Virus Research 102 (2004) 97108

Plant viral suppressors of RNA silencing

Braden M. Roth, Gail J. Pruss, Vicki B. Vance
Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA

Abstract RNA silencing is an ancient eukaryotic pathway in which double stranded RNA (dsRNA) triggers destruction of related RNAs in the cell. Early studies in plants pointed to a role for this pathway as a defense against viruses. Most known plant viruses have RNA genomes and replicate via dsRNA intermediates, thereby serving as potent inducers of RNA silencing early in replication and as silencing targets later in infection. Because RNA silencing is an antiviral mechanism, it is not surprising that many plant viruses encode suppressors of RNA silencing. This review focuses on the currently known plant virus encoded suppressors of silencing and the functional assays used to identify these proteins. Because they interfere with silencing at different points in the pathway, these viral suppressors are powerful tools to help unravel the mechanism of RNA silencing in plants. 2004 Elsevier B.V. All rights reserved.
Keywords: RNA silencing; RNAi; Viral suppressor protein; HC-Pro; CMV 2b

1. Introduction RNA silencing is an adaptive defense mechanism that is triggered by double stranded RNA (dsRNA). Three initially unrelated lines of research led to the recognition of RNA silencing as an important means of defense against viruses and other nucleic acid invaders. The discovery that plant viruses encode suppressors of RNA silencing is a key element in the story. The rst line of research led to the discovery of transgene-induced RNA silencing. Attempts to over-express endogenous genes by introducing additional copies resulted instead in turning off the endogenous gene as well as the transgene (Napoli et al., 1990; Smith et al., 1990; van der Krol et al., 1990). The next piece of the puzzle came from studies of pathogen-derived resistance in which RNA silencing directed against a viral transgene provided resistance to any virus carrying the targeted sequence (Baulcombe, 1996; Dougherty and Parks, 1995; English et al., 1996; Goodwin et al., 1996; Lindbo et al., 1993; Smith et al., 1994). Thus, viruses could be targets of RNA silencing. The third clue came from studies of synergistic viral diseases caused by certain pairs of co-infecting viruses. A viral protein called helper component proteinase (HC-Pro) was shown to mediate one class of viral synergistic disease (Shi et al., 1997;

Vance et al., 1995). Expression of HC-Pro in transgenic plants allowed a broad range of heterologous viruses to accumulate beyond the normal level, suggesting that HC-Pro blocked a general plant defense mechanism (Pruss et al., 1997). Remarkably, the mechanism blocked by HC-Pro was found to be RNA silencing (Anandalakshmi et al., 1998; Brigneti et al., 1998; Kasschau and Carrington, 1998). Since the initial demonstration that HC-Pro blocks RNA silencing, many other plant viral suppressors of silencing have been identied (see Table 1).

2. RNA silencing The term RNA silencing refers to a set of related pathways found in a broad range of eukaryotic organisms. Genetic and biochemical experiments have established a general mechanistic model for these related pathways and identied factors that are required for RNA silencing in a variety of organisms (Fig. 1). The process is initially triggered by dsRNA, which can be introduced experimentally or arise from endogenous transposons, replicating RNA viruses, or the transcription of transgenes. The dsRNA trigger is cleaved by a ribonuclease III (RNAse III)-like enzyme termed Dicer into 2124 nucleotide duplexes termed short-interfering RNAs (siRNAs) (Bernstein et al., 2001; Hamilton and Baulcombe, 1999; Zamore et al., 2000). The production of siRNAs by Dicer is an ATP-dependent step (Bernstein et al., 2001;

Corresponding author. E-mail address: vance@biol.sc.edu (V.B. Vance).

0168-1702/$ see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2004.01.020

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Table 1 Plant viral suppressors of RNA silencing Genus Carmovirus Virus Turnip crinkle virus (TCV) Suppressor CP Evidence TCV infection does not reverse silencing. In agro-coinltration assay, CP blocks sense and antisense induced local silencing and prevents systemic silencing. Suppresses inverted repeat (IR) induced local silencing in agro-coinltration assay. BYV p21 corresponds to BYSV p22. Infection with CMV or with PVX-2b vector blocks silencing. Interferes with systemic signal (see text). Agro-coinltration assay with sense induced silencing. BNYVV P14 corresponds to PCV P15. Infection with ACMV, PVX-AC2, or PVX-C2 reverses silencing. Blocks sense induced silencing in agro-coinltration assay. AC2 and C2 are homologs. RNA mediated cross protection between PVX-GFP and TMV-GFP vectors is eliminated when b is expressed from the PVX vector. PCV infection blocks silencing. p15 blocks local and delays systemic sense-induced silencing in agro-coinltration assay. BWYV PO suppresses local but not systemic sense-induced silencing in agro-coinltration assay. CABYV PO tested only on local silencing. PVX infection does not suppress silencing. In agro-coinltration, p25 blocks systemic but not always local silencing (see text). Evidence from multiple types of assay. Does not block systemic silencing in stable expression grafting assay, but does in agro-coinltration assay (see text). Infection with PVX-P1 viral vector reverses silencing. Agro-coinltration assay of sense induced local silencing. Limited activity in reversal of silencing; strong activity in agro-coinltration (see text). AMCV (artichoke mottled crinkle virus) P19 also works as a suppressor. TSWV infection reverses silencing. In agro-coinltration, NSs suppressed sense, but not IR, induced local and systemic silencing. Reference Qu et al. (2003) and Thomas et al. (2003)

