transiently expressed or transiently active. Whether present in an extra-chromosomal non-replicating vector or a vector that is integrated into a chromosome, the nucleic acid molecule can be present in a plant expression construct.

[0238] To confirm the presence of the constructs in the regenerating plants, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, Western blotting, enzyme assays and PCR.

[0239] 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

INPACT-BASED CONSTRUCT FOR EXPRESSING DECOY RNA

[0240] An exemplary expression system for high level expression decoy RNA is suitably based on the INPACT expression system (see, Figure 12). This system comprises a proreplicon in which inverted repeat DNA sequences forming the stem of the double stranded decoy RNA molecule are split into two exons, of which the promoter and 5' end of the split gene is positioned downstream of the 3' end of the gene and terminator. The split cassette is flanked by the TYDV large intergenic regions (LIRs) which contain the viral genomic c/^'s-acting elements necessary for first-strand synthesis and these LIRs, in turn, are embedded within an intron. The cassette also contains the TYDV small intergenic region (SIR) within which reside c/^'s-acting elements necessary for host- mediated second-strand synthesis. As an integrated sequence, the INPACT cassette cannot be expressed. However, in the presence of the virus-encoded activation proteins, TYDV Rep and RepA, the integrated INPACT cassette serves as a template for duplication by a process known as rolling circle replication (RCR), resulting in the production of a circular, extra-chromosomal, ssDNA copy of the INPACT cassette. This episome is then converted to a dsDNA molecule via the SIR, and host polymerases. This molecular form is both transcriptionally active and can serve as a template for further amplification and as such is termed a replisome (replicating episome). Finally, the transgene mRNA is processed to remove the intron (and embedded LIR) and form the dsRNA hairpin.

Therefore, transgene amplification and expression from the INPACT cassette is strictly dependent on TYDV Rep/RepA abundance. In order to regulate both Rep and RepA proteins, the bicistronic Rep/RepA coding sequence is placed under the transcriptional control of the alcohol-inducible AlcA:ALCR gene switch. This system provides for temporal, spatial and dose-dependent control of target gene expression. The LIR was tailored to remove non-essential sequences by deleting 56 bp from the 3' end and corrupting two potential intron splice sites. No changes were made to the 5' end of the LIR which contains a CA motif and iterated sequences thought to be essential for cognate Rep recognition/replication nor to the stem-loop sequence which contains the origin of first strand synthesis. A small synthetic intron termed a "syntron" was also designed to specifically house the LIR. The syntron sequence contains consensus plant donor

(AG/GTAAG) and acceptor (TGCAG/GT) splice sites (Goodall and Filipowicz, 1989), U-rich tracts repeated within proximity to the 5' and 3' splice sites (Baynton et a/., 1996), and a branch point consensus sequence appropriately located upstream of the 3' splice junction. [0241] The double stranded decoy RNA is suitably designed so that it has little or no homology to a transgene that is introduced into the same host (see, Figure 13). Upon transcription of the inverted repeat, a dsRNA hairpin (the decoy RNA molecule) is formed, which permeates the RNA silencing pathway and competes with other substrates of that pathway to thereby reduce PTGS of the transgene.

[0242] Alternatively, or in addition, the decoy RNA can be designed so that it has little or no homology to an endogenous gene in the same host in to which the INPACT expression system is introduced (Figure 14). Upon transcription of the inverted repeat, a dsRNA hairpin is formed, which permeates the RNA silencing pathway and competes with other substrates of that pathway to thereby reduce PTGS of the endogenous gene.

EXPERIMENTAL GENERATION OF TRANSGENIC PLANTS

Enhanced reporter gene expression by co-expression of a non-specific hpRNA

GFP

[0243] The transient expression of a GFP reporter was assessed in co- infiltrations with non-specific hpRNA expression vectors and compared to three described VSR proteins, P19, PI and PO. Hairpin RNA expression vectors used for this analysis, p35S-DCLlhp and pSSU-CHShp (Figure 15), were designed with homology to their corresponding target genes in Arabidopsis thaliana (DICER-LIKEl (DCLl, Atl G01040) and CHALCONE SYNTHASE (CHS, At5G13930) respectively). These hpRNAs display limited homology to N. benthamiana transcripts from the draft transcriptome.

Furthermore, Arabidopsis DCLl is involved in microRNA biogenesis and is not expected to participate in the production of siRNAs triggered by GFP transgene expression. CHS is required for anthocyanin production and is not thought to have a role in RNA silencing.

