Systemic antiviral silencing in plants

Virus Research 118 (2006) 1–6

Review

Systemic antiviral silencing in plants Qi Xie a , Hui-Shan Guo b,∗ a

National Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, PR China b National Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China Received 29 September 2005 Available online 20 December 2005

Abstract RNA silencing controls numerous developmental processes in eukaryotic organisms from fungi, plants, to animals. In plants as well as in animals, this system of RNA regulation functions as part of an immune response against invading viruses. From transitive RNA silencing to virus-induced gene silencing (VIGS), the systemic effects are proven to be the core of RNA silencing. This article reviews the latest advances in view of the effect of cellular RDR6, an RNA-dependent RNA polymerase (RdRp), on systemic RNA silencing, systemic virus silencing, and discusses the abilities of viral suppressors in modulating RNA silencing efficiency to establish effective infection. © 2005 Elsevier B.V. All rights reserved. Keywords: Systemic RNA silencing; RDR; Viral suppressor

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Systemic RNA silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Systemic virus silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suppression and “escape” of systemic silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Recently, tremendous progress has been made in understanding the various pathways of RNA silencing. RNA silencing controls numerous developmental processes in eukaryotic organisms through post-transcriptional gene silencing (PTGS) in plants, quelling in fungi, and RNA interference (RNAi) in animals (Baulcombe, 2004, 2005; Meister and Tuschl, 2004). In plants as well as in animals, this system of RNA regulation controls virus accumulation, and functions as part of an immune response against invading viruses (Voinnet, 2001; Li et al., 2002; Bennasser et al., 2005; Lu et al., 2005; Wilkins et al., 2005). Double-stranded RNAs (dsRNAs), which can be produced by the virus or the host, or present in stretches of single-stranded target (Molnar et al., 2005), act as a common silencing trigger and are cleaved by Dicer, a type III RNase. As a result small interfer-

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Corresponding author. Tel.: +86 10 62635685; fax: +86 10 62548243. E-mail address: [email protected] (H.-S. Guo).

0168-1702/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2005.11.012

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ing RNAs (siRNAs) of 21–25 nt in length are produced, which are a unifying feature of RNA silencing in all organisms. These siRNAs are then incorporated into a multi-component RNAinduced silencing complex (RISC), to promote cognate mRNA degradation, and/or loaded onto the RNA-induced transcriptional silencing complex (RITS) to direct chromatin silencing including DNA and histone methylation (Verdel et al., 2004). A fascinating feature of RNA silencing in plants is its ability to spread between cells through plasmodesmata and over long distances via phloem (Mlotshwa et al., 2002; Ruiz-Medrano et al., 2004). The mobile silencing signal trafficking resembles the viral movement (Mlotshwa et al., 2002), and the fact that many viral silencing suppressors are typically required for longdistance virus spread in the infected plant (Voinnet et al., 2000; Fagard and Vaucheret, 2000; Li and Ding, 2001; Kasschau and Carrington, 2001; Ding et al., 2004b), suggests that the signal is a crucial component of the antiviral defense system. Particularly important proteins required for systemic silencing are host

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RNA-dependent RNA polymerases (RdRp or RDR) (Dalmay et al., 2000; Mourrain et al., 2000). Many excellent reviews on RNA silencing have been recently published (Baulcombe, 2004, 2005; Ding et al., 2004a; Voinnet, 2005a; Wang and Metzlaff, 2005). In this update article, we review the latest information on how (a) host RDR functions during systemic RNA and virus silencing and (b) virally encoded suppressors control host silencing factors to establish infection.

component. The PSRP1 binds selectively to and mediates the cell-to-cell trafficking of 25 nt ssRNA species (Yoo et al., 2004) supporting the widespread thought that the long small RNAs (24–26 nt) are the strongest candidate as systemic silencing signal (Mlotshwa et al., 2002). The two strands of a siRNA duplex are not equally eligible for assembly into RISC (Khvorova et al., 2003; Schwarz et al., 2003) and it is possible that, likewise, a single-stranded small RNA is selected by the PSRP1 for transport through the vascular transport system.