Closterovirus Cucumovirus Furovirus Geminivirus

Beet yellows virus (BYV) Beet yellow stunt virus (BYSV) Cucumber mosaic virus (CMV) Tomato aspermy virus (TAV) Beet necrotic yellow vein virus (BNYVV) African cassava mosaic virus (ACMV) Tomato yellow leaf curl virus-China (TYLCV-C) Barley stripe mosaic virus (BSMV) Poa semilatent virus (PSLV) Peanut clump virus (PCV) Beet western yellows virus (BWYV) Cucurbit aphid-borne yellows virus (CABYV) Potato virus X (PVX) Potato virus Y (PVY) Tobacco etch virus (TEV) Rice yellow mottle virus (RYMV) Rice hoja blanca virus (RHBV) Tomato bushy stunt virus (TBSV) Cymbidium ringspot virus (CymRSV)

p21 p22 2b P14 AC2 C2 b P15 PO

Reed et al. (2003) Li et al. (2002), see text Dunoyer et al. (2002) Dong et al. (2003), Voinnet et al. (1999) and van Wezel et al. (2002) Yelina et al. (2002) Dunoyer et al. (2002) Pfeffer et al. (2002)

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Hordeivirus Pecluvirus Polerovirus

Potexvirus Potyvirus

p25 HC-Pro

See text See text

Sobemovirus Tenuivirusa Tombusvirus

P1 NS3 P19

Voinnet et al. (1999) Bucher et al. (2003) Voinnet et al. (2003), Qu and Morris (2002) and Takeda et al. (2002), see text

Tospovirusa
a

Tomato spotted wilt virus (TSWV)

NSs

Bucher et al. (2003) and Takeda et al. (2002)

Tospoviruses and tenuiviruses replicate in their insect vectors and in plants.

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One fascinating aspect of RNA silencing is that it is non-cell-autonomous, and this feature may reect the antiviral nature of the process (for a recent review, see Mlotshwa et al., 2002b). Virus infection usually starts with entry via a small wound. The virus replicates in the initially infected cell and then moves into adjacent cells, spreading from cell to cell until it enters the vascular system, which allows rapid movement to distant parts of the plant. In response, the host plant initiates RNA silencing against the viral RNA and produces the mobile silencing signal. The mobile signal moves along the same route the virus takes. Thus, the plant and the virus enter a race. If the virus moves ahead of the signal, it can establish infection when it enters the distant cells. However, if the mobile silencing signal gets there rst, the virus will enter the distant cell only to nd itself targeted by RNA silencing, and the infection will fail to become systemic. One major class of viral suppressors comprises proteins that block systemic silencing, suggesting the co-evolution of defense and counter-defense between the host plant and the invading virus at the level of systemic spread.

3. Functional assays used to identify suppressors of RNA silencing Three major approaches have been widely used to identify plant viral suppressors of RNA silencing: (1) transient expression assays, (2) the reversal of silencing assay, and (3) stable expression assays. These assays are described below and shown in cartoon form in Figs. 2 through 4. 3.1. Transient expression assaysAgrobacterium co-inltration
Fig. 1. RNA silencing pathway.

Zamore et al., 2000) and probably involves interactions with other proteins, including an argonaute-like protein, a dsRNA binding protein, and an RNA helicase (Tabara et al., 2002). There are four Dicer-like (DCL) homologs in Arabidopsis; however, the specic enzyme responsible for siRNA production in plants has not yet been identied. The siRNAs produced from a fully double-stranded RNA substrate by Dicer have distinctive characteristics: they represent both polarities and have two nucleotide 3 overhangs with 5 phosphate and 3 hydroxyl groups (Elbashir et al., 2001a,b). In another ATP-dependent step (Nykanen et al., 2001), the siRNAs are denatured and incorporated into a multi-subunit endonuclease silencing complex called RNA-induced silencing complex (RISC; Hammond et al., 2000). Within the activated RISC, single-stranded siRNAs act as guides to bring the complex into contact with complementary mRNAs and thereby cause their degradation (Bernstein et al., 2001; Elbashir et al., 2001a; Hammond et al., 2000, 2001; Zamore et al., 2000).

