Roth, Pruss, and Vance 2004

Virus Research 102 (2004) 97–108

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 andreplicate via dsRNA intermediates, thereby serving as potent inducers of RNA silencing early in replication and as silencing targets later ininfection. 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 theseproteins. Because they interfere with silencing at different points in the pathway, these viral suppressors are powerful tools to help unravelthe 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 istriggered by double stranded RNA (dsRNA). Three initiallyunrelated lines of research led to the recognition of RNA si-lencing as an important means of defense against viruses andother nucleic acid invaders. The discovery that plant virusesencode suppressors of RNA silencing is a key element inthe story. The first line of research led to the discovery oftransgene-induced RNA silencing. Attempts to over-expressendogenous genes by introducing additional copies resultedinstead in turning off the endogenous gene as well as thetransgene (Napoli et al., 1990; Smith et al., 1990; van derKrol et al., 1990). The next piece of the puzzle came fromstudies of pathogen-derived resistance in which RNA silenc-ing directed against a viral transgene provided resistance toany virus carrying the targeted sequence (Baulcombe, 1996;Dougherty and Parks, 1995; English et al., 1996; Goodwinet al., 1996; Lindbo et al., 1993; Smith et al., 1994). Thus,viruses could be targets of RNA silencing. The third cluecame from studies of synergistic viral diseases caused bycertain pairs of co-infecting viruses. A viral protein calledhelper component proteinase (HC-Pro) was shown to medi-ate one class of viral synergistic disease (Shi et al., 1997;

∗ Corresponding author.E-mail address:[email protected] (V.B. Vance).

Vance et al., 1995). Expression of HC-Pro in transgenicplants allowed a broad range of heterologous viruses to ac-cumulate beyond the normal level, suggesting that HC-Problocked a general plant defense mechanism (Pruss et al.,1997). Remarkably, the mechanism blocked by HC-Pro wasfound to be RNA silencing (Anandalakshmi et al., 1998;Brigneti et al., 1998; Kasschau and Carrington, 1998). Sincethe initial demonstration that HC-Pro blocks RNA silencing,many other plant viral suppressors of silencing have beenidentified (seeTable 1).

2. RNA silencing

The term RNA silencing refers to a set of related pathwaysfound in a broad range of eukaryotic organisms. Genetic andbiochemical experiments have established a general mecha-nistic model for these related pathways and identified factorsthat are required for RNA silencing in a variety of organ-isms (Fig. 1). The process is initially triggered by dsRNA,which can be introduced experimentally or arise from en-dogenous transposons, replicating RNA viruses, or the tran-scription of transgenes. The dsRNA trigger is cleaved by aribonuclease III (RNAse III)-like enzyme termed Dicer into21–24 nucleotide duplexes termed short-interfering RNAs(siRNAs) (Bernstein et al., 2001; Hamilton and Baulcombe,1999; Zamore et al., 2000). The production of siRNAs byDicer is an ATP-dependent step (Bernstein et al., 2001;

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Table 1Plant viral suppressors of RNA silencing

Genus Virus Suppressor Evidence Reference

Carmovirus Turnip crinkle virus (TCV) CP TCV infection does not reverse silencing. In agro-coinfiltrationassay, CP blocks sense and antisense induced local silencing andprevents systemic silencing.

Qu et al. (2003)and Thomas et al. (2003)

Closterovirus Beet yellows virus (BYV) p21 Suppresses inverted repeat (IR) induced local silencing inagro-coinfiltration assay. BYV p21 corresponds to BYSV p22.

Reed et al. (2003)Beet yellow stunt virus (BYSV) p22

Cucumovirus Cucumber mosaic virus (CMV) 2b Infection with CMV or with PVX-2b vector blocks silencing.Interferes with systemic signal (see text).

Li et al. (2002), see textTomato aspermy virus (TAV)

Furovirus Beet necrotic yellow vein virus (BNYVV) P14 Agro-coinfiltration assay with sense induced silencing. BNYVVP14 corresponds to PCV P15.

Dunoyer et al. (2002)

Geminivirus African cassava mosaic virus (ACMV) AC2 Infection with ACMV, PVX-AC2, or PVX-C2 reverses silencing.Blocks sense induced silencing in agro-coinfiltration assay. AC2and C2 are homologs.

Dong et al. (2003), Voinnet et al. (1999)and van Wezel et al. (2002)Tomato yellow leaf curl virus-China (TYLCV-C) C2

Hordeivirus Barley stripe mosaic virus (BSMV) �b RNA mediated cross protection between PVX-GFP and TMV-GFPvectors is eliminated when�b is expressed from the PVX vector.

