Viral suppressors of RNA silencing

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1 Review Viral suppressors of RNA silencing József Burgyán 1,2 and Zoltán Havelda 2 1 Istituto di Virologia Vegetale, CNR, Strada Delle Cacce 73, Torino, Italy 2 Agricultural Biotechnology Center, Szent-Gyorgyi A. 4. Gödöllö, Hungary The infection and replication of viruses in the host induce diverse mechanisms for combating viral infection. One of the best-studied antiviral defence mechanisms is based on RNA silencing. Consistently, several viral suppressors of RNA silencing (VSRs) have been identified from almost all plant virus genera, which are surprisingly diverse within and across kingdoms, exhibiting no obvious sequence similarities. VSRs efficiently inhibit host antiviral responses by interacting with the key components of cellular silencing machinery, often mimicking their normal cellular functions. Recent findings have revealed that the impact of VSRs on endogenous pathways is more complex and profound than had been estimated thus far. This review highlights the current understanding of and new insights into the mechanisms and functions of plant VSRs. Antiviral RNA silencing RNA silencing is a conserved sequence-specific gene regulation system, which has an essential role in the development and maintenance of genome integrity in a wide variety of organisms. In higher plants and insects, RNA silencing also operates as an adaptive inducible antiviral defence mechanism [1,2]. The silencing of RNA relies on host- or virus-derived nucleotide long srna molecules, which are the key mediators of RNA silencing-related pathways in plants and other eukaryotic organisms [3 6]. In plants, similar to other eukaryotic organisms, there are two main types of srnas, mirnas and sirnas, but the sirna class contains several different types [7,8]. These srnas are produced from double-stranded RNA (dsrna) or from folded structures by Dicer-like proteins (DCLs), and they guide Argonaute (AGO) proteins to target cognate RNA or DNA sequences [5]. These endogenous srnas play important roles in many aspects of gene regulation in plants, controlling developmental programming or biotic and abiotic stress responses [5,9]. Both cellular and antiviral sirna biogenesis often requires RNA-dependent RNA polymerases (RDRs). In the model plant Arabidopsis (Arabidopsis thaliana), there are four DCLs, ten AGOs and six RDRs [7], which are specialized for different silencingrelated pathways. It is not yet known whether all plant species have the same number of AGO family members [7]. One of the first discovered and well-studied functions of RNA silencing is the host defence against invading viruses [10]. The hallmark of its adaptive antiviral function is the accumulation of virus-derived sirnas (vsirnas) at a high level during viral infection [5,11 17]. Importantly, these Corresponding author: Burgyán, J. (J.Burgyan@ivv.cnr.it). vsirnas were found to be associated with AGO1, the slicer component of the plant RNA-induced silencing complex () effector [18,19]. As a counter defensive strategy, many plant viruses have evolved viral suppressors of RNA silencing (VSRs) to counteract antiviral silencing [1,20,21], providing strong evidence for the antiviral nature of RNA silencing. In addition, the lack or inactivation of VSRs leads to the recovery of plants from viral infections, demonstrating the efficient antiviral response of the plant [10,22,23]. Mechanism of silencing-based antiviral plant response The pathway of antiviral silencing can be divided into three major steps: (i) sensing and processing viral RNAs to viral sirnas; (ii) amplifying vsirnas; and (iii) assembling antiviral and targeting viral RNAs. The silencing-based antiviral plant response starts with the recognition of ds or structured single-stranded (ss) viral RNA by one or more members of plant Dicers [9,24]. The recognized viral RNAs are then processed by Dicers into vsirnas [1,5,9,12,17,25,26]. In plants, two distinct classes of vsirnas have been identified: primary sirnas, which result from the DCLmediated cleavage of an initial trigger RNA, and secondary sirnas, which require an RDR enzyme for their biogenesis [5,16,17,25,27 29]. In the Arabidopsis model plant, DCL4 and DCL2 are the most important DCLs involved in virusinduced RNA silencing and they can process ds or hairpin viral RNAs into vsirnas of 21 and 22 nt, respectively [5,26,30,31]. The amplification and high level of vsirna accumulation in many