This approach provides a rapid and easy test of suppressor activity and is currently the technique most commonly used to identify viral suppressors (Llave et al., 2000; Voinnet et al., 2000). The method makes use of a commonly used bacterial pathogen of plants, Agrobacterium tumefaciens. The Agrobacterium serves two purposes: one strain is used to induce RNA silencing of a reporter gene (usually green uorescent protein (GFP)), and another strain is used to express the candidate suppressor. The overall strategy is to co-inltrate mixtures of the two bacterial strains (one acting to induce silencing, the other to suppress it) into a plant leaf and then examine the inltrated patch over time for silencing of the reporter (Fig. 2A). Nicotiana benthamiana is well suited to these assays because the leaves are easily inltrated and produce high quantities of protein in response to agro-inltration. Non-transgenic N. benthamiana as well as N. benthamiana expressing the reporter gene can be used. In a typical experiment with Agrobacterium expressing GFP as the inducer, the inltrated patch initially expresses high levels of GFP and glows bright green under ultraviolet (UV) light (Fig. 3A, rst panel). However, in three to ve days, the procedure triggers local RNA silencing, and the patch

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Fig. 2. Cartoon guide to transient expression assays. (A) Assay for suppressors of local silencing. (B) Assay for suppressors of systemic silencing.

Fig. 3. Agrobacterium-induced systemic silencing (A) and cartoon guide to reversal of silencing assay (B).

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becomes a dirty red color under UV light due to a mixture of green from residual GFP and red from chlorophyll in the leaves (Fig. 3A, second panel). If the candidate suppressor expressed from the co-inltrated Agrobacterium interferes with RNA silencing, the patch will remain bright green: if it does not, the patch will turn red (Fig. 2A). Variations of this technique include using Agrobacterium expressing inverted repeat (IR) or viral cDNA constructs to induce silencing in combination with or in place of Agrobacterium expressing a sense gene construct (Johansen and Carrington, 2001; Voinnet et al., 2000). Another variation of the transient expression approach has been widely used to investigate the effect of silencing suppressors on the mobile silencing signal (Fig. 2B). For this purpose, inltration is performed on a transgenic N. benthamiana line that expresses GFP (line 16C, Ruiz et al., 1998). Early experiments using an Agrobacterium strain expressing a sense GFP construct as the inducer of silencing demonstrated that local induction of GFP silencing produced a mobile silencing signal that moved out of the inltrated spot, along veins, and into surrounding tissues, leaving a trail of red marking its path (Fig. 3A, third panel) and eventually silencing GFP throughout the whole plant (Fig. 3A, fourth panel) (Voinnet and Baulcombe, 1997). Thus, the ability of suppressors to interfere with systemic silencing can be tested in this system by co-inltrating the inducer together with a putative suppressor of silencing and observing long enough to determine whether the plant becomes systemically silenced (Fig. 2B). 3.2. Reversal of silencing assay This versatile assay can be used to rst identify candidate viruses that may suppress silencing as a major line of counter-defense (Fig. 3B; Brigneti et al., 1998). A modication of the same technique can then be used to identify the specic viral gene product that suppresses silencing. The overall strategy is to infect a silenced plant with the candidate virus and determine whether the silenced phenotype is reversed (Fig. 3B). The most common version of this technique uses the GFP-expressing transgenic N. benthamiana line 16C described above. Soon after germination (at about the four-leaf stage), the plant is inltrated with Agrobacterium expressing GFP, which triggers local silencing and then systemic silencing as described above. Ultimately, the plant becomes completely silenced for GFP (red in UV light) (Fig. 3A, fourth panel). At this point, the plant is inoculated with the virus being tested for suppressor activity. If virus infection allows the plant to express GFP, the infecting virus likely encodes a suppressor of silencing (Voinnet et al., 1999). Individual genes can be assayed for suppressor activity using a modication of the reversal of silencing technique that employs potato virus X (PVX). PVX is an efcient vector for systemic expression of heterologous genes in N. benthamiana and does not, itself, encode a suppressor of silencing that works in the reversal of silencing assay.