Yelina et al. (2002)Poa semilatent virus (PSLV)

Pecluvirus Peanut clump virus (PCV) P15 PCV infection blocks silencing. p15 blocks local and delayssystemic sense-induced silencing in agro-coinfiltration assay.

Dunoyer et al. (2002)

Polerovirus Beet western yellows virus (BWYV) PO BWYV PO suppresses local but not systemic sense-inducedsilencing in agro-coinfiltration assay. CABYV PO tested only onlocal silencing.

Pfeffer et al. (2002)Cucurbit aphid-borne yellows virus (CABYV)

Potexvirus Potato virus X (PVX) p25 PVX infection does not suppress silencing. In agro-coinfiltration,p25 blocks systemic but not always local silencing (see text).

See text

Potyvirus Potato virus Y (PVY) HC-Pro Evidence from multiple types of assay. Does not block systemicsilencing in stable expression grafting assay, but does inagro-coinfiltration assay (see text).

See textTobacco etch virus (TEV)

Sobemovirus Rice yellow mottle virus (RYMV) P1 Infection with PVX-P1 viral vector reverses silencing. Voinnet et al. (1999)

Tenuivirusa Rice hoja blanca virus (RHBV) NS3 Agro-coinfiltration assay of sense induced local silencing. Bucher et al. (2003)

Tombusvirus Tomato bushy stunt virus (TBSV) P19 Limited activity in reversal of silencing; strong activity inagro-coinfiltration (see text). AMCV (artichoke mottled crinklevirus) P19 also works as a suppressor.

Voinnet et al. (2003), Qu and Morris(2002) and Takeda et al. (2002), see textCymbidium ringspot virus (CymRSV)

Tospovirusa Tomato spotted wilt virus (TSWV) NSs TSWV infection reverses silencing. In agro-coinfiltration, NSs

suppressed sense, but not IR, induced local and systemic silencing.Bucher et al. (2003)and Takeda et al. (2002)

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

B.M. Roth et al. / Virus Research 102 (2004) 97–108 99

Fig. 1. RNA silencing pathway.

Zamore et al., 2000) and probably involves interactionswith other proteins, including an argonaute-like protein,a dsRNA binding protein, and an RNA helicase (Tabaraet al., 2002). There are fourDicer-like (DCL) homologsin Arabidopsis; however, the specific enzyme responsiblefor siRNA production in plants has not yet been identified.The siRNAs produced from a fully double-stranded RNAsubstrate by Dicer have distinctive characteristics: theyrepresent both polarities and have two nucleotide 3′ over-hangs with 5′ phosphate and 3′ hydroxyl groups (Elbashiret al., 2001a,b). In another ATP-dependent step (Nykanenet al., 2001), the siRNAs are denatured and incorporatedinto a multi-subunit endonuclease silencing complex calledRNA-induced silencing complex (RISC;Hammond et al.,2000). Within the activated RISC, single-stranded siRNAsact as guides to bring the complex into contact with com-plementary mRNAs and thereby cause their degradation(Bernstein et al., 2001; Elbashir et al., 2001a; Hammondet al., 2000, 2001; Zamore et al., 2000).

One fascinating aspect of RNA silencing is that it isnon-cell-autonomous, and this feature may reflect the antivi-ral nature of the process (for a recent review, seeMlotshwaet al., 2002b). Virus infection usually starts with entry viaa small wound. The virus replicates in the initially infectedcell and then moves into adjacent cells, spreading from cellto cell until it enters the vascular system, which allows rapidmovement to distant parts of the plant. In response, the hostplant initiates RNA silencing against the viral RNA and pro-duces the mobile silencing signal. The mobile signal movesalong the same route the virus takes. Thus, the plant and thevirus enter a race. If the virus moves ahead of the signal, itcan establish infection when it enters the distant cells. How-ever, if the mobile silencing signal gets there first, the viruswill enter the distant cell only to find itself targeted by RNAsilencing, and the infection will fail to become systemic.One major class of viral suppressors comprises proteins thatblock systemic silencing, suggesting the co-evolution of de-fense and counter-defense between the host plant and theinvading virus at the level of systemic spread.

3. Functional assays used to identify suppressors ofRNA silencing

Three major approaches have been widely used to iden-tify plant viral suppressors of RNA silencing: (1) transientexpression assays, (2) the reversal of silencing assay, and (3)stable expression assays. These assays are described belowand shown in cartoon form inFigs. 2 through 4.