but not all virus infections depend on the combined activity of the host-encoded RDRs such as RDR1, RDR2 and RDR6. These findings suggest that aberrant viral ssrnas lacking quality control marks might be converted by RDR enzymes to dsrnas, which could serve as a substrate for secondary vsirna production [16,17,29,32,33].This model, however, is not fully supported by the previous observations that the majority of vsirnas are derived from the plus (mrna sense) viral strand [12,13,23,34,35]. The generated vsirnas are loaded into distinct AGO-containing effector complexes to guide them to their RNA target molecules [1,36,37]. In plants, the loading of sirnas into a particular AGO complex is preferentially, but not exclusively, dictated by their 5 0 terminal nucleotides [38]. It has been shown that both AGO1 and AGO7 function to ensure the efficient clearance of viral RNAs, and that AGO7 seems to work as a surrogate slicer in the absence of AGO1 [39]. Moreover, it is probable that AGO1 is capable of targeting viral RNAs with more compact structures, whereas AGO7 favours less struc /$ see front matter ß 2011 Elsevier Ltd. All rights reserved. doi: /j.tplants Trends in Plant Science, May 2011, Vol. 16, No

2 tured RNA targets [39]. Unfortunately, our knowledge is still limited about plant si/mirna assembly, although a recently developed in vitro system has provided further insights into plant development and RNA targeting mediated by this effector [40]. Suppressing the silencing-based antiviral response and suppressor strategies Plant viruses are efficient pathogens, which are able to infect and invade distinct plant species. They often cause severe symptoms and damage, which suggests an efficient counter defensive strategy against the antiviral silencing response. Many VSRs have been identified since the discovery of the first VSRs more than a decade ago [41 43]. The fact that most viruses have evolved VSRs underlines the antiviral nature of RNA silencing and reveals a pathogen counter defensive strategy with the active suppression of host surveillance [1,20,44]. The VSRs are considered the outcome of recent evolutionary processes and they are surprisingly diverse within and across kingdoms, with no obvious sequence homology. The various VSRs are able to target all effectors of the silencing pathway, such as viral RNA recognition, dicing, assembly, RNA targeting and amplification (Figure 1). Since the discovery of silencing suppressor proteins, a lot of effort has been made to understand the molecular basis of silencing suppression. However, the molecular mechanisms that underlie the actions of VSRs have only been resolved in a few cases. Most identified VSRs are multifunctional: besides being RNA-silencing suppressors, they often perform essential roles by functioning as coat proteins, replicases, movement [()TD$FIG] Viral RNAs proteins, helper components for viral transmission, proteases or transcriptional regulators. This multifunctional nature of VSRs often causes serious difficulties in the exploration of their mechanisms because the inactivation of VSRs often leads to a loss of viability of the given virus. Viral suppressors inhibiting viral RNA sensing and dicing The inhibition of viral RNA recognition and the subsequent dicing by plant Dicer effectors is not a frequent strategy of known VSRs. Two viral proteins have been identified that were shown to inhibit the processing of dsrna to sirnas in agroinfiltration assays: P14 of Pothos latent aureusvirus and P38 of Turnip crinkle virus (TCV). In addition, P38 and P14 have been shown to bind dsrna in a size-independent way [45,46]. Genetic evidence has shown that P38 specifically inhibits DCL4 activity, which has been shown to be the primary antiviral Dicer in the Arabidopsis model plant [30]. Recently, it was discovered that the action of the P38 protein occurs through AGO1 binding and that it interferes with the AGO1-dependent homeostatic network, which leads to the inhibition of Arabidopsis DCLs [47]. In addition to P14 and P38, the P6 VSR of the Cauliflower mosaic virus (CaMV) [48] has been shown to interfere with vsirna processing. P6 was previously described as a viral translational transactivator protein essential for virus biology. Importantly, P6 has two importin-alphadependent nuclear localization signals, which are mandatory for CaMV infectivity. A recent discovery showed that one of the nuclear functions of P6 is to suppress RNA silencing by interacting with dsrna-binding