Thus, candidate suppressors from heterologous viruses can be tested in the reversal of silencing assay by expressing them from a PVX vector (Fig. 3B). Many viral suppressors have been identied by expression from PVX in this manner (Table 1; Voinnet et al., 1999). 3.3. Stable expression assays In this approach, a stable transgenic line expressing a candidate suppressor of silencing (usually initially identied in one of the previous two assays) is crossed to a series of well-characterized transgenic lines silenced for a reporter gene (Fig. 4A; Anandalakshmi et al., 1998; Kasschau and Carrington, 1998). An advantage of this approach is that it offers the opportunity to examine the effect of the suppressor on different well-dened types of transgene-induced RNA silencing, thus, providing information about the mechanism of suppression. The stable expression assays are also well suited to investigate the role of suppressors in systemic silencing using grafting (Fig. 4B; Guo and Ding, 2002; Mallory et al., 2001, 2003). In these assays, the ability of a plant to send a mobile silencing signal is assayed by grafting an expressing line onto the top of it (the bottom plant is called the rootstock and the expressing plant grafted onto the top is called the scion) (Palauqui et al., 1997). If the suppressor of silencing in the rootstock blocks either the production or movement of the systemic silencing signal, then the transgene in the scion will continue to be expressed. If a suppressor does not block the systemic silencing signal in either of these ways, then the transgene in the scion will become silenced (Fig. 4B).

4. Mechanism of suppression The assays discussed above have proven extremely useful as rapid and sensitive methods to identify suppressors of silencing. They have also enabled rudimentary characterization of suppressor mechanism, but conicting results from different assays have made it difcult to draw rm conclusions for many suppressors. Interestingly, the currently known suppressors share no obvious similarities at either the nucleic acid or the protein level, perhaps reecting differences at the mechanistic level as well. At present, two major classes of suppressor action have been identied. 4.1. Suppressors that affect small RNA metabolism Many suppressors reduce the accumulation of siRNAs, raising the possibility that silencing is blocked at the step at which Dicer processes the dsRNA that triggers silencing. Thus, it may be that many suppressors prevent silencing by blocking production of the siRNAs that provide the sequence specicity of the process. A second mechanism involving siRNAs is exemplied by the suppressor P19, which has been shown to bind siRNAs, perhaps sequestering them and

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Fig. 4. Cartoon guide to stable expression assays. (A) Genetic crosses with silenced transgenic lines. (B) Grafting assay for suppression of systemic silencing.

thereby blocking their function (Silhavy et al., 2002). Interestingly, the effect of suppressors on small RNA metabolism may extend to other types of small regulatory RNAs. For example the silencing suppressor HC-Pro affects the accumulation not only of the siRNAs that mediate silencing but also of endogenous microRNAs (miRNAs), which have been implicated in development (Kasschau et al., 2003; Mallory et al., 2002b). Surprisingly, although HC-Pro blocks the accumulation of siRNAs, it enhances the accumulation of miRNAs (Mallory et al., 2002b). Even more interesting, there is evidence that HC-Pro might block the function of miRNAs (Kasschau et al., 2003). It remains to be seen if other viral suppressors also affect the miRNA pathway, perhaps using that pathway to turn off expression of genes required for silencing. 4.2. Suppressors that affect systemic silencing Systemic silencing can be assayed in either transient expression experiments or in experiments with stably transformed transgenic lines (Figs. 2B and 4B). Many suppressors have been demonstrated to block systemic silencing in at least one of these assays (Table 1). Because of the conicting results sometimes obtained with different assays, the most successful attempts to determine whether a suppressor primarily affects systemic silencing have used a multi-pronged approach rather than relying on just one type of assay. For example, although HC-Pro did not block systemic silencing in grafting experiments using stable transgenic lines

(Mallory et al., 2001, 2003), it interfered with systemic silencing in the transient expression assay (Hamilton et al., 2002). Because HC-Pro is a powerful suppressor of local silencing in all assays, these results suggest that HC-Pro primarily affects local silencing, but also has a smaller effect on systemic silencing. In contrast, CMV 2b primarily targets systemic silencing because it blocks movement of the signal in many assays, but has a lesser effect on local silencing (Brigneti et al., 1998; Bucher et al., 2003; Guo and Ding, 2002). It may not be surprising that a primary effect of a particular suppressor on one aspect of silencing could lead, perhaps through feedback mechanisms, to secondary effects on other parts of the pathway, thereby making it appear that the suppressor works at multiple points. 4.3. Conicting results using different assays Why do different assays give different results when looking at effects of viral suppressors on systemic silencing? The major conicts are seen with stable expression assays versus transient ones and probably reect a number of intrinsic differences between the systems. Transient expression assays and the reversal of silencing assay use Agrobacterium to induce silencing. Because Agrobacterium is a plant pathogen, inltration likely induces plant defensive and bacterial counter-defensive interactions, which might modify the activity of some viral suppressors. Thus, attempts to compare suppressor activities or to understand the mechanism of action of the different suppressors are complicated