3.1. Transient expression assays—Agrobacteriumco-infiltration

This approach provides a rapid and easy test of sup-pressor activity and is currently the technique most com-monly used to identify viral suppressors (Llave et al., 2000;Voinnet et al., 2000). The method makes use of a commonlyused bacterial pathogen of plants,Agrobacterium tumefa-ciens. The Agrobacteriumserves two purposes: one strainis used to induce RNA silencing of a reporter gene (usuallygreen fluorescent protein (GFP)), and another strain is usedto express the candidate suppressor. The overall strategy isto co-infiltrate mixtures of the two bacterial strains (one act-ing to induce silencing, the other to suppress it) into a plantleaf and then examine the infiltrated patch over time for si-lencing of the reporter (Fig. 2A). Nicotiana benthamianaiswell suited to these assays because the leaves are easily in-filtrated and produce high quantities of protein in responseto agro-infiltration. Non-transgenicN. benthamianaas wellasN. benthamianaexpressing the reporter gene can be used.In a typical experiment withAgrobacteriumexpressing GFPas the inducer, the infiltrated patch initially expresses highlevels of GFP and glows bright green under ultraviolet (UV)light (Fig. 3A, first panel). However, in three to five days,the procedure triggers local RNA silencing, and the patch

100 B.M. Roth et al. / Virus Research 102 (2004) 97–108

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).


becomes a dirty red color under UV light due to a mixtureof green from residual GFP and red from chlorophyll in theleaves (Fig. 3A, second panel). If the candidate suppressorexpressed from the co-infiltratedAgrobacteriuminterfereswith RNA silencing, the patch will remain bright green: if itdoes not, the patch will turn red (Fig. 2A). Variations of thistechnique include usingAgrobacteriumexpressing invertedrepeat (IR) or viral cDNA constructs to induce silencingin combination with or in place ofAgrobacteriumexpress-ing a sense gene construct (Johansen and Carrington, 2001;Voinnet et al., 2000).

Another variation of the transient expression approach hasbeen widely used to investigate the effect of silencing sup-pressors on the mobile silencing signal (Fig. 2B). For thispurpose, infiltration is performed on a transgenicN. ben-thamiana line that expresses GFP (line 16C,Ruiz et al.,1998). Early experiments using anAgrobacteriumstrain ex-pressing a sense GFP construct as the inducer of silencingdemonstrated that local induction of GFP silencing produceda mobile silencing signal that moved out of the infiltratedspot, along veins, and into surrounding tissues, leaving a trailof red marking its path (Fig. 3A, third panel) and eventuallysilencing GFP throughout the whole plant (Fig. 3A, fourthpanel) (Voinnet and Baulcombe, 1997). Thus, the abilityof suppressors to interfere with systemic silencing can betested in this system by co-infiltrating the inducer togetherwith a putative suppressor of silencing and observing longenough to determine whether the plant becomes systemi-cally silenced (Fig. 2B).

3.2. Reversal of silencing assay

This versatile assay can be used to first identify candi-date viruses that may suppress silencing as a major line ofcounter-defense (Fig. 3B; Brigneti et al., 1998). A modifi-cation of the same technique can then be used to identifythe specific viral gene product that suppresses silencing. Theoverall strategy is to infect a silenced plant with the candi-date virus and determine whether the silenced phenotype isreversed (Fig. 3B). The most common version of this tech-nique uses the GFP-expressing transgenicN. benthamianaline 16C described above. Soon after germination (at aboutthe four-leaf stage), the plant is infiltrated withAgrobac-terium expressing GFP, which triggers local silencing andthen systemic silencing as described above. Ultimately, theplant becomes completely silenced for GFP (red in UV light)(Fig. 3A, fourth panel). At this point, the plant is inocu-lated with the virus being tested for suppressor activity. Ifvirus infection allows the plant to express GFP, the infect-ing virus likely encodes a suppressor of silencing (Voinnetet al., 1999). Individual genes can be assayed for suppres-sor activity using a modification of the reversal of silencingtechnique that employs potato virus X (PVX). PVX is an ef-ficient vector for systemic expression of heterologous genesin N. benthamianaand does not, itself, encode a suppres-sor of silencing that works in the reversal of silencing assay.