1. Systemic RNA silencing 2. Systemic virus silencing The Arabidopsis RDR6, also known as SDE1/SGS2, was earlier shown to be essential for sense transgene-induced PTGS (S-PTGS) but not required for inverted repeat dsRNA-induced PTGS (IR-PTGS), possibly by contributing to production of dsRNA from the mRNA of the sense transgene (Dalmay et al., 2000; Mourrain et al., 2000; Vance and Vaucheret, 2001). siRNAs were shown to directly trigger RNA silencing in plants, as it has been also demonstrated in animals, and to induce transgene silencing in systemic leaves, distant from the original site of induction (Klahre et al., 2002; Lu et al., 2004b). Moreover, using viral vectors carrying parts of the GFP transgene, RNA silencing has been shown to spread from the initial target region to the entire GFP RNA (Vaistij et al., 2002). In both cases, secondary siRNAs were detected from regions 5 and 3 of the primary trigger sequence, and the spread of silencing to these adjacent sequences was transcript-dependent and required host RDR6 (Vaistij et al., 2002). Spreading of silencing both through the plant and along the transgene, and the strict requirement of RDR6 for this process was further demonstrated by Voinnet and co-workers (Parizotto et al., 2004). Plants transformed with a GFP sensor construct, in which the GFP was transcriptionally fused to a microRNA171 (miR171) target sequence (GFP-171.1), become silenced throughout the plant due to cleavage of GFP-171.1 guided by miR171. Taking into account that miR171 accumulates exclusively in flowers (Llave et al., 2002), the extension of GFP silencing to the whole plant likely resulted from the spread of silencing from the original site in the flower, where miR171 triggered transitive RNA silencing of the GFP sequence within GFP-171.1, to systemic tissue through the amplification and movement of secondary siRNAs, or some other signal containing the GFP sequence, via the vascular tissue. Secondary siRNAs were not detected in an rdr6 (originally called sde1) mutant in which GFP-171.1 transfomants displayed highly vein-specific GFP fluorescence in leaves (Parizotto et al., 2004) suggesting that RDR6 is responsible for the amplification of transitive RNA silencing. The movement and RDR6-dependent amplification of systemic silencing signal is also consistent with the observation of non-cell autonomous silencing. It can be initiated in a specific area of the plant and then spread via limited and extensive cell-to-cell movement and systemic transport (Himber et al., 2003; Voinnet et al., 2000; for review of non-cell autonomous silencing, see Voinnet, 2005b). The sequence specificity of RNA silencing and the recent detection of small RNA-binding protein (PSRP1) in phloem sap from cucumber and lupin (Yoo et al., 2004) support the idea that the mobile silencing signal could be the siRNAs-charged

An elegant and pioneering experiment determined that a systemic silencing signal comes from plant infected with movement-defective forms of Potato virus X (PVX) (Voinnet et al., 2000). This was done by disabling the PVX p25 protein to allow virus replication but no movement (Voinnet et al., 2000). Virus-induced gene silencing (VIGS) often involves the production of virus-derived siRNAs in plants infected by RNA as well as DNA viruses (Hamilton and Baulcombe, 1999; Chellappan et al., 2004). VIGS results in specific targeting of the viral and homologous host RNAs for degradation. Cleavage of viral dsRNA replicative intermediates by Dicer resulting in accumulation of primary siRNAs is considered to be primary VIGS (Voinnet, 2005a). Virus-derived siRNAs originated predominately in the most folded regions of the positive strand of the viral RNA have also been reported recently (Molnar et al., 2005). Primary VIGS must be further amplified to achieve an efficient antiviral defense response. Arabidopsis RDR6 was shown to regulate the accumulation of some viruses in plants through the analysis of the rdr6 mutant in Arabidopsis (Mourrain et al., 2000). Direct evidence of involvement of the host RDR6 in amplification of primary VIGS derived signal comes from the recent investigation by Baulcombe and co-workers (Schwach et al., 2005). The RDR6 homologue in N. benthamiana (NbRDR6) was determined to limit PVX systemic spreading, but not contribute significantly to the formation of virus-derived siRNA in PVX-infected plants (Schwach et al., 2005). There was no difference in PVX:GFP infection foci in inoculated leaves of WT and RDR6i plants, in which NbRDR6 had been silenced by an RNAi hairpin construct. This observation suggests that RDR6 does not compete with the viral RdRp during virus replication, and supports the idea that RDR6 is not required for VIGS initiation. In an assay of de novo induction of GFP silencing, no evidence was observed for spread of GFP silencing signal from the veins and to systemic young leaves in PVX:GFP-infected GFP16c/RDR6i plants in contrast to PVX:GFP-infected GFP16c plants, suggesting that there is an effect of RDR6 on the silencing signal at the systemic infection front. Via grafting experiments, Schwach et al. (2005) further confirmed that RDR6 activity is required for the cell to respond to the initial silencing signal, presumably siRNAs or some derivation resulting from primary VIGS, to generate many secondary siRNAs. These molecules would then trigger a more efficient antiviral secondary VIGS response in this tissue. Together with the observation that PVX:GFP spread more extensively into young tissue in RDR6i than in