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by the largely unknown effects of Agrobacterium on the system. Similarly, the reversal of silencing assay involves virus infection and likely induces accompanying defense and counter-defensive responses that complicate the interpretation of results. Further sources of variation include differences in the level and time course of expression of the silencing suppressor and silencing inducer in the different systems. Of the three widely used types of assay (Section 3), stable expression assays are the ones most free of complications due to extraneous pathogens. In addition, use of the same well-characterized silenced transgenic line to test the effects of different viral suppressors affords a degree of standardization from lab to lab not yet found with transient expression assays. It is important to use well-characterized lines to compare suppressor activities because different types of transgenes trigger silencing in different ways (Vance and Vaucheret, 2001). Understanding the basis of the conicting results given by the currently widely used assays will likely offer considerable insight into the mechanisms of suppressor action. Alternative approaches such as yeast two-hybrid, biochemical, and localization studies (see Section 5) are increasingly being used to investigate the mechanisms of action of the different viral suppressors of silencing and will undoubtedly help clear up the confusion.

5. Better studied suppressors of silencing 5.1. Cucumber mosaic virus (CMV) 2b The CMV 2b protein was one of the rst identied suppressors of RNA silencing and also one of the best studied from a mechanistic standpoint. The initial indication that CMV 2b suppressed silencing came from the reversal of silencing assay in which 2b expressed from PVX could prevent the initiation of silencing but could not reverse silencing that was already established (Brigneti et al., 1998). That early result raised the possibility that 2b might block systemic silencing. Subsequently, stable expression assays and grafting experiments provided an elegant demonstration that 2b blocks the movement of the systemic silencing signal (Guo and Ding, 2002). The stable expression assays made use of a well-characterized silenced tobacco line called 6b5 (Elmayan and Vaucheret, 1996), which is silenced for the reporter gene GUS and is a model for sense-transgene induced RNA silencing. The 6b5-GUS locus triggers silencing even when it is hemizygous, produces high levels of GUS siRNAs, and is highly effective in the production and transmission of a systemic silencing signal as demonstrated by grafting experiments. A genetic cross between line 6b5 and a stable transgenic tobacco line expressing CMV 2b established that 2b could partially suppress local sense-transgene induced silencing: offspring of the cross accumulated GUS mRNA, but GUS siRNA accumulation,

although reduced, was not eliminated. However, grafting experiments denitively demonstrated that CMV 2b blocks the movement of the systemic silencing signal. In the rst protocol, 6b5 rootstocks suppressed for silencing by CMV 2b did not silence grafted GUS-expressing scions, showing that expression of CMV 2b in the rootstocks prevented production or transmission of the systemic silencing signal (Fig. 5A, rst panel). In the second protocol, a small spacer of transgenic tobacco expressing CMV 2b, grafted between a GUS-silenced 6b5 rootstock and a GUS-expressing scion, blocked systemic silencing (Fig. 5A, second panel). Thus, CMV 2b prevents transmission of the systemic silencing signal in a stable expression assay. The experiments described above provide compelling evidence that the mechanism of action of CMV 2b, at least in part, is to block systemic silencing. Guo and Ding (2002) also report similar conclusions based on Agrobacterium co-inltration experiments. Paradoxically, the same type of co-inltration experiments in another laboratory produced a different result: CMV 2b delayed but did not block systemic silencing (Hamilton et al., 2002). A possible cause of this discrepancy is that the two labs were using different ratios of silencing inducer to suppressor in the co-inltrations (S.W. Ding, personal communication), suggesting that a standardized methodology for co-inltration assays would help resolve some of the current inconsistencies. How does CMV 2b protein block the movement of the mobile silencing signal? The ability of 2b protein to prevent the transmission of the signal suggests that it either sequesters or inactivates the signal in the phloem stream. One possibility is that 2b acts directly by binding to the signal. However, the nding that 2b localizes to the nucleus (Lucy et al., 2000) suggests that the suppressor acts indirectly, perhaps by activating one or more processes that subsequently affect the signal. 5.2. Potyviral helper-component protease (HC-Pro) HC-Pro was the rst identied suppressor of RNA silencing. The original reports demonstrated that it suppresses both transgene- and virus-induced silencing (Anandalakshmi et al., 1998; Kasschau and Carrington, 1998). In contrast to CMV 2b protein, it is able to reverse established silencing in the reversal of silencing assay (Brigneti et al., 1998), suggesting that the two suppressors work at different steps in the silencing pathway. Although HC-Pro alone has suppressor activity (Anandalakshmi et al., 1998; Brigneti et al., 1998), it is frequently expressed as the proteinase 1 (P1)/HC-Pro polyprotein in suppression of silencing studies. The P1/HC-Pro construct is the N-terminal portion of the natural viral polyprotein and allows HC-Pro to be proteolytically processed just as when expressed from the viral genome. Some evidence suggests that P1 might enhance HC-Pro activity as a suppressor of silencing (Pruss et al., 1997). A variety of approaches, including both transient and stable expression assays, have been used to investigate how

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Fig. 5. Cartoon guide to (A) CMV 2b and (B) HC-Pro grafting experiments.