Thus, candidate suppressors from heterologous viruses canbe tested in the reversal of silencing assay by expressingthem from a PVX vector (Fig. 3B). Many viral suppressorshave been identified 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 can-didate suppressor of silencing (usually initially identified inone of the previous two assays) is crossed to a series ofwell-characterized transgenic lines silenced for a reportergene (Fig. 4A; Anandalakshmi et al., 1998; Kasschau andCarrington, 1998). An advantage of this approach is thatit offers the opportunity to examine the effect of the sup-pressor on different well-defined types of transgene-inducedRNA silencing, thus, providing information about the mech-anism of suppression. The stable expression assays are alsowell suited to investigate the role of suppressors in sys-temic silencing using grafting (Fig. 4B; Guo and Ding, 2002;Mallory et al., 2001, 2003). In these assays, the ability of aplant to send a mobile silencing signal is assayed by graft-ing an expressing line onto the top of it (the bottom plantis called the rootstock and the expressing plant grafted ontothe top is called the scion) (Palauqui et al., 1997). If the sup-pressor of silencing in the rootstock blocks either the pro-duction or movement of the systemic silencing signal, thenthe transgene in the scion will continue to be expressed. Ifa suppressor does not block the systemic silencing signalin either of these ways, then the transgene in the scion willbecome silenced (Fig. 4B).

4. Mechanism of suppression

The assays discussed above have proven extremely use-ful as rapid and sensitive methods to identify suppressorsof silencing. They have also enabled rudimentary charac-terization of suppressor mechanism, but conflicting resultsfrom different assays have made it difficult to draw firm con-clusions for many suppressors. Interestingly, the currentlyknown suppressors share no obvious similarities at eitherthe nucleic acid or the protein level, perhaps reflecting dif-ferences at the mechanistic level as well. At present, twomajor classes of suppressor action have been identified.

4.1. Suppressors that affect small RNA metabolism

Many suppressors reduce the accumulation of siRNAs,raising the possibility that silencing is blocked at the stepat which Dicer processes the dsRNA that triggers silencing.Thus, it may be that many suppressors prevent silencing byblocking production of the siRNAs that provide the sequencespecificity of the process. A second mechanism involvingsiRNAs is exemplified by the suppressor P19, which hasbeen shown to bind siRNAs, perhaps sequestering them and


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

thereby blocking their function (Silhavy et al., 2002). Inter-estingly, the effect of suppressors on small RNA metabolismmay extend to other types of small regulatory RNAs. Forexample the silencing suppressor HC-Pro affects the accu-mulation not only of the siRNAs that mediate silencing butalso of endogenous microRNAs (miRNAs), which have beenimplicated in development (Kasschau et al., 2003; Malloryet al., 2002b). Surprisingly, although HC-Pro blocks the ac-cumulation of siRNAs, it enhances the accumulation of miR-NAs (Mallory et al., 2002b). Even more interesting, thereis evidence that HC-Pro might block the function of miR-NAs (Kasschau et al., 2003). It remains to be seen if otherviral suppressors also affect the miRNA pathway, perhapsusing that pathway to turn off expression of genes requiredfor silencing.

4.2. Suppressors that affect systemic silencing

Systemic silencing can be assayed in either transient ex-pression experiments or in experiments with stably trans-formed transgenic lines (Figs. 2B and 4B). Many suppressorshave been demonstrated to block systemic silencing in atleast one of these assays (Table 1). Because of the conflictingresults sometimes obtained with different assays, the mostsuccessful attempts to determine whether a suppressor pri-marily affects systemic silencing have used a multi-prongedapproach rather than relying on just one type of assay.

For example, although HC-Pro did not block systemic si-lencing in grafting experiments using stable transgenic lines

(Mallory et al., 2001, 2003), it interfered with systemic si-lencing in the transient expression assay (Hamilton et al.,2002). Because HC-Pro is a powerful suppressor of localsilencing in all assays, these results suggest that HC-Pro pri-marily affects local silencing, but also has a smaller effecton systemic silencing. In contrast, CMV 2b primarily targetssystemic silencing because it blocks movement of the sig-nal 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 aparticular suppressor on one aspect of silencing could lead,perhaps through feedback mechanisms, to secondary effectson other parts of the pathway, thereby making it appear thatthe suppressor works at multiple points.

4.3. Conflicting results using different assays

Why do different assays give different results when look-ing at effects of viral suppressors on systemic silencing?The major conflicts are seen with stable expression assaysversus transient ones and probably reflect a number of in-trinsic differences between the systems. Transient expres-sion assays and the reversal of silencing assay useAgrobac-teriumto induce silencing. BecauseAgrobacteriumis a plantpathogen, infiltration likely induces plant defensive and bac-terial counter-defensive interactions, which might modifythe activity of some viral suppressors. Thus, attempts tocompare suppressor activities or to understand the mecha-nism of action of the different suppressors are complicated


by the largely unknown effects ofAgrobacteriumon thesystem. Similarly, the reversal of silencing assay involvesvirus infection and likely induces accompanying defenseand counter-defensive responses that complicate the inter-pretation of results. Further sources of variation include dif-ferences in the level and time course of expression of thesilencing suppressor and silencing inducer in the differentsystems.