protein 4, which is required for the functioning of DCL4 [49]. Aberrant/cleaved viral RNA P14, p38 DCL2/3/4 Viral RNA recognition and dicing SGS3 V2,2b RDRs SD5 Amplification P19, HC-Pro, P21, P0, 2b, P38 AGO1/7 GW/WG? HSP90? assembly P1 AGO1/7 AGO1/7 RNA targeting Translational inhibition Slicing TRENDS in Plant Science Figure 1. Current model of antiviral RNA silencing in plants and its suppression by virus-encoded silencing suppressors. RNA silencing is initiated by the recognition of viral dsrnas or partially ds hairpin RNAs, which are processed to vsirnas by dsrna-specific RNases called DCLs (DCL2/3/4). In the next step, HSP90-activated AGO1/7 [40] are loaded with vsirna, thereby forming large s, which probably also incorporate other unidentified proteins (e.g. GW/WG motifs containing AGO interactor proteins). Afterwards, the vsirna-loaded targets viral RNAs by slicing or translational arrest. Secondary vsirnas are produced in an amplification loop through the actions of RDRs and their cofactors (SGS3 and SD5) [90]. Viral-silencing suppressors can disrupt these pathways at multiple points, thereby preventing the assembly of different effectors or inhibiting their actions. The points at which certain VSRs (i.e. P14, P38, V2, 2b, P19, HC-Pro, P21, P0 and P1) interact with the silencing pathways are depicted. 266

3 Viral suppressors preventing assembly The VSRs are able to prevent assembly by targeting one of its essential known or unknown components. The VSRs identified thus far are able to target sirnas and mirnas or AGO proteins in different ways. In addition, it is probable that other yet undiscovered components of effectors are also targets of the viral suppressors, which might possess a wide variety of suppressor strategies. The most common suppression strategy, evolved by several viral genera, is ds sirna sequestration [1,45,50 53], which prevents the assembly of the effector. Importantly, these sirna-binding VSRs are completely unrelated proteins, although they share analogous biochemical properties, suggesting their independent evolution in different viruses. The P19 protein of tombusviruses, probably the best known VSR thus far, prevents RNA silencing by sirna sequestration through binding ds sirna with a high affinity [54]. Crystallographic studies have shown that P19 forms a tail-to-tail homodimer, which acts like a molecular calliper, measuring the length of sirna duplexes and binding them in a sequence-independent way, selecting for the 19 bp long dsrna region of the typical sirna [55,56]. Thus, the P19 VSR evolved to bind and inactivate vsirnas, which are the most conserved key elements of the RNA-silencing pathway. Indeed, the P19 VSR was also able to inhibit sirna-directed RNA cleavage and to suppress the assembly of the in the heterologous in vitro system based on Drosophila embryo extracts [50]. In addition, recent findings have also demonstrated that P19 inhibits the spread of the ds sirna duplex identified as the signal of RNA silencing [57]. Other VSRs, such as the Tomato aspermy cucumovirus 2b protein or B2 of the insect-infecting Flock house virus, also bind ds sirna in a size-specific manner; however, structural studies have shown that their modes of binding sirnas do not share any similarity with P19 [58,59]. It is noteworthy that two sirna binding VSRs (HC-Pro and P38) require the RAV2 transcription factor for the suppression of RNA silencing, although the mechanistic role of this plant cofactor is unclear [60]. The 2 0 -O methylation step is essential in the biogenesis of mirnas and sirnas [61], and the sirna-binding VSRs (Carnation Italian ringspot virus P19, Tobacco etch virus HC-Pro, Tobamovirus P122/ P130) also compromise this step by preventing si/mirna assembly [51,62 65]. It is probable that these sirnabinding VSRs have a higher affinity to sirna and mirnas than to HEN1 methyltransferase. However, the inhibition of the methylation step also requires the temporal and spatial coexpression of the suppressor, endogenous or viral sirnas and mirnas [64]. The VSR of Potato chlorotic stunt crinivirus (SPCSV) uses a completely different strategy to prevent assembly. The SPCSV-encoded RNase3 endonuclease cleaves 21, 22 and 24 vsirnas into 14 bp products, which are inactive in the RNA-silencing pathways [66]. In the presence of sirna-binding/targeting VSRs, plants are not able to confine the spread of the viral infection because vsirnas are sequestered and inactivated before they can be incorporated