[0244] The lone 35S-GFP infiltration resulted in limited GFP expression with fluorescence decreasing rapidly from 3 DPI and falling below detection at 8DPI (data not shown and Figure 16. A-l). Co-infiltration of 35S-GFP with VSR proteins yielded strong green fluorescence (Figure 16. A-2, 3 and 6) reflecting the limitation of reporter transgene expression by RNA silencing. As previously described, TBSV P19 displayed the greatest RNA silencing suppressor activity (Fusaro et al., 2012). Co-infiltration of 35S- GFP and either of the non-specific hpRNAs enhanced reporter transgene expression as evidenced by fluorescence levels similar to those observed with either PO or PI VSR proteins (Figure 16. A-4 and 5). GUS reporter

[0245] β-glucuronidase (GUS) reporter transgenes were similarly co-infiltrated to quantify the expression levels obtained with each suppressor system. Two reporter constructs were used; 35S_GUS, a conventional gene expression vector containing the uidA reporter gene encoding GUS under the control of the CaMV 35S promoter, and 35S_CPMV-GUS containing the same expression cassette with CPMV 5' and 3' enhancer elements. The pSSU-CHShp vector was again used in these experiments alongside a 35S promoter driven hpRNA specific to GFP p35S-hpGFP).

[0246] Histochemical GUS staining of N. benthamiana whole leaves was performed 8 DPI in leaves co-infiltrated with each GUS reporter construct and the candidate RNA silencing suppressors. As expected, both GUS reporter constructs were vulnerable to RNA silencing (Figures 16. B,C,D) with visibly enhanced expression by co- infiltration with P19 (Figures 16. B-2, C-2 and D-2). GUS reporter protein from both constructs also accumulated to higher levels in tissues co- infiltrated with the SSU_hpCHS or the 35S_hpGFP (Figures 16. B-3, C-3 and B-l, C-l respectively).

[0247] GUS-ELISA assays reported maximal GUS activity at 3 DPI in control infiltrations (Figure 17). When GUS was flanked with the CPMV enhancer elements the activity was higher and decreased at a slower rate (Figure 17. B). In both cases, GUS expression was enhanced by co-infiltration with P19 (Figure 17. A and B) with enzymatic activity detectably higher for the entire period assessed (18 days). In contrast, co- infiltration with PO only elevated expression levels significantly at 3 and 5 DPI. Co- infiltration with PI had little observable effect.

[0248] Co-infiltration of either non-specific hpRNA construct (SSU_hpCHS or 35S_hpGFP) with each of the GUS reporter constructs similarly enhanced reporter gene activity at 3 DPI, with GUS levels reaching up to 80% of those achieved by co- infiltration with P19. Interestingly, the highly active 35S promoter driven hpRNA

(35S_hpGFP) did not sustain comparable silencing suppressor activity beyond 3-5 DPI. However, the SSU_hpCHS driven by a relatively weak Arabidopsis SSU promoter (McHale 2013) sustained expression to an average of 80% that of co-expression with P19.

Non-specific hairpin RNA expression limits reporter gene specific small -interfering

RNA accumulation

[0249] The present inventors theorized that co-expression of the non-specific hpRNAs with desired recombinant proteins, in this case GFP or GUS, provides a decoy for the RNA silencing machinery. To test this hypothesis, they performed northern blotting for sRNA accumulation at 8 DPI in tissues from co-infiltration of either the 35S_GUS (Figure 18. A) or 35S_CPMV-GUS (Figure 18. B) with the P19 VSR or either of the nonspecific hpRNAs. [0250] In the absence of a silencing suppressor, abundant siRNAs were detected for both the 35S_GUS and the 35S_CPMV-GUS. As expected, siRNAs specific to the GUS transcript accumulated to a lower level in the presence of the P19 VSR. Co- expression of the 35S_hpGFP or SSU_hpCHS resulted in reduced GUS specific siRNA accumulation, although not to the same extent as P19. The predicted hpRNA derived siRNAs accumulated to readily detectable levels from each of the hpRNA expression constructs (Figure 18 - bottom lane). This result suggests that siRNA production from non-specific hairpin RNA limits the production of reporter gene specific siRNAs rather than directing suppression of the silencing machinery.

Co-infiltration of a non-specific hpRNA with VSR proteins suggests silencing suppressor activity by competitive loading of silencing machinery

[0251] To further test whether silencing suppression by co-expression of a nonspecific hpRNA is mediated by competitive siRNA production, GUS activity was analyzed after co-infiltration of the GFP hairpin with each of the described VSR proteins (P19, PO or PI in Figure 19. C).

[0252] Interestingly, co-expression of both the 35S_hpGFP and either PO or PI VSR proteins achieved higher levels of reporter gene expression than co-expression with the 35S_hpGFP and P19 (Figure 19. C). As both PO and PI reduce silencing activity by reducing the abundance of effector proteins, these suppressors would are expected to support cumulative suppression of silencing with the proposed competitive siRNA production. This competitive siRNA accumulation would however be expected to interfere with P19 directed suppression as the capacity of P19 to sequester reporter gene derived siRNAs would be limited by competitive loading of hpRNA derived siRNAs.

Transient expression of 35S>GUS, co-infiltrated with pINPACT-GFPhp +

35S>REP/REPA (+ controls).

[0253] Wild-type Nicotiana tabacum plants were co-infiltrated by Agrobacterium infiltration with a construct comprising a coding sequence for GUS under the control of the CaMV 35S promoter (35S>GUS) and with an INPACT expression system that comprises a proreplicon from which a double stranded decoy RNA specific for GFP

(INPACT-GFPhp) is conditionally expressible in the presence of a Rep protein that is produced from an ancillary construct in which a coding sequence or Rep/RepA is under the control of the CaMV 35S promoter (35S>REP>REPA).