Of the three widely used types of assay (Section 3), stableexpression assays are the ones most free of complicationsdue to extraneous pathogens. In addition, use of the samewell-characterized silenced transgenic line to test the effectsof different viral suppressors affords a degree of standard-ization from lab to lab not yet found with transient expres-sion assays. It is important to use well-characterized linesto compare suppressor activities because different types oftransgenes trigger silencing in different ways (Vance andVaucheret, 2001).

Understanding the basis of the conflicting results givenby the currently widely used assays will likely offer con-siderable insight into the mechanisms of suppressor action.Alternative approaches such as yeast two-hybrid, biochemi-cal, and localization studies (seeSection 5) are increasinglybeing used to investigate the mechanisms of action of thedifferent viral suppressors of silencing and will undoubtedlyhelp 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 first identified sup-pressors of RNA silencing and also one of the best studiedfrom a mechanistic standpoint. The initial indication thatCMV 2b suppressed silencing came from the reversal ofsilencing assay in which 2b expressed from PVX couldprevent the initiation of silencing but could not reverse si-lencing that was already established (Brigneti et al., 1998).That early result raised the possibility that 2b might blocksystemic silencing. Subsequently, stable expression assaysand grafting experiments provided an elegant demonstrationthat 2b blocks the movement of the systemic silencing signal(Guo and Ding, 2002). The stable expression assays madeuse of a well-characterized silenced tobacco line called6b5 (Elmayan and Vaucheret, 1996), which is silenced forthe reporter gene GUS and is a model for sense-transgeneinduced RNA silencing. The 6b5-GUS locus triggers si-lencing even when it is hemizygous, produces high levelsof GUS siRNAs, and is highly effective in the productionand transmission of a systemic silencing signal as demon-strated by grafting experiments. A genetic cross betweenline 6b5 and a stable transgenic tobacco line expressingCMV 2b established that 2b could partially suppress localsense-transgene induced silencing: offspring of the crossaccumulated GUS mRNA, but GUS siRNA accumulation,

although reduced, was not eliminated. However, graftingexperiments definitively demonstrated that CMV 2b blocksthe movement of the systemic silencing signal. In the firstprotocol, 6b5 rootstocks suppressed for silencing by CMV2b did not silence grafted GUS-expressing scions, showingthat expression of CMV 2b in the rootstocks preventedproduction or transmission of the systemic silencing signal(Fig. 5A, first panel). In the second protocol, a small spacerof transgenic tobacco expressing CMV 2b, grafted betweena GUS-silenced 6b5 rootstock and a GUS-expressing scion,blocked systemic silencing (Fig. 5A, second panel). Thus,CMV 2b prevents transmission of the systemic silencingsignal in a stable expression assay.

The experiments described above provide compelling ev-idence that the mechanism of action of CMV 2b, at least inpart, is to block systemic silencing.Guo and Ding (2002)also report similar conclusions based onAgrobacteriumco-infiltration experiments. Paradoxically, the same type ofco-infiltration experiments in another laboratory produced adifferent result: CMV 2b delayed but did not block systemicsilencing (Hamilton et al., 2002). A possible cause of thisdiscrepancy is that the two labs were using different ratiosof silencing inducer to suppressor in the co-infiltrations(S.W. Ding, personal communication), suggesting that astandardized methodology for co-infiltration assays wouldhelp resolve some of the current inconsistencies.

How does CMV 2b protein block the movement of themobile silencing signal? The ability of 2b protein to pre-vent the transmission of the signal suggests that it either se-questers or inactivates the signal in the phloem stream. Onepossibility is that 2b acts directly by binding to the signal.However, the finding that 2b localizes to the nucleus (Lucyet al., 2000) suggests that the suppressor acts indirectly, per-haps by activating one or more processes that subsequentlyaffect the signal.

5.2. Potyviral helper-component protease (HC-Pro)

HC-Pro was the first identified suppressor of RNA silenc-ing. The original reports demonstrated that it suppresses bothtransgene- and virus-induced silencing (Anandalakshmiet al., 1998; Kasschau and Carrington, 1998). In contrast toCMV 2b protein, it is able to reverse established silencingin the reversal of silencing assay (Brigneti et al., 1998), sug-gesting that the two suppressors work at different steps in thesilencing pathway. Although HC-Pro alone has suppressoractivity (Anandalakshmi et al., 1998; Brigneti et al., 1998),it is frequently expressed as the proteinase 1 (P1)/HC-Propolyprotein in suppression of silencing studies. TheP1/HC-Pro construct is the N-terminal portion of the naturalviral polyprotein and allows HC-Pro to be proteolyticallyprocessed just as when expressed from the viral genome.Some evidence suggests that P1 might enhance HC-Proactivity as a suppressor of silencing (Pruss et al., 1997).