into the. According to the model suggested previously [67,68] in the absence of sirna-binding VSRs, virus-specific vsirnas act as a systemic signal, moving faster than the virus in the infected plant and thereby establishing antiviral silencing in cells ahead of the infection front. Thus, the s already activated by vsirnas destroy the entering viral RNA, resulting in the fast recovery of the plant [23,67,68]. Indeed, sirna duplexes, as opposed to their precursor molecules, act as mobile silencing signals between plant cells [57,69]. AGO proteins targeting VSRs The prevention of assembly could also occur through direct or indirect interactions between VSRs and the protein components of. The 2b protein of Cucumber mosaic virus (CMV) was one of the first described VSRs [42], and it prevents the spread of the long-range silencing signal facilitating systemic virus infection [70]. Moreover, the 2b protein of Fny-CMV has been found to physically interact with the PAZ domain and part of the PIWI-domain of AGO1, inhibiting the slicing activity of AGO1 [18]. The Fny-CMV 2b was found to preferentially colocalize with the AGO1 protein in the nucleus of the cell and also in the cytoplasmic foci [71]. The Fny-CMV 2b protein expression phenocopies the ago1-27 mutant phenotype [18]. The VSR mutant CMV-D2b can be rescued by the dcl2/dcl4 host double mutant, which is impaired in vsirna production, suggesting that 2b is dispensable for infection and spread in a host defective in srna-directed immunity [32]. A crystallographic study showed that the 2b protein of Tomato aspermy virus (TAV), a cucumovirus related to CMV, binds sirna duplexes [58]. The analysis of the crystal structure of TAV-2b-siRNA showed that 2b adopts an alpha helix structure to form a homodimer and binds to sirna by measuring its length. The 2b protein is also known to bind long dsrna [72] and to inhibit the production of viral secondary sirnas [32]. Thus, cucumovirus 2b proteins have a dual mode of silencing inhibition, either by sequestering sirnas or by interacting with AGO1 and preventing assembly. The P0 protein of the phloem-limited poleroviruses also targets the AGO protein, the core component of the and induces its degradation [73 75]. TheP0proteinis indispensable for viral infection because null mutations of P0 in Beet western yellows virus and Potato leafroll virus strongly diminish or completely abolish virus accumulation [76]. In contrast to the RNA-binding VSRs, P0 has no RNA-binding activity [18,19]. Instead,itinteractswith the SCF family of E3-ligase S-phase kinase-related protein-1 components, orthologous to Arabidopsis ASK1 and ASK2, by means of its minimal F-box motif and thereby promotes AGO degradation [73 75]. The mutation of the F-box motif of P0 leads to the loss of silencing suppressor activity, suggesting the involvement of the proteasome pathway in AGO degradation. However, P0-mediated AGO1 degradation is insensitive to proteasome inhibitors, which is inconsistent with the idea that AGO1 is targeted by P0 for ubiquitination and proteasome-dependent degradation. Furthermore, P0 does not seem to interact directly with AGO1 [19,74]. Indeed, it has been found that P0 cannot interfere with the slicer activity of preprogrammed sirna/mirna containing AGO1, but can prevent the de novo formation of sirna/mirna-loaded AGO1 [19]. 267

4 The transgenic expression of P0 in Arabidopsis leads to severe developmental abnormalities, similar to those induced by mutants affecting mirna pathways. This effect of P0 is accompanied by AGO1 protein decay in planta and enhanced levels of several mirna target transcripts [74]. Preventing -mediated targeting by mimicking the glycine/tryptophan (GW/WG) host proteins required for AGO binding Two recent studies have illustrated a new strategy for VSR binding to AGO whereby the viral proteins mimic the cellular GW/WG repetitive motif [47,77]. The GW/WG linear peptide motif has been identified in different silencing-related proteins and it functions as AGO hooks to interact with AGO proteins [78,79]. The AGO-interacting WG/GW motif has been identified in the largest subunit NRPD1b of the RNA polymerase V in plants [80], in human GW182 protein [81 83] and in the Tas3 homologue of the GW182 RITS complex component in yeast [84,85]. In addition, the RdDM effector KTF1 contains several GW/ WG motifs and has been identified as an AGO4-binding element [86,87]. It was shown that the P38 VSR protein contains two GW repeats and interacts with