[0254] The results of these experiments shown in Figure 20 reveal that accumulation of the GUS protein was enhanced 2.5-fold in plants co-infiltrated with 35S>GUS, 35S>REP>REPA and INPACT-GFPhp (the decoy). There was also an enhancement, albeit a lesser one when the decoy was co- infiltrated with INPACT-GFPhp and pBIN+ (which should be un-inactivated INPACT). This was probably due to low levels of recombination, which is known to occur when the INPACT cassette is infiltrated without REP/REPA, allowing some decoy expression.

Stable expression of 35S>GUS, co-infiltrated with pINPACT-GFPhp +

35S>REP/REPA (+ controls).

[0255] Wild-type Nicotiana tabacum plants were co-infiltrated by Agrobacterium infiltration with a construct comprising a coding sequence for GUS under the control of the CaMV 35S promoter (35S>GUS) and with an INPACT expression system that comprises a proreplicon from which a double stranded decoy RNA specific for GFP (INPACT-GFPhp) is conditionally expressible in the presence of a Rep protein that is produced from an ancillary construct in which a coding sequence or Rep/RepA is under the control of the CaMV 35S promoter (35S>REP>REPA). Transgenic plants were selected in which these constructs were stably introduced and GUS protein accumulation was quantified prior to infiltration (Day 0), and 4 days post infiltration (Day 4) by ELISA. Leaf tissue was independently collected from infiltrated leaves and un-infiltrated leaves from the top or bottom of each plant on day 4 post infiltration.

[0256] The results of these experiments shown in Figure 21 indicate that GUS protein accumulation was enhanced 3.5-fold in stable transgenic plants following infiltration with the decoy (INP-GFPhp + REP>REPA). GUS protein accumulation was not enhanced in stable transgenic plants infiltrated without the decoy (35S>REP/REPA, pBIN+), or infiltrated with the un-activated decoy (INPACT-GFP-hp + pBIN+).

[0257] The apparent enhancement conferred by low level decoy expression, via recombination of the INPACT cassette, in the previous experiment (where plants were infiltrated with un-activated INPACT-GFPhp), probably wasn't seen in this experiment as PTGS of transgenes is much higher when the GOI is expressed transiently (rather than stably. Thus, any reduction in PTGS (e.g., through low level expression of the decoy via recombination) is more likely to result in a bump in protein accumulation where the GOI is expressed transiently, rather than stably (as was observed).

[0258] This experiment also illustrates, for the first time, that the double stranded decoy RNA can enhance expression of a stably expressed gene.

EXAMPLE 2

EFFECT OF DECOY RNA EXPRESSION LEVELS ON INPACT-DIRECTED EXPRESSION OF A TARGET

GENE

[0259] Transgenic tobacco (Nicotiana tabacum cv. Samsun) parent line SRN6; a parent line containing a single copy of the Rep/RepA genes required to activate the INPACT system under the transcriptional control of an ethanol gene switch, was super- transformed with INPACT-GUS plus one of three different hairpin 1 (hpl) constructs (i-iii, below), in which hpl refers to a double stranded hairpin RNA decoy molecule with specificity to the movement protein of Tobacco yellow leaf curl virus (also referred to as "MPhp"). These three cassettes represent three different levels of dsRNA hpl decoy expression. Plants were regenerated, EtOH activated and GUS accumulation measured. GUS levels were compared to baseline control plants containing INPACT-GUS alone.

(i) AlcA-hpl (weak inducible decoy expression)

(ii) 35S-hpl (strong constitutive decoy expression)

(iii) INPACT-hpl (strong inducible amplified decoy expression)

Results

Tobacco transformation, regeneration and PCR screening

[0260] The above constructs were singly or in combinations used for transformation of tobacco (cv. Samsun NN) parent line SRN6 via Agrobacterium- mediated infection of leaf disks. The cassette combinations used were: 1) INPACT-GUS (no decoy) 2) INPACT-GUS + Alc-hpl, 3) INPACT-GUS + 35S-hpl, 4) INPACT-GUS + INPACT-hpl .

[0261] Transgenic tobacco plants were regenerated on media containing the appropriate antibiotic. Leaf samples were harvested, snap frozen in liquid nitrogen and total gDNA extracted using a rapid release method. PCRs with primers specific to sections of the GUS, or hpl nucleotide sequences were used to determine whether plants contained the appropriate cassettes. PCR products were electrophoresed throug h agarose. Plasmid positive (+ve) and wild-type genomic DNA negative (-ve) controls were included for each primer set. The results are summarized in Table 8. No lines for construct combination 4 have yet been generated; however explants at the callus stage are still under regeneration.