HC-Pro suppresses RNA silencing. Despite some conicting results, at least one common nding has emerged: HC-Pro affects small RNA metabolism. Exactly how HC-Pro exerts its effect on small RNAs and how this leads to suppression of silencing are questions that remain to be resolved. The interaction(s) of HC-Pro and its cellular effector(s) probably occur in the cytoplasm because HC-Pro is found primarily in cytoplasm (Mlotshwa et al., 2002a). 5.2.1. HC-Pro and small RNAs HC-Pro has been reported to alter the accumulation of several classes of small RNAs: the siRNAs that direct RNA degradation during silencing, a novel class of slightly-larger small RNAs of unknown function, and the endogenous microRNAs (miRNAs) that have been implicated in regulation of development. In stable expression assays in tobacco, HC-Pro has been reported to suppress three classes of transgene-induced RNA silencing, in each case interfering with the accumulation of siRNAs (Mallory et al., 2001, 2002b). Similarly, siRNA accumulation is dramatically reduced during HC-Pro suppression of silencing in transient expression assays (Johansen and Carrington, 2001; Llave et al., 2000). In stable expression assays in tobacco, HC-Pro suppression of IR transgene-induced silencing or amplicon-transgene-induced silencingbut not sense transgene-induced silencingresulted in the accumulation of a novel class of slightly-larger small RNAs (Mallory et al., 2002b). Similar slightly-larger small RNAs have also been observed in transient expression assays (Hamilton et al., 2002). The function of this class of small RNAs is not clear; however, they do not appear to act as siRNAs because their presence is not correlated with RNA degradation (Hamilton et al., 2002; Mallory et al., 2002b). They

correlate with systemic silencing in transient expression assays (Hamilton et al., 2002) but not in stable expression assays (Mallory et al., 2002b). Finally and surprisingly, the accumulation of endogenous miRNAs is increased in both tobacco and Arabidopsis transgenic lines that express HC-Pro (Kasschau et al., 2003; Mallory et al., 2002b), suggesting a more general role in the biogenesis of small regulatory RNAs. A recent report that HC-Pro enhances the stability of several miRNA target messages raises the possibility that HC-Pro affects the function as well as the biogenesis of small RNAs (Kasschau et al., 2003). 5.2.2. HC-Pro and systemic silencing In stable expression experiments utilizing grafting, HC-Pro failed to block systemic silencing (Fig. 5B, rst panel; Mallory et al., 2001, 2003). Three different transgenic tobacco lines were used as rootstocks, representing three different means of inducing silencing: sense transgene, inverted repeat transgene, and amplicon transgene. In the case of the amplicon-transgene induced silencing, in which the transgene is the cDNA of a replication competent virus, HC-Pro actually promoted systemic silencing (Mallory et al., 2003). These results suggest that production and transmission of the systemic silencing signal are largely unaffected by HC-Pro, implying that HC-Pro suppression of silencing occurs downstream of the signal. To test that hypothesis, the effect of HC-Pro on perception of and/or response to the silencing signal was tested in additional grafting experiments (Fig. 5B, second panel; Mallory et al., 2001). A silenced GUS line previously shown to silence GUS systemically across a graft junction was used as rootstock, and the scion was a line that expressed both GUS and HC-Pro. The scion failed to become silenced for GUS,