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WT plants, the fact that the growing tip including the meristem of the PVX:GFP-infected RDR6i plant shows GFP fluorescence evenly distributed throughout, strongly suggests that RDR6 is implicated in the development of systemic RNA silencing in response to a primary VIGS signal received either with or ahead of the systemic infection front. As a consequence of the silencing, systemic spreading of the virus slows down and the virus is unable to reach the meristematic tissue (Schwach et al., 2005). Recovery from virus infection in plants (Ratcliff et al., 1997, 1999; Covey et al., 1997) is enhanced in plants carrying virusderived transgenes (Lindbo et al., 1993; Smith et al., 1995; Guo and Garcia, 1997). The RDR6-based amplification of silencing signals and meristem exclusion of virus infection (Schwach et al., 2005) can now explain this transgene-enhanced recovery phenotype. Viruses themselves can be the source of primary siRNAs, and secondary siRNAs can be produced from additional dsRNA derived from RDR6 synthesis using the virus-derived transgene RNA as templates. This leads to the rapid amplification of antiviral secondary VIGS, limiting systemic spread of the virus more effectively than in non-transgenic plants. In this respect, it seems that a portion of viral sequence, when expressed as transgene, become a more suitable RNA template for RDR6 than in the context of the viral genome. Support for this idea comes from the observation that endogenous gene sequences that are not susceptible to systemic silencing can be systemically silenced when expressed as transgenes (Palauqui et al., 1997). This suggests that RDR6 activity could be much more dependent on the quality than on the mere concentration of template to respond to an incoming silencing signal. Plant DNA virus, geminivirus, for example, genomes replicate in the nucleus and generate dsDNA intermediates, which are the templates for both replication and transcription. The transcription processes might provide a source of dsRNA to trigger RNA silencing in this system. In VIGS induced by a geminivirus, Cabbage leaf curl virus, only weak silencing was observed in rdr6 Arabidopsis, in which severe viral symptom were displayed; overexpressing RDR6 mRNA to complement the rdr6 mutation, the VIGS level was restored to as the control plants and viral symptoms reduced, this indicated that RDR6 protein was also playing a decisive role in amplification or stabilization of DNA VIGS (Muangsan et al., 2004). The RDR6-based amplification of VIGS signal resulting in antiviral defense, however, may have specificity for particular viral RNAs. The RDR6i plants were hypersensitive to PVX, Potato virus Y (PVY) and Cucumber mosaic virus (CMV) in combination with its Y satellite, but not to Tobacco mosaic virus (TMV), Tobacco rattle virus (TRV), Turnip crinkle virus (TCV) or CMV alone (Schwach et al., 2005). A previous study on Arabidopsis rdr6 mutant showed hypersensitivity to CMV, but not to TMV and TRV (Dalmay et al., 2000; Mourrain et al., 2000). A possible explanation for these observations could be that some viral RNAs may have specific sequences or structures that make them more suitable as templates for RDR6. Moreover, other RDR proteins, for example, RDR1, also appear to have a role during virus challenge. Tobacco and Arabidopsis plants with reduced levels of RDR1 showed enhanced susceptibility to TMV and PVX (Xie et al., 2001; Yu et al., 2003). A natural truncated