Table 8. Summary of transgenic tobacco lines regenerated for Example 2

#PCR #PCR +ve xl #PCR +ve x2

Construct combination Parent screened cassette cassettes

INPACT-GUS SRN6 Π Π

INPACT-GUS-Alc-hpl SRN6 13 11

INPACT-GUS-35S-hpl SRN6 15 12 INPACT-GUS + INPACT-hpl SRN6 37 37 0 GUS expression analysis

[0262] Ten lines each of construct combinations 1, 2 and 3 were EtOH activated (5% EtOH activation of tissue culture plants). For screening purposes a single replicate of each respective line was activated and tissue harvested on days 0 and 4 post EtOH activation from each plant. Total soluble protein (TSP) was extracted and GUS expression for each tissue sample was measured by 4-methylumbelliferyl β-D-glucuronide (MUG) analysis on day 4 post EtOH activation to identify elite lines, as illustrated in Figure 22.

[0263] The addition of Alc-hpl or 35S-hpl does not appear to increase INPACT driven GUS expression day 4, post ethanol activation, indicating that stronger expression of hpl by a replicon based expression system such as, for example, the INPACT vector system, is desirable to permeate the RNA silencing pathway and compete with other RNA silencing pathway substrates to thereby induce silencing of hpl expression, with reduced silencing of GUS expression.

EXAMPLE 3

EFFECT OF DIFFERENT DECOY RNAS ON TARGET GENE EXPRESSION

[0264] Transgenic tobacco parent line SRN6 was super-transformed with INPACT-GUS plus one of two vectors (i-ii below) containing different inverted repeat DNA sequences (hairpins).

(i) INPACT-hpl

(ii) INPACT-hp2

[0265] Hairpin 2 (hp2) is a double stranded hairpin RNA decoy molecule with specificity to GFP (also referred to herein as "GFPhp").

[0266] For transient expression analysis, N. benthamiana (Benthi) plants were co-infiltrated with recombinant Agrobacteria harboring combinations of the two INPACT- hp cassettes above, and a non-replicating GUS expression vector. GUS expression was compared to plants infiltrated with the GUS construct and no INPACT-hp cassette.

Results

Stable transformations, regeneration PCR screening

[0267] As for Example 2, the above constructs were singly or in combinations used for transformation of tobacco (cv. Samsun NN) parent line SRN6 via Agrobacterium- mediated infection of leaf disks.

[0268] Transgenic tobacco plants were regenerated on media containing the appropriate antibiotic. Leaf samples were harvested, snap frozen in liquid nitrogen and total gDNA extracted using a rapid release method. PCRs with primers specific to sections of the GUS, hpl or hp2 nucleotide sequences were used to determine whether plants contained the appropriate cassettes. PCR products were electrophoresed through agarose. Plasmid positive (+ve) and wild-type genomic DNA negative (-ve) controls were included for each primer set. The results are summarized in Table 9; plants containing construct combination 3 are still being regenerated .

Table 9 Summary of transgenic tobacco lines regenerated for Example 3

PCR PCR +ve xl PCR +ve x2

Construct combination Parent screened cassette cassettes

INPACT-GUS Reported in Table 8

INPACT-GUS + INPACT-hpl Reported in Table 8

INPACT-GUS + INPACT-hp2 SRN6 56 47 9

Transient transformations and gene expression analysis

[0269] Tobacco and N. benthamiana were infiltrated for transient

Agrobacterium mediated transformation. Three leaves of three individual plants per construct combination were infiltrated and tissue collected on day 4 post infiltration.

[0270] The construct combinations used and respective results are detailed below.

1) Empty-vector + 35S>Rep/RepA (control infiltration)

2) 35S-GUS-NOS + Empty Vector + 35S>Rep/RepA

3) 35S-GUS-NOS + INPACT-hpl + 35S>Rep/RepA

4) 35S-GUS-NOS + INPACT-hp2 + 35S>Rep/RepA

[0271] Tissue was collected day 4 post infiltration, TSP was extracted and analyzed by MUG analysis.

[0272] The results presented in Figure 23 show that transient GUS expression levels in tobacco were enhanced ~4 fold by the addition of either INPACT-hpl or INPACT hp2.

[0273] The results presented in Figure 24 show that transient GUS expression in N. benthamiana was enhanced ~4 fold by the addition of INPACT-hpl and ~3 fold by the addition of INPACT-hp2.

[0274] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety. [0275] 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.

[0276] 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.

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Claims

WHAT IS CLAIMED IS:

1. An expression system for expressing a target nucleic acid sequence in a host cell with reduced RNA silencing of the target nucleic acid sequence, the expression system comprising a first expression system component (e.g., comprising one or more expression cassettes or constructs) and a second expression system component (e.g., comprising one or more expression cassettes or constructs), wherein the target nucleic acid sequence is expressible from the first expression system component, wherein a modulator nucleic acid sequence is expressible from the second expression system component, and wherein expression of the modulator nucleic acid sequence in the host cell produces a double stranded decoy RNA molecule that is a substrate of the RNA silencing pathway and that is non-homologous with a RNA expression product of the target nucleic acid sequence, wherein the decoy RNA molecule competes with other substrates of the RNA silencing pathway to thereby induce silencing of the modulator nucleic acid sequence with reduced silencing of the target nucleic acid sequence.

2. An expression system according to claim 1, wherein the double stranded decoy RNA molecule has 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 the RNA expression product of the target nucleic acid sequence.