A variety of approaches, including both transient and sta-ble expression assays, have been used to investigate how


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

HC-Pro suppresses RNA silencing. Despite some conflictingresults, at least one common finding has emerged: HC-Proaffects small RNA metabolism. Exactly how HC-Pro exertsits effect on small RNAs and how this leads to suppressionof silencing are questions that remain to be resolved. Theinteraction(s) of HC-Pro and its cellular effector(s) probablyoccur in the cytoplasm because HC-Pro is found primarilyin cytoplasm (Mlotshwa et al., 2002a).

5.2.1. HC-Pro and small RNAsHC-Pro has been reported to alter the accumulation of

several classes of small RNAs: the siRNAs that direct RNAdegradation during silencing, a novel class of slightly-largersmall RNAs of unknown function, and the endogenous mi-croRNAs (miRNAs) that have been implicated in regulationof development. In stable expression assays in tobacco,HC-Pro has been reported to suppress three classes oftransgene-induced RNA silencing, in each case interferingwith the accumulation of siRNAs (Mallory et al., 2001,2002b). Similarly, siRNA accumulation is dramaticallyreduced during HC-Pro suppression of silencing in tran-sient expression assays (Johansen and Carrington, 2001;Llave et al., 2000). In stable expression assays in tobacco,HC-Pro suppression of IR transgene-induced silencingor amplicon-transgene-induced silencing—but not sensetransgene-induced silencing—resulted in the accumulationof a novel class of slightly-larger small RNAs (Malloryet al., 2002b). Similar slightly-larger small RNAs have alsobeen observed in transient expression assays (Hamiltonet al., 2002). The function of this class of small RNAs isnot clear; however, they do not appear to act as siRNAsbecause their presence is not correlated with RNA degra-dation (Hamilton et al., 2002; Mallory et al., 2002b). They

correlate with systemic silencing in transient expressionassays (Hamilton et al., 2002) but not in stable expressionassays (Mallory et al., 2002b). Finally and surprisingly,the accumulation of endogenous miRNAs is increased inboth tobacco andArabidopsistransgenic lines that expressHC-Pro (Kasschau et al., 2003; Mallory et al., 2002b),suggesting a more general role in the biogenesis of smallregulatory RNAs. A recent report that HC-Pro enhancesthe stability of several miRNA target messages raises thepossibility that HC-Pro affects the function as well as thebiogenesis of small RNAs (Kasschau et al., 2003).

5.2.2. HC-Pro and systemic silencingIn stable expression experiments utilizing grafting,

HC-Pro failed to block systemic silencing (Fig. 5B, firstpanel; Mallory et al., 2001, 2003). Three different trans-genic tobacco lines were used as rootstocks, representingthree different means of inducing silencing: sense trans-gene, 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 com-petent virus, HC-Pro actually promoted systemic silencing(Mallory et al., 2003). These results suggest that productionand transmission of the systemic silencing signal are largelyunaffected by HC-Pro, implying that HC-Pro suppressionof silencing occurs downstream of the signal. To test thathypothesis, the effect of HC-Pro on perception of and/orresponse to the silencing signal was tested in additionalgrafting experiments (Fig. 5B, second panel;Mallory et al.,2001). A silenced GUS line previously shown to silenceGUS systemically across a graft junction was used as root-stock, and the scion was a line that expressed both GUSand HC-Pro. The scion failed to become silenced for GUS,


showing definitively that HC-Pro works downstream of thesystemic silencing signal: The rootstock is known to senda signal; therefore, in the presence of HC-Pro, the scioneither fails to perceive the signal or fails to respond to it.

Although the grafting experiments described above makea convincing case that HC-Pro does not block the systemicsilencing signal, results fromAgrobacteriumco-infiltrationassays suggest that this suppressor does interfere with sys-temic silencing (Hamilton et al., 2002). There are a num-ber of possible reasons for obtaining different results withthe two assays. First, the stable transgenic lines express theP1/HC-Pro polyprotein, whereas the transient assay exper-iments used a construct that encodes only HC-Pro and hasa methionine substituted for the natural N-terminal aminoacid of the protein. The protein produced in the transient as-say, therefore, is not identical to HC-Pro produced from thevirus. Thus, it is possible that the difference between the twoassays reflects 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 reporteda dramatic difference in the effect on PVX replication (Prusset al., 1997). A second possibility is that the result in thetransient assay depends on the ratio of inducer to suppres-sor, as suggested for CMV 2b protein (seeSection 5.1), andthis possibility could be tested by using a range of ratios.A more exciting possibility is that the discrepancy reflectsan intrinsic difference between the two assays, as discussedin Section 4.3. Thus, HC-Pro might block systemic silenc-ing in some circumstances, but not in others, and identify-ing the important variables will help in our understandingof HC-Pro suppression of silencing.