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loss-of-function mutation of RDR1 is associated with increased susceptibility to certain viruses by N. benthamiana, and overexpressing a functional RDR1 mRNA in N. benthamiana increased resistance against viruses belonging to tobamoviruses genus but not against CMV or PVX (Yang et al., 2004). It seems, therefore, the antiviral effect of RDRs has specificity for different viral RNAs. In addition to its influence on long-distance movement of viruses, the silencing signal has recently been shown to influence short-range PVX movement between cells (Bayne et al., 2005). Short-distance movement of the virus-induced silencing signal has been observed in foci surrounding the single epidermal cells in which virus cell-to-cell movement-defective Turnip crinkle virus (TCV) mutant replicates (Ryabov et al., 2004). If the short-distance movement signal moved ahead of the virus, spread of virus to adjacent cells would be prevented or slowed down, as it has been demonstrated for PVX (Bayne et al., 2005). Short-distance movement of transgene silencing signal has been shown not to depend on RDR6 (Himber et al., 2003), assuming this is also true for cell-to-cell movement of virus-induced silencing signal, short-distance movement of virus may be not affected by RDR6, which is required for the amplification of the long-distance silencing signal and meristem exclusion of virus infection (Schwach et al., 2005). A silencingassociated protein DCL4 (Dicer-like-4), which is involved in biogenesis of trans-acting siRNAs (Xie et al., 2005; Gasciolli et al., 2005; Yoshikawa et al., 2005), has recently been shown to produce the 21 nt siRNA component of the plant cell-to-cell silencing signal induced by IR-PTGS (Dunoyer et al., 2005). At least three Silencing Movement Deficient genes (SMD1, SMD2 and SMD3) are required for cell-to-cell trafficking of silencing induced by transgene (Dunoyer et al., 2005). It will be of significance to verify the effects of these silencing signal macromolecules including DCL4 on the short-distance movement of silencing induced by virus infection and antiviral defense. 3. Suppression and “escape” of systemic silencing The most convincing argument that PTGS acts as an antiviral defense in plants and animals is that viruses evolve proteins that can function as suppressors of PTGS (Li and Ding, 2001; Ding et al., 2004a; Qu and Morris, 2005). About 30 viral suppressors have been identified in both plant and animal viruses (Voinnet, 2005a). Results from some of the well-studied plant viral suppressors indicate many suppressors interfere with RNA silencing directly by one way or another. Some interfere with systemic signaling for silencing (Mlotshwa et al., 2002). For instance, the CMV 2b protein inhibits the activity of the mobile systemic silencing signal (Guo and Ding, 2002). The PVX p25 prevents spread of the systemic silencing signal but not the local silencing mediated by virus RNA (Voinnet et al., 2000). In transgene-induced silencing assays, however, p25 was shown to be able to prevent both local and systemic silencing (Voinnet et al., 2000). These observations suggest that p25 interferes specifically with RDR6-dependent amplification of secondary dsRNA or secondary VIGS (Voinnet et al., 2000; Hamilton et al., 2002). A very recent report by Baulcombe and co-workers

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(Bayne et al., 2005) shows that p25 is also necessary, although not sufficient, to suppress short-distance movement of silencing signal to allow PVX cell-to-cell movement. p25 suppresses IR-PTGS but not eliminates siRNA accumulation, suggesting that p25 may interfere with the assembly or activity of effector complexes of silencing (Bayne et al., 2005). Suppressors acting upstream of the siRNA production or blocking the activity of siRNA may therefore prevent both the local and the systemic signaling. The P1/HC-Pro suppressor from potyviruses may prevent the accumulation or destabilize siRNAs (Mallory et al., 2002) or interfere with the incorporation of siRNA into RISC (Chapman et al., 2004). An interesting finding is that P1/HCPro blocked the accumulation of siRNA but did not prevent systemic silencing in a grafting assay (Mallory et al., 2001). A possible explanation is that a very low (undetectable) level of siRNA might exist and spread to the grafted scion and be amplified in RDR6-based manner. The tombusvirus p19 protein sequesters siRNAs (Dunoyer et al., 2004) and hence prevents their incorporation into RISC (Lakatos et al., 2004) and their possible function as systemic silencing signal, and this is supported by findings on the protein crystal of p19 (Vargason et al., 2003; Ye et al., 2003). Recently, the p69 encoded by Turnip yellow mosaic virus has been identified as silencing suppressor by preventing host RDR-dependent secondary dsRNA synthesis (Chen et al., 2004). P14 protein encoded by aureusviruses is an efficient suppressor of both virus- and transgene-induced silencing by sequestering both long dsRNA and siRNAs without size specificity (Merai et al., 2005). The potential that viruses have multiple suppressors was validated when three silencing suppressors, p20, p23 and CP, encoded by Citrus tristeza virus (CTV) were identified (Lu et al., 2004a). Of them, p20 and CP were able to suppress the export of the silencing signal, while p20 and p23 inhibited intracellular silencing (Lu et al., 2004a). Multiple viral components, which include viral RNAs and putative RNA replicase proteins, are required for a silencing suppression mechanism recently reported for Red clover necrotic mosaic virus (Takeda et al., 2005). In this case, depriving the RNA silencing machinery of Dicer-like enzymes by the viral replication complexes appears to be the cause of the suppression. Also recently, a protoplast-based transient RNA silencing system has been used to dissect novel effects of viral suppressors (Qi et al., 2004). The CMV 2b protein, for example, showed to be a very potent suppressor of silencing in single cells (Qi et al., 2004) in addition to inhibit the activity of the mobile silencing signal. This indicates that a viral suppressor can affect multiple steps of the RNA silencing pathway. The first silencing suppressor, Pns10, encoded by a plant double-stranded RNA (dsRNA) virus, Rice dwarf virus (RDV), has recently been identified (Cao et al., 2005). Pns10 suppressed local and systemic S-PTGS but not IR-PTGS, suggesting that Pns10 also targets an upstream step of dsRNA formation in the RNA silencing pathway (Cao et al., 2005). It seems, therefore, the RNA components, such as single-strand template RNA, dsRNA and/or siRNA, of the silencing pathway are preference targets of most viral suppressors. The suppressors described above have direct effects on the RNA silencing pathway. Silencing suppressors may also