3. An expression system according to claim 1 or claim 2, wherein the double stranded decoy RNA molecule has 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 or more endogenous RNA expression products in the host cell .

4. An expression system according to any one of claims 1 to 3, wherein the double stranded decoy RNA molecule is at least 17 nucleotides and as much as 3000 nucleotides in length (and all integer nucleotide lengths in between) .

5. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 80% sequence identity over a subseq uence of the RNA expression product.

6. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 75% sequence identity over a subsequence of the RNA expression product.

7. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 70% sequence identity over a subsequence of the RNA expression product.

8. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 65% sequence identity over a subsequence of the RNA expression product.

9. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 60% sequence identity over a subsequence of the RNA expression product.

10. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 55% sequence identity over a subsequence of the RNA expression product.

11. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 50% sequence identity over a subsequence of the RNA expression product.

12. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 45% sequence identity over a subsequence of the RNA expression product.

13. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 40% sequence identity over a subsequence of the RNA expression product.

14. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 35% sequence identity over a subsequence of the RNA expression product.

15. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 30% sequence identity over a subsequence of the RNA expression product.

16. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 25% sequence identity over a subsequence of the RNA expression product.

17. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 20% sequence identity over a subsequence of the RNA expression product.

18. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 15% sequence identity over a subsequence of the RNA expression product.

19. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 10% sequence identity over a subsequence of the RNA expression product.

20. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 5% sequence identity over a subsequence of the RNA expression product.

21. An expression system according to claim 2 or claim 4, wherein the decoy RNA molecule has no more than about 1% sequence identity over a subsequence of the RNA expression product.

22. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 80% sequence identity over a subsequence of an individual endogenous RNA expression product.

23. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 75% sequence identity over a subsequence of an individual endogenous RNA expression product.

24. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 70% sequence identity over a subsequence of an individual endogenous RNA expression product.

25. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 65% sequence identity over a subsequence of an individual endogenous RNA expression product.

26. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 60% sequence identity over a subsequence of an individual endogenous RNA expression product.

27. An expression system according to claim 3 or clai m 4, wherein the decoy RNA molecule has no more than 55% sequence identity over a subsequence of an individual endogenous RNA expression product.

28. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 50% sequence identity over a subsequence of an individual endogenous RNA expression product.

29. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 45% sequence identity over a subsequence of an individual endogenous RNA expression product.

30. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 40% sequence identity over a subsequence of an individual endogenous RNA expression product.

31. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 35% sequence identity over a subsequence of an individual endogenous RNA expression product.

32. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 30% sequence identity over a subsequence of an individual endogenous RNA expression product.

33. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 25% sequence identity over a subsequence of an individual endogenous RNA expression product.

34. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 20% sequence identity over a subsequence of an individual endogenous RNA expression product.

35. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 15% sequence identity over a subsequence of an individual endogenous RNA expression product.

36. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 10% sequence identity over a subsequence of an individual endogenous RNA expression product.

37. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 5% sequence identity over a subsequence of an individual endogenous RNA expression product.

38. An expression system according to claim 3 or claim 4, wherein the decoy RNA molecule has no more than 1% sequence identity over a subsequence of an individual endogenous RNA expression product.

39. An expression system according to any one of claims 5 to 38, wherein the subsequence is 17 nucleotides in length.

40. An expression system according to any one of claims 5 to 38, wherein the subsequence is 18 nucleotides in length.

41. An expression system according to any one of claims 5 to 38, wherein the subsequence is 19 nucleotides in length.

42. An expression system according to any one of claims 5 to 38, wherein the subsequence is 20 nucleotides in length.

43. An expression system according to any one of claims 5 to 38, wherein the subsequence is 21 nucleotides in length.

44. An expression system according to any one of claims 5 to 386, wherein the subsequence is 22 nucleotides in length.

45. An expression system according to any one of claims 5 to 38, wherein the subsequence is 23 nucleotides in length.

46. An expression system according to any one of claims 5 to 38, wherein the subsequence is 24 nucleotides in length.

47. An expression system according to any one of claims 5 to 38, wherein the subsequence is 25 nucleotides in length.

48. An expression system according to any one of claims 1 to 47, wherein the double stranded decoy RNA molecule is selected from long dsRNA (e.g. , a precursor sRNA that is suitably a substrate for DICER or a DICER-like protein), siRNA and shRNA.

49. An expression system according to any one of claims 1 to 48, wherein the double stranded decoy RNA molecule 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.

50. An expression system according to any one of claims 1 to 49, wherein the host cell is selected from eukaryotic cells in which RNA-mediated gene silencing occurs.

51. An expression system according to claim 50, wherein the host cell is an animal (e.g., mammalian) host cell .

52. An expression system according to claim 50, wherein the host cell is a plant host cell .

53. An expression system according to any one of claims 1 to 52, wherein the modulator nucleic acid sequence is, or corresponds to, a heterologous nucleic acid sequence.

54. An expression system according to any one of claims 1 to 52, wherein the modulator nucleic acid sequence is, or corresponds to, an endogenous nucleic acid sequence.