5.2.3. HC-Pro interacting proteinsIf HC-Pro works by interacting with components or regu-

lators of the silencing machinery, identification of plant pro-teins that interact with HC-Pro should provide clues aboutits mechanism of action. Using the yeast two-hybrid sys-tem, an HC-Pro-interacting protein called regulator of genesilencing calmodulin-like protein (rgsCaM) was identified(Anandalakshmi et al., 2000). Like HC-Pro, rgsCaM couldreverse silencing when expressed from PVX in the rever-sal of silencing assay. In addition, when over-expressed instable expression experiments, rgsCaM caused a develop-mental phenotype typical of HC-Pro-expressing transgeniclines and interfered with virus induced gene silencing. Be-cause calmodulins are classic signaling molecules, these re-sults raise the possibility that HC-Pro suppresses silencingindirectly via a signal cascade involving rgsCaM.

5.3. Tombusvirus P19

This protein has been an exciting addition to the reper-toire of plant viral suppressors. Initially described in the re-versal of silencing assay (Voinnet et al., 1999), it appearedto be a weak suppressor, only reversing silencing in the re-gion of veins. Similarly, P19 delayed but did not prevent

virus-induced gene silencing and did not suppress silencinginduced by a defective interfering RNA that accumulates tohigh levels (Qiu et al., 2002). In transient expression as-says, however, P19 is a star, blocking both local and sys-temic 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 stud-ies have shown that P19 binds siRNAs and that binding de-pends on characteristics of RNase III products (dsRNAs withtwo nucleotide 3′ overhangs) (Silhavy et al., 2002). This re-sult raises the possibility that P19 suppresses silencing bysequestering siRNAs, thereby preventing their incorporationinto the RISC complex to serve as guides. This is a novelmechanism among suppressors, and because it theoreticallystems from an intrinsic property of the protein to bind func-tional siRNAs, it is possible that P19 could interfere withsilencing in a broad range of different plants (and even otherorganisms).

5.4. Potato virus X (PVX) p25

Three of five potexviruses tested in the reversal of silenc-ing assay suppressed RNA silencing, although PVX wasone that did not show suppressor activity (Voinnet et al.,1999). Paradoxically, however, PVX is the only potexvirusfor which a suppressor of RNA silencing has subsequentlybeen characterized. In grafting experiments designed to sep-arate movement of the systemic silencing signal from thatof the virus in virus induced silencing (VIGS),Voinnet et al.(2000)unexpectedly found that PVX infection failed to pro-duce systemic silencing independent of the presence of virus,suggesting that a PVX-encoded protein interferes with pro-duction or transmission of the systemic signal. Protein p25,which is required for cell-to-cell movement of potexviruses,was identified as the culprit in a set of experiments in whichdeletion derivatives of replication competent PVX-GFP viralvector constructs were agro-infiltrated into plants express-ing GFP. The PVX-GFP constructs were restricted to ini-tially infiltrated cells because they all lacked coat protein,which is also required for potexviral movement. Inductionof systemic silencing in these experiments, therefore, de-pends entirely on the systemic signal. Only viral constructsfrom which p25 expression had been eliminated inducedwidespread systemic silencing in all plants.

Agro-coinfiltration experiments showed that p25, with-out any other PVX protein, was sufficient to block sys-temic silencing. Moreover, p25 blocked systemic silencingwhether silencing was induced by a simple transgene con-struct (35S-GFP) or by a replication competent PVX-GFPconstruct. The effect of p25 on local silencing in these ex-periments, however, depended on the nature of the constructused to induce silencing. Although p25 suppressed local si-lencing in agro-coinfiltration assays with 35S-GFP, it didnot suppress local silencing induced by infiltration of repli-cation competent PVX-GFP constructs. p25 suppression ofsystemic silencing in agro-coinfiltration assays is correlated


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 aconvincing case that PVX p25 blocks systemic silencing,a different result was obtained in experiments using trans-genic plants constitutively expressing white clover mosaicpotexvirus (WClMV) p25. Agro-infiltration of a 35S-IR con-struct systemically silenced these plants (Foster et al., 2002).Moreover, the systemic silencing reverted a severe develop-mental phenotype, indicating that the systemic signal wasable to enter the shoot apical meristem. Thus, WClMV p25did not prevent systemic silencing in this experimental sys-tem. Whether the difference in the results obtained with thePVX and WClMV p25 proteins reflects differences in theassays or in the activities of the proteins has not yet beendetermined.