A. thaliana AGO1 but not AGO4 [47]. Direct AGO1 binding to the individual N terminal and C terminal GW motifs of P38 in vitro was also detected, and the interaction was abolished by changing GW to GA. Point mutations in the P38 GW residues are sufficient to abolish TCV virulence, which is restored in the A. thaliana AGO1 hypomorphic mutant, uncovering both physical and genetic interactions between the two proteins. It has been suggested that P38 compromises srna loading into AGO1 by interacting with nonloaded AGO1 [47]. It has also been shown that AGO1 inactivation by P38 profoundly impacts the cellular availability of the four Arabidopsis Dicers, uncovering an AGO1-dependent homeostatic network that functionally connects these factors [47]. The P1 VSR of Sweet potato mild mottle ipomovirus also uses the GW/WG AGO hook to inhibit si/mirna-loaded activity [77]. It has been shown that the interaction between P1 and srna-loaded AGO1 is specific and direct. The suppression activity mapped to the N terminal part of P1 containing three GW/WG motifs and site-directed mutagenesis proved that these motifs are essential for both the binding and suppression of AGO1 functions. The action of the P1 protein is unique and different from that of P38. P1 inhibits both existing srna-loaded and de novo-formed s [77], whereas P38 cannot inhibit the existing si/mirna-loaded activity in plants (J. Burgyan et al., personal communication). However, P1 cannot bind RNA, whereas P38 binds dsrna in a size-independent manner [45,77]. An attractive model has been suggested for the mechanism of silencing suppression mediated by P1. This model can mimic essential AGO1 interactors and outcompete them from the, although these hypothetical AGO1 interactors have not yet been identified in plants. Alternatively P1 can compromise base-pairing between the srna-loaded AGO1 and target RNA [77]. The actions of P38 and P1 VSRs indicate a new way for the suppression of RNA silencing by mimicking the plant GW/WG proteins that hook up to AGOs, and this approach might represent a widespread strategy by pathogens to counteract RNA silencing-based host defences. VSRs targeting the amplification of antiviral silencing In virus-infected plants, host RDRs such as RDR1 and RDR6 have been shown to be involved in the synthesis of vsirnas. These secondary vsirnas play an essential role in silencing-based antiviral immunity against CMV [5,25,29]. Importantly, the 2b VSR inhibits the production of secondary vsirnas [32]. However, the mechanism by which 2b inhibits secondary vsirna production through AGO1 and/or vsirna binding is still unclear [53]. Previous studies have shown that the V2 protein from Tomato yellow leaf curl virus is an efficient suppressor of RNA silencing [88,89], and it has been proposed that V2 interacts with the tomato homologue of Arabidopsis suppressor of gene silencing-3 (SGS3) [89], which is involved in the RDR6-mediated silencing amplification pathway [90]. Another in vitro study on V2 showed that it competes with the SGS3 protein for binding a dsrna with 5 0 ssrna overhangs, whereas a V2 mutant lacking the suppressor function in vivo cannot efficiently outcompete SGS3 binding [91]. This finding might also reveal a new RNA intermediate essential for SGS3/RDR6-dependent sirna formation in plants [92,93]. Surprisingly, the plant RDR itself can function as a VSR, because a recent report showed that Nicotiana tabacum RDR1 antagonizes RDR6-mediated antiviral RNA silencing in Nicotiana benthamiana [94]. However, the mechanism by which RNA silencing suppression is mediated by Nt-RDR1 is still not well understood. Repressing AGO1 through the specific induction of mir168 by the tombusvirus P19 protein Recent work has revealed a new strategy for antiviral silencing suppression through the specific induction of mir168, which is ubiquitous in plant-virus infections [74] (Figure 2). The authors showed that in Cymbidium ringspot virus-infected plants the enhanced expression of AGO1 mrna is not accompanied by an increased AGO1 protein accumulation and that the P19 VSR of the virus specifically increased the expression of mir168, which controls the level of AGO1. By contrast, the inactivation of P19 resulted in a lack of mir168 induction by the P19 mutant virus, with an enhanced accumulation of the AGO1 protein. It was also shown that the transient expression of P19 mediates the induction of mir168 and that it downregulates the endogenous AGO1 level. These experiments revealed