- I l l -

55. An expression system according to any one of claims 1 to 54, wherein one or both of the target nucleic acid sequence and the modulator nucleic acid sequence is conditionally expressible.

56. An expression system according to any one of claims 1 to 54, wherein one or both of the target nucleic acid sequence and the modulator nucleic acid sequence is constitutively expressible.

57. An expression system according to any one of claims 1 to 54, wherein the target nucleic acid sequence is constitutively expressible and the modulator nucleic acid sequence is conditionally expressible.

58. An expression system according to any one of claims 1 to 54, wherein the target nucleic acid sequence and the modulator nucleic acid sequence are both conditionally expressible.

59. An expression system according to any one of claims 1 to 54, wherein the target nucleic acid sequence is conditionally expressible and the modulator nucleic acid sequence is constitutively expressible.

60. An expression system according to any one of claims 1 to 54, wherein the target nucleic acid sequence is conditionally expressible and the modulator nucleic acid sequence is constitutively and optionally conditionally expressible.

61. An expression system according to any one of claims 1 to 60, wherein 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 operably connected to at least one transcriptional control sequence (e.g. , a promoter, transcription terminator, c/^'s-acting sequence, etc.), wherein the effector nucleic acid sequence is selected from a target nucleic acid sequence or a modulator nucleic acid sequence.

62. An expression system according to claim 61, wherein the effector nucleic acid sequence is in the form of a contiguous sequence.

63. An expression system according to claim 61, wherein the effector nucleic acid sequence is in the form of a plurality of non-contiguous sequences that can conditionally form a contiguous sequence.

64. An expression system according to claim 61, wherein one or both of the first expression system component and the second expression system component comprises an inactive replicon that comprises replicase c/^'s-acting elements, which facilitate, in the presence of a replicase, circularization and release from the inactive replicon 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 an effector nucleic acid sequence of the invention (i.e., a target or modulator nucleic acid sequence) is expressible.

65. An expression system according to claim 64, wherein one or both of the first expression system component and the second expression system component 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, 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 an effector nucleic acid sequence of the invention (i.e., a target or modulator nucleic acid sequence) is expressible.

66. An expression system according to claim 64 or claim 65, wherein the inactive replicon or proreplicon comprises an effector nucleic acid sequence, which is in the form of a contiguous sequence and which is operably connected to at least one tra nscriptional control sequence (e.g. , a promoter, transcription terminator, c/^'s-acting sequence, etc.).

67. An expression system according to claim 66, wherein the contiguous sequence is operably linked to a constitutive promoter for constitutively expressing the contiguous sequence.

68. An expression system according to claim 67, wherein expression of the effector nucleic acid sequence is optionally boosted in the presence of a Rep protein .

69. An expression system according to claim 68, wherein the Rep protein is produced from a rep gene in an ancillary construct.

70. An expression system according to claim 69, 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 effector nucleic acid sequence.

71. An expression system according to claim 66, wherein the contiguous sequence is operably linked to a regulated promoter for conditionally expressing the contiguous sequence.

72. An expression system according to claim 71, wherein expression of a rep gene from an ancillary construct and the effector 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 effector nucleic acid sequence.

73. An expression system according to claim 65, wherein the proreplicon comprises an effector 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 effector nucleic acid sequence and a downstream member of the non-contiguous sequences corresponds to a 5' portion of the effector 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.).

74. An expression system according to claim 73, 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 effector nucleic acid sequence (i.e., a contiguous nucleic acid entity).

75. An expression system according to claim 74, 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 effector 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 effector nucleic acid sequence.

76. An expression system according to claim 75, wherein the effector nucleic acid sequence is a modulator nucleic acid sequence, and wherein the modulator nucleic acid sequences encodes a double stranded decoy 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.

77. An expression system according to any one of claims 61 to 76, wherein the effector 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 effector nucleic acid sequence.

78. An expression system according to claim 77, wherein an effector nucleic acid sequence is in the form of non-contiguous sequences, wherein an individual noncontiguous sequence is sepa rated from an upstream or downstream Rep recognition element by a non-coding sequence (e.g., an intron) .

79. An expression system according to claim 78, wherein the 3' portion of the effector nucleic acid sequence is separated from an upstream Rep recog nition element by a 3' portion of an intron and the 5' portion of the effector nucleic acid sequence is sepa rated 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 effector nucleic acid sequence, which comprises in operable linkage, from 5' to 3', the 5' portion of the effector 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 effector nucleic acid sequence.

80. An expression system according to claim 79, wherein a promoter (e.g., regulated or constitutive) is operably connected upstream of the 5' portion of the effector nucleic acid sequence to form an expression cassette.

81. An expression system according to claim 65, 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 target nucleic acid sequence, a 5' portion of the target nucleic acid sequence, and the second Rep recognition element.

82. An expression system according to claim 81, wherein a promoter is operably linked to the 5' portion of the target nucleic acid sequence and a transcription terminator is optionally operably linked to the 3' portion of the target nucleic acid sequence.