6. Do all plant viruses have suppressors of silencing?

The discovery that plants have a generalized antiviral de-fense mechanism triggered by dsRNA has revolutionizedour thinking about plant–virus interactions. In the euphoricaftermath of the initial identification of plant viral suppres-sors of silencing, a popular expectation was that most or allplant viruses would encode a suppressor of RNA silencing.Although many different viral suppressors have been iden-tified, the fact that HC-Pro helps so many different virusessuggests that a lot of viruses do not effectively suppress si-lencing. Such viruses may have evolved other ways to tryto avoid silencing, such as by replicating within spherulesin the ER (Schwartz et al., 2002), where the dsRNA is hid-den, or by replicating and moving rapidly enough to outrunthe mobile silencing signal. Furthermore, plants have otherdefense mechanisms, and silencing might not be the majorthreat for all viruses. Some viruses, therefore, may well havesuppressors of other defense pathways.

7. Suppressors of silencing as tools

The finding that certain viral proteins suppress RNA si-lencing has provided a new tool for technologies utilizinggenetically modified plants and is, therefore, of practical sig-nificance. Many biotechnological applications are impairedby RNA silencing, and suppressors of silencing can be usedto attain consistent, high-level expression of transgenes inplants (Mallory et al., 2002a; Voinnet et al., 2003). With si-lencing under control, transgenic plants can be engineered toproduce a range of transgene expression: moderate levels ofexpression to produce desired plant traits or very high-levelexpression to use the plant as a factory producing pharma-ceuticals, vaccines or other high-value gene products.

Perhaps more importantly, viral suppressors of silencingalso provide unique tools to understand the mechanism of

RNA silencing. Much of what is currently known aboutthe RNA silencing pathway comes from elegant in vitroand genetic studies in organisms other than plants (for arecent review seeTijsterman et al., 2002). In plants, tra-ditional genetic approaches have led to the identificationof a number of cellular genes required for RNA silencing(Dalmay et al., 2000, 2001; Fagard et al., 2000; Mourrainet al., 2000). Surprisingly, however, all of these genes arerequired for sense-, but not IR-transgene induced silencing(Boutet et al., 2003). The plant viral suppressors, many ofwhich appear to work downstream of dsRNA, provide anovel means of entry into parts of the silencing pathwaythat are not easily accessible by genetic means. The cur-rently known suppressors appear to work at different stepsin silencing, thereby providing access to a number of pointsin the pathway where silencing can be controlled.

Identifying host proteins that interact with a viral sup-pressor of RNA silencing is one very promising approachthat is being used to take advantage of viral suppressorsto elucidate the silencing pathway. The yeast two-hybridsystem has been used to find tobacco proteins that in-teract with HC-Pro, identifying a calmodulin-related pro-tein called rgsCaM that suppresses RNA silencing whenover-expressed (Anandalakshmi et al., 2000). This resultsuggests that a calcium controlled signal transduction path-way involving rgsCaM is one of the mechanisms regulatingRNA silencing. Intriguingly, in the case of geminiviruses,yeast two-hybrid studies have identified SNF1 kinase as acellular interactor of the tomato golden mosaic virus AL2protein (Hao et al., 2003). AL2 is a homologue of theACMV and TYLCV-C suppressors of silencing. Whetherthe interaction of AL2 with SNF1, which is a regulator ofmetabolism in response to stress, plays any role in suppres-sion of silencing is unknown as yet.

The potential of using viral suppressors to help understandthe mechanism of RNA silencing in plants is largely un-tapped, and these studies promise to be an exciting and fer-tile area of research. The recent identification of a viral sup-pressor that works in animal cells (Li et al., 2002) offers thepossibility that such proteins may provide a similar tool tounderstand the silencing pathway in other organisms as well.

Note added in proof

The tomato mosaic virus replication protein has recentlybeen reported to suppress RNA silencing (Kubota, K., Tsuda,S., Tamai, A., Meshi, T., 2003. Tomato mosaic virus repli-cation protein suppresses virus-targeted posttranscriptionalgene silencing. J. Virol. 77, 11016–11026).

Acknowledgements

VBV gratefully acknowledges support from the USDACompetitive Grants Program, NIH, and Dow AgroSciencesLLC.


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Roth, Pruss, and Vance 2004