that the virus expressing P19 VSR inhibits the translation of AGO1 mrna by enhancing the endogenous mir168 level to alleviate the antiviral function of the AGO1 protein [95]. Importantly, it has also been demonstrated that in virus-infected plants P19 is not able to efficiently bind mirna duplexes, including mir168 [64,95], suggesting that the specific induction of mir168 by P19 is not mediated through mir168 binding. The drastic downregulation of AGO1 observed in some virus infections can have additional lateral effects similar to the recently described AGO1 quenching by P38 that impacts the cellular availability of the four Arabidopsis Dicers, compromising the AGO1-dependent homeostatic network [47]. Another consequence of AGO1 protein deficiency in 268

5 [()TD$FIG] Review Trends in Plant Science May 2011, Vol. 16, No. 5 Virus infection Virus counter defence P19 DCLs vsirnas Host defence response AGO1 translation AGO1 mrna mir168 induction AGO1/7 Viral RNA targeting AGO10 TRENDS in Plant Science Figure 2. Model for the regulation of the level of AGO1 mediated by P19-induced mir168. The virus infection triggers the enhanced expression of AGO1 mrna and, consequently, the AGO1 proteins as a part of the host defence respond. The enhanced AGO1 level facilitates the formation of vsirna-loaded s. However, when the P19 VSR is expressed by the virus it induces the overexpression of mir168, which presumably loads into AGO10 [95,109] and arrests the translation AGO1 mrna. Thus, P19 VSR, in addition to its sirna sequestering ability, ensures that the loading of viral sirnas onto AGO1 is efficiently inhibited, thereby allowing successful systemic infection. virus-infected plants can be the misregulation of mirna targets, resulting in disturbed gene expression, which can lead to the development of viral symptoms. VSRs interfering with the epigenetic modification of the viral genome Suppressors from the Geminiviridae family modulate endogenous biochemical pathways for the benefit of viruses. The Tomato golden mosaic virus (TGMV)-encoded AL2 protein and the closely related Beet curly top virus (BCTV) L2 interact with and inactivate adenosine kinase (ADK), a cellular enzyme important for adenosine salvage and the methyl cycle. Plants infected with the l2 mutant BCTV and other unrelated viruses display increased ADK activity, suggesting that ADK could be a part of the response of a plant to viral infection [96]. ADK plays a role in sustaining the methyl cycle. By inhibiting ADK, the AL2 and L2 proteins indirectly block this cycle and thereby could interfere with the epigenetic modification of the viral genome [97,98]. It has previously been shown that in vitro methylated TGMV cannot replicate in protoplasts [97], suggesting that the methylation of the viral genome could be a valid mode for combating geminivirus infections. Evidence for the transcription-dependent activity of Mungbean yellow mosaic virus and African cassava mosaic virus protein AC2 has also been found. This suggests that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes can code for components of an endogenous network that controls silencing [99]. Viral RNA replication-mediated silencing suppression Host factors involved in both RNA silencing suppression and viral replication have been proposed as playing roles in RNA silencing suppression during infection by the Red clover necrotic mosaic virus (RCNMV). The putative host factor involved in both processes could be the DCL1 protein because mirna biogenesis is inhibited by virus replication and dcl1 mutant plants show reduced susceptibility to RCNMV infection [100]. In the suggested scenario, DCL1 and its homologues are recruited by the viral replication complex and are, therefore, depleted from the silencing pathways. Side effects of VSRs Many VSRs have been identified as the pathogenic determinants largely responsible for virus-induced symptoms [101]. It is well established that the antiviral and endogenous silencing pathways share common elements, and VSRs have been shown to interfere with these pathways. Indeed, sirna-binding VSRs (e.g. HC-Pro and P122) can interact with sirna and mirna biogenesis [51,64, ] and compromise these srna-regulated plant gene expressions. Similarly, long dsrna-binding VSRs (e.g. P38 and P14) can compromise the activity of DCLs, and AGO1-targeting VSRs (e.g. 2b, P0, P1 and P38) inhibit s, which in turn can alter the expression of an unpredictable number of genes involved in plant development. Indeed, the