83. An expression system according to claim 82, 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 target nucleic acid sequence and the second Rep recognition element become operably connected to form a contiguous target nucleic acid sequence comprising, from 5' to 3', the 5' portion of the target nucleic acid sequence, the second Rep recognition element and the 3' portion of the target nucleic acid seq uence, and wherein a utonomous episomal replication of the target replicon results in amplification of the target replicon with expression of the contig uous target nucleic acid sequence.

84. An expression system according to claim 65, wherein the second construct the second expression system component that comprises a proreplicon (a "modulator 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 "modulator 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 modulator nucleic acid sequence, a 5' portion of the modulator nucleic acid sequence, and the second Rep recognition element.

85. An expression system according to claim 82, wherein a promoter is operably linked to the 5' portion of the modulator nucleic acid sequence and a transcription terminator is optionally operably linked to the 3' portion of the modulator nucleic acid sequence.

86. An expression system according to claim 85, wherein a Rep protein interacts with the Rep recognition element(s) in the modulator proreplicon to facilitate

circularization, release and autonomous episomal replication of the modulator replicon, wherein circularization of the modulator replicon results in rearrangement of the construct such that the 3' and 5' portions of the modulator nucleic acid sequence and the second Rep recognition element become operably connected to form a contiguous modulator nucleic acid sequence and comprising, from 5' to 3', the 5' portion of the modulator nucleic acid sequence, the second Rep recognition element and the 3' portion of the modulator nucleic acid sequence, and wherein a utonomous episomal replication of the modulator replicon results in amplification of the modulator replicon with expression of the contiguous modulator nucleic acid sequence.

87. An expression system according to claim 65, wherein the second expression system component comprises a biphasic expression system component for constitutively expressing the decoy RNA molecule and for optionally conditionally boosting expression of the decoy RNA molecule, wherein the biphasic expression system component comprises ( 1) a proreplicon comprising an upstream first Rep recognition element, a downstream second Rep recognition element and an intervening construct from which a modulator nucleic acid sequence is conditional expressible, wherein the first and second Rep recognition elements mediate circularization, release and autonomous episomal replication of a correspond ing replicon in the presence of a Rep protein, and (2) a constitutive expression cassette for constitutively expressing a modulator nucleic acid sequence, wherein the intervening construct of the proreplicon includes, from 5' to 3', a first 3' portion of a modulator nucleic acid sequence, a constitutive promoter, a 5' portion of the modulator nucleic acid sequence and the second Rep recognition element, and wherein the constitutive expression cassette comprises, from 5' to 3', the constitutive promoter, the 5' portion of the modulator nucleic acid sequence, the second Rep recognition element, and a second 3' portion of the modulator nucleic acid sequence.

88. An expression system according to claim 87, wherein in the absence of the Rep protein, the constitutive promoter of the constitutive expression cassette drives expression of a modulator nucleic acid sequence that comprises in operable connection, from 5' to 3', the 5' portion of the modulator nucleic acid sequence, the second Rep recognition element and the second 3' portion of the modulator nucleic acid sequence, which leads to constitutive production of a decoy RNA molecule.

89. An expression system according to claim 88, wherein production of decoy RNA is boosted in the presence of a Rep protein that is produced from an ancillary expression cassette.

90. An expression system according to claim 89, wherein the Rep protein interacts with the Rep recognition elements of the proreplicon to facilitate circularization and release of a corresponding replicon and rearrangement of the intervening construct to form a contiguous modulator nucleic acid sequence that comprises in operable connection, from 5' to 3', the 5' portion of the modulator nucleic acid sequence, the second Rep recognition element, and the first 3' portion of the modulator nucleic acid sequence, wherein interaction of the Rep protein with the Rep recognition elements results in autonomous episomal replication (e.g. , rolling circle replication) of the replicon, to thereby amplify the replicon with expression of the contiguous modulator nucleic acid sequence and boosted production of decoy RNA molecule.

91. An expression system according to any one of claims 1 to 90, wherein the first expression system component comprises a proreplicon for expressing the target nucleic acid sequence and the second expression system component comprises a proreplicon for expressing modulator nucleic acid sequence.

92. An expression system according to any one of claims 1 to 90, wherein the first expression system component comprises a proreplicon for expressing the target nucleic acid sequence and the second expression system component is in the form of a biphasic expression system component for expressing modulator nucleic acid sequence.

93. An expression system according to claim 65 and any claim dependent thereon, wherein 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 host cell.

94. An expression system according to claim 93, wherein the rep gene is selected from among geminivirus (e.g., Mastrevirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Topocuvirus, Turncurtovirus), nanovirus {e.g., Nanovirus, Babuvirus), circovirus (e.g., Circovirus), and bacterial rep genes.

95. An expression system according to claim 65 and any claim dependent thereon, wherein the Rep recognition elements of a proreplicon are virus intergenic regions (IRs).

96. An expression system according to claim 95, wherein the virus intergenic regions are selected from long intergenic regions (LIRs) and short intergenic regions (SIRs).

97. An expression system according to claim 95, wherein 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.

98. An expression system according to claim 95, wherein the Rep recognition elements in a proreplicon are selected from Mastrevirus IRs.