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showing denitively that HC-Pro works downstream of the systemic silencing signal: The rootstock is known to send a signal; therefore, in the presence of HC-Pro, the scion either fails to perceive the signal or fails to respond to it. Although the grafting experiments described above make a convincing case that HC-Pro does not block the systemic silencing signal, results from Agrobacterium co-inltration assays suggest that this suppressor does interfere with systemic silencing (Hamilton et al., 2002). There are a number of possible reasons for obtaining different results with the two assays. First, the stable transgenic lines express the P1/HC-Pro polyprotein, whereas the transient assay experiments used a construct that encodes only HC-Pro and has a methionine substituted for the natural N-terminal amino acid of the protein. The protein produced in the transient assay, therefore, is not identical to HC-Pro produced from the virus. Thus, it is possible that the difference between the two assays reects this small difference in the proteins. Indeed, earlier experiments expressing either P1/HC-Pro or HC-Pro (with an N-terminal methionine) from a PVX vector reported a dramatic difference in the effect on PVX replication (Pruss et al., 1997). A second possibility is that the result in the transient assay depends on the ratio of inducer to suppressor, as suggested for CMV 2b protein (see Section 5.1), and this possibility could be tested by using a range of ratios. A more exciting possibility is that the discrepancy reects an intrinsic difference between the two assays, as discussed in Section 4.3. Thus, HC-Pro might block systemic silencing in some circumstances, but not in others, and identifying the important variables will help in our understanding of HC-Pro suppression of silencing. 5.2.3. HC-Pro interacting proteins If HC-Pro works by interacting with components or regulators of the silencing machinery, identication of plant proteins that interact with HC-Pro should provide clues about its mechanism of action. Using the yeast two-hybrid system, an HC-Pro-interacting protein called regulator of gene silencing calmodulin-like protein (rgsCaM) was identied (Anandalakshmi et al., 2000). Like HC-Pro, rgsCaM could reverse silencing when expressed from PVX in the reversal of silencing assay. In addition, when over-expressed in stable expression experiments, rgsCaM caused a developmental phenotype typical of HC-Pro-expressing transgenic lines and interfered with virus induced gene silencing. Because calmodulins are classic signaling molecules, these results raise the possibility that HC-Pro suppresses silencing indirectly via a signal cascade involving rgsCaM. 5.3. Tombusvirus P19 This protein has been an exciting addition to the repertoire of plant viral suppressors. Initially described in the reversal of silencing assay (Voinnet et al., 1999), it appeared to be a weak suppressor, only reversing silencing in the region of veins. Similarly, P19 delayed but did not prevent

virus-induced gene silencing and did not suppress silencing induced by a defective interfering RNA that accumulates to high levels (Qiu et al., 2002). In transient expression assays, however, P19 is a star, blocking both local and systemic silencing and apparently eliminating all small RNAs (Hamilton et al., 2002; Silhavy et al., 2002; Takeda et al., 2002; Voinnet et al., 2003). Interestingly, biochemical studies have shown that P19 binds siRNAs and that binding depends on characteristics of RNase III products (dsRNAs with two nucleotide 3 overhangs) (Silhavy et al., 2002). This result raises the possibility that P19 suppresses silencing by sequestering siRNAs, thereby preventing their incorporation into the RISC complex to serve as guides. This is a novel mechanism among suppressors, and because it theoretically stems from an intrinsic property of the protein to bind functional siRNAs, it is possible that P19 could interfere with silencing in a broad range of different plants (and even other organisms). 5.4. Potato virus X (PVX) p25 Three of ve potexviruses tested in the reversal of silencing assay suppressed RNA silencing, although PVX was one that did not show suppressor activity (Voinnet et al., 1999). Paradoxically, however, PVX is the only potexvirus for which a suppressor of RNA silencing has subsequently been characterized. In grafting experiments designed to separate movement of the systemic silencing signal from that of the virus in virus induced silencing (VIGS), Voinnet et al. (2000) unexpectedly found that PVX infection failed to produce systemic silencing independent of the presence of virus, suggesting that a PVX-encoded protein interferes with production or transmission of the systemic signal. Protein p25, which is required for cell-to-cell movement of potexviruses, was identied as the culprit in a set of experiments in which deletion derivatives of replication competent PVX-GFP viral vector constructs were agro-inltrated into plants expressing GFP. The PVX-GFP constructs were restricted to initially inltrated cells because they all lacked coat protein, which is also required for potexviral movement. Induction of systemic silencing in these experiments, therefore, depends entirely on the systemic signal. Only viral constructs from which p25 expression had been eliminated induced widespread systemic silencing in all plants. Agro-coinltration experiments showed that p25, without any other PVX protein, was sufcient to block systemic silencing. Moreover, p25 blocked systemic silencing whether silencing was induced by a simple transgene construct (35S-GFP) or by a replication competent PVX-GFP construct. The effect of p25 on local silencing in these experiments, however, depended on the nature of the construct used to induce silencing. Although p25 suppressed local silencing in agro-coinltration assays with 35S-GFP, it did not suppress local silencing induced by inltration of replication competent PVX-GFP constructs. p25 suppression of systemic silencing in agro-coinltration assays is correlated

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with the absence of a slightly-larger class of small RNAs, possibly indicating the step in the pathway affected by p25 (Hamilton et al., 2002). Although these transient expression experiments make a convincing case that PVX p25 blocks systemic silencing, a different result was obtained in experiments using transgenic plants constitutively expressing white clover mosaic potexvirus (WClMV) p25. Agro-inltration of a 35S-IR construct systemically silenced these plants (Foster et al., 2002). Moreover, the systemic silencing reverted a severe developmental phenotype, indicating that the systemic signal was able to enter the shoot apical meristem. Thus, WClMV p25 did not prevent systemic silencing in this experimental system. Whether the difference in the results obtained with the PVX and WClMV p25 proteins reects differences in the assays or in the activities of the proteins has not yet been determined.