expression of VSRs in transgenic plants leads to phenotypes that mimic virus symptoms [102,103,105]. However, the transgenic expression of VSRs does not necessary reflect the effects of viral infection on endogenous silencing pathways because in natural viral infections the expression of VSRs is restricted to virus-infected tissues and compartments, and it is also limited by time. A surprising effect of 2b VSR has been demonstrated recently. It has been shown that the 2b protein of CMV facilitates epigenetic modifications through the transport of sirna to the nucleus [106]. Future directions Viral suppressors often target conserved core elements of RNA-silencing pathways such as sirnas or effectors such as AGO and DCL proteins; in some cases, a single VSR can target more than one element in the silencing pathway 269

6 (Figure 1). However, the molecular bases of the silencing suppression of VSRs are more complex than has been suggested thus far. Indeed, the recently discovered action of P38 exemplified a complex interaction between VSR and plant silencing-regulated networks [47]. Similarly, the P19 protein of tombusviruses, in addition to sequestering sirna duplexes, specifically controls antiviral AGO1 expression through enhanced mir168 expression, which arrests AGO1 translation. It is possible that many other VSRs interact in multiple ways with RNA-silencing pathways, which remain to be discovered. There are still several gaps in our knowledge regarding the effectors of plant silencing machinery. For example, until recently, little was known about the mechanisms of plant si/mirna assembly or the possible components of the plant, which could also be potential targets of VSRs. We can predict that the recently developed plant in vitro system [40] will accelerate the exploration of plant assembly and RNA-targeting mechanisms mediated by this effector. Using this in vitro system, we will have a better possibility of exploring the mechanisms of VSR interactions with one of the components and preventing its assembly. To better understand the molecular mechanisms of VSR-mediated silencing suppression we need more detailed information about the replication, subcellular localization and regulation of the expressions of viral genes including VSRs, which are still not known for many plant viruses. Many VSRs have multiple functions in the virus life cycle, thereby the separate analysis of their silencing suppressor activities can lead to misinterpretations. Indeed, several proteins of mammalian viruses are able to inhibit RNA silencing in plants; however, it is not entirely clear whether the silencing suppression of these proteins is their real biological function in naturally infected mammalian cells [53]. Therefore, an analysis of VSRs in their natural virus backgrounds is essential. Well-described VSRs can also be used as powerful tools for better understanding the silencing pathways because they target specific steps of silencing machinery. Indeed, the P19 protein was recently used to demonstrate that sirna duplexes function as mobile silencing signals between plant cells [107] and that the P1, P38 and P0 proteins could be powerful tools for studying the still unknown components of s. Moreover, VSRs have been used for the cross-kingdom suppression of RNA silencing in mammalian cells, as exemplified by NS3 of the Rice hoja blanca virus [108], although the antiviral RNA-silencing response in mammalian cells remains controversial. Acknowledgements We apologize to those colleagues whose work we were unable to cite because of space and reference restrictions. József Burgyán and Zoltán Havelda are supported by a bilateral research program between Consiglio Nazionale delle Ricerche (CNR, Italy) and Magyar Tudományos Akadémia (MTA, Hungary). József Burgyán is also funded by the European Commission (FP6 Integrated Project SIROCCO LSHG-CT ). References 1 Ding, S.W. and Voinnet, O. (2007) Antiviral immunity directed by small RNAs. Cell 130, Ding, S.W. (2010) RNA-based antiviral immunity. Nat. Rev. Immunol. 10, Phillips, J.R. et al. (2007) The role of small RNAs in abiotic stress. FEBS Lett. 581, Voinnet, O. (2009) Origin, biogenesis, and activity of plant micrornas. Cell 136, Ruiz-Ferrer, V. and Voinnet, O. (2009) Roles of plant small RNAs in biotic stress responses. Annu. Rev. Plant Biol. 60, Llave, C. (2010) Virus-derived small interfering RNAs at the core of plant virus interactions. 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