99. An expression system according to claim 95, wherein the Rep recognition elements in a proreplicon are selected from Segomow^'rivs-associated DNA-β satellite IRs.

100. An expression system according to claim 95, wherein the Rep recognition elements in a proreplicon are Mastrevirus LIRs and the first and/or second expression cassette further comprises a Mastrevirus SIR.

101. An expression system according to any one of claims 1 to 100, wherein the target nucleic acid sequence is expressed at a level that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% higher than the expression level of the target nucleic acid sequence in the absence of the decoy RNA molecule

102. An expression system according to any one of claims 1 to 101, wherein expression of the modulator nucleic acid sequence and production of the decoy RNA molecule in the host cell reduces silencing or attenuation of expression of the target nucleic acid sequence to a level that is no more than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of the silencing or attenuation of expression of the target nucleic acid sequence in the absence of decoy RNA molecule.

103. An expression system according to any one of claims 1 to 102, wherein one or both of the first expression system component and the second expression system component is/are stably introduced into the genome of the host cell.

104. An expression system according to any one of claims 1, 2 and 48 to 103, wherein the decoy RNA molecule has at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity over the entire sequence or over a subsequence (e.g., a subsequence of at least 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides in length) of an endogenous RNA expression product.

105. A expression system according to any one of claims 1, 2 and 48 to 104, wherein the decoy RNA molecule is unable to hybridize under medium or high stringency conditions, as defined for example herein, to any sequence of nucleotides located within a RNA expression product of the target nucleic acid sequence but is able to hybridize to a sequence of nucleotides located within an endogenous RNA expression product of the host cell under the same conditions.

106. An expression system according to claim 104 or claim 105, wherein the endogenous RNA expression product is an expression product of a gene involved in the RNA silencing pathway.

107. An expression system according to claim 106, wherein the gene is selected from Agola, Agolb, Ago2, Ago4a, Ago4b, Ago5, Ago6, Ago7, AgolO, Cmt3a, Cmt3b, Dell , Dcl2, Dcl3, Dcl4, Drbla, Drblb, Drb2a, Drb2b, Drb3a, Drb3b, Drb4, Drb5, Drdl, Drm3, Henl, Metl, Nrpdla, Nrpdlb, Nrpd2a, Rdrl, Rdr2, Rdr5, Rdr6a, Rdr6b* and Sgs3.

108. A host cell that contains an expression system according to any one of claims 1 to 107.

109. A host cell according to claim 108, wherein one or both of the first and second expression system components are stably introduced in the genome of the host cell.

110. A host cell according to claim 108 or claim 109, wherein the host cell is an animal host cell.

111. A host cell according to claim 108 or claim 109, wherein the host cell is a plant host cell.

112. A host cell according to claim 111, wherein the plant host cell is selected from monocotyledonous or dicotyledonous host cells.

113. A transgenic organism or part thereof including organs and tissues, which comprises a host cell according to any one of claims 108 to 112.

114. A transgenic plant that comprises a plant host cell containing a n expression system according to any one of claims 1 to 107, wherein the plant is selected from among Gossypium spp. (cottons), Nicotiana tabacum (tobacco), Ananas comosus (pineapple), Saccharum spp. (sugar cane), Musa spp. (banana), Lycopersicon

esculentum (tomato) and Solanum tuberosum (potato), 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) and Fabaceae family (legumes) plants.

115. A method for enhancing expression of a target nucleic acid sequence in a host cell, the method comprising, consisting or consisting essentially of co-expressing the target nucleic acid sequence and a modulator nucleic acid sequence in the host cell, wherein expression of the modulator nucleic acid sequence produces a double stranded decoy RNA molecule that is a substrate of the RNA silencing pathway and that is nonhomologous with a RNA expression product of the target nucleic acid sequence, wherein the decoy RNA molecule competes with other substrates of the RNA silencing pathway to thereby induce silencing of the modulator nucleic acid sequence with reduced silencing of the target nucleic acid sequence.

116. A method according to claim 115, further comprising introducing into the host cell at least one expression system component from which the target nucleic acid sequence and/or the modulator nucleic acid sequence is/are expressible.

117. A method according to claim 116, wherein the target nucleic acid sequence and/or the modulator nucleic acid sequence is/are stably introduced in the genome of the host cell.

118. A method according to any one of claims 115 to 117, wherein the host cell is plant host cell.

119. A method according to claim 118, wherein the plant host cell is selected from monocotyledonous or dicotyledonous host cells.

120. A method according to any one of claims 115 to 119, wherein the host cell is a non-plant eukaryotic cell.

121. A method according to claim 120, wherein the non-plant eukaryotic cell is selected from yeast, fungus and animal cells (e.g., mammalian cells such as primate cells including human cells), in which RNA silencing occurs.

122. A method according to any one of claims 115 to 121, further comprising exposing the host cell to one or more stimuli that stimulate or enhance expression of the target nucleic acid sequence, the modulator sequence or both the target nucleic acid sequence and the modulator sequence.