6. Do all plant viruses have suppressors of silencing? The discovery that plants have a generalized antiviral defense mechanism triggered by dsRNA has revolutionized our thinking about plantvirus interactions. In the euphoric aftermath of the initial identication of plant viral suppressors of silencing, a popular expectation was that most or all plant viruses would encode a suppressor of RNA silencing. Although many different viral suppressors have been identied, the fact that HC-Pro helps so many different viruses suggests that a lot of viruses do not effectively suppress silencing. Such viruses may have evolved other ways to try to avoid silencing, such as by replicating within spherules in the ER (Schwartz et al., 2002), where the dsRNA is hidden, or by replicating and moving rapidly enough to outrun the mobile silencing signal. Furthermore, plants have other defense mechanisms, and silencing might not be the major threat for all viruses. Some viruses, therefore, may well have suppressors of other defense pathways.

7. Suppressors of silencing as tools The nding that certain viral proteins suppress RNA silencing has provided a new tool for technologies utilizing genetically modied plants and is, therefore, of practical signicance. Many biotechnological applications are impaired by RNA silencing, and suppressors of silencing can be used to attain consistent, high-level expression of transgenes in plants (Mallory et al., 2002a; Voinnet et al., 2003). With silencing under control, transgenic plants can be engineered to produce a range of transgene expression: moderate levels of expression to produce desired plant traits or very high-level expression to use the plant as a factory producing pharmaceuticals, vaccines or other high-value gene products. Perhaps more importantly, viral suppressors of silencing also provide unique tools to understand the mechanism of

RNA silencing. Much of what is currently known about the RNA silencing pathway comes from elegant in vitro and genetic studies in organisms other than plants (for a recent review see Tijsterman et al., 2002). In plants, traditional genetic approaches have led to the identication of a number of cellular genes required for RNA silencing (Dalmay et al., 2000, 2001; Fagard et al., 2000; Mourrain et al., 2000). Surprisingly, however, all of these genes are required for sense-, but not IR-transgene induced silencing (Boutet et al., 2003). The plant viral suppressors, many of which appear to work downstream of dsRNA, provide a novel means of entry into parts of the silencing pathway that are not easily accessible by genetic means. The currently known suppressors appear to work at different steps in silencing, thereby providing access to a number of points in the pathway where silencing can be controlled. Identifying host proteins that interact with a viral suppressor of RNA silencing is one very promising approach that is being used to take advantage of viral suppressors to elucidate the silencing pathway. The yeast two-hybrid system has been used to nd tobacco proteins that interact with HC-Pro, identifying a calmodulin-related protein called rgsCaM that suppresses RNA silencing when over-expressed (Anandalakshmi et al., 2000). This result suggests that a calcium controlled signal transduction pathway involving rgsCaM is one of the mechanisms regulating RNA silencing. Intriguingly, in the case of geminiviruses, yeast two-hybrid studies have identied SNF1 kinase as a cellular interactor of the tomato golden mosaic virus AL2 protein (Hao et al., 2003). AL2 is a homologue of the ACMV and TYLCV-C suppressors of silencing. Whether the interaction of AL2 with SNF1, which is a regulator of metabolism in response to stress, plays any role in suppression of silencing is unknown as yet. The potential of using viral suppressors to help understand the mechanism of RNA silencing in plants is largely untapped, and these studies promise to be an exciting and fertile area of research. The recent identication of a viral suppressor that works in animal cells (Li et al., 2002) offers the possibility that such proteins may provide a similar tool to understand the silencing pathway in other organisms as well. Note added in proof The tomato mosaic virus replication protein has recently been reported to suppress RNA silencing (Kubota, K., Tsuda, S., Tamai, A., Meshi, T., 2003. Tomato mosaic virus replication protein suppresses virus-targeted posttranscriptional gene silencing. J. Virol. 77, 1101611026).

Acknowledgements VBV gratefully acknowledges support from the USDA Competitive Grants Program, NIH, and Dow AgroSciences LLC.

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