function by protecting the viral RNA from the RNA silencing machinery (Schwartz et al., 2002). A viral protein interacting with viral RNA to form cytoplasmic filamentous ribonucleoprotein (RNP) complexes involved in protection or stabilization of the viral RNA in all infected tissues and possibly in movement of the viral RNA through the phloem has been reported (Taliansky et al., 2003). Recently, Ding et al. (2004b) found that TMV 126 kDa protein, known to function as an RNA silencing suppressor, formed viral replication complexes (VRCs). The size of the VRCs correlated with the suppressor activity of the 126 kDa protein that formed it (Liu et al., 2005). However, it was determined that suppressor activity occurred only where virus was actively accumulating, thus the 126 kDa suppressor protein had no long-term effect on the silencing pathway, further supporting the idea that this suppressor may help to avoid, rather than to inactivate, the silencing system (Ding et al., 2004b). An interesting finding is that the VRCs traffic along microfilaments (Liu et al., 2005). This suggests that protection against silencing is afforded as the replicating viral RNA moves within the cell. Silencing suppression linked to viral transport was also observed with potexviruses, which required three triple gene block (TGB) proteins, TGBp1, TGBp2 and TGBp3 and CP for cell-to-cell movement. Of these, TGBp1 is a multifunctional protein that suppresses RNA silencing (Lough et al., 2001; Verchot-Lubicz, 2005). Tomato bushy stunt virus p19 protein is found to form dimers and interact with host RNA-binding proteins (Hin19) (Park et al., 2004). The requirement of the p19 suppressor activity for binding to itself and to Hin19 suggests that virus spread through suppression of RNA silencing could involve the formation of a complex that includes p19 dimers and host RNA-binding protein (Park et al., 2004). Another strategy for viral suppressors to modulate RNA silencing is highlighted by studies of geminiviruses suggesting that the viral gene AC2, which encodes a transcription activator protein (TrAP), suppresses RNA silencing by controlling the expression of host genes coding for positive or negative effectors of RNA silencing (Hartitz et al., 1999; Trinks et al., 2005; Vanitharani et al., 2005). However, suppressor AC2 (also known as AL2) from Tomato golden mosaic virus (TGMV) has recently been shown to interact with and inactivate adenosine kinase (ADK), a cellular enzyme important for adenosine salvage and methyl cycle maintenance, ADK activity was shown to be required to support RNA silencing. This indicates that AC2 might also suppress silencing by a novel mechanism that involves ADK inhibition (Wang et al., 2005). In summary, RNA silencing induced by either transgenes or viruses must be further amplified and spread to gain efficient systemic transitive RNA silencing or systemic antiviral silencing. Both processes required host RDR6 for generation of secondary siRNAs and secondary VIGS. The RDR6-independent initiation of VIGS in virus-infected plant (Schwach et al., 2005) reflects the plant ability to recognize dsRNA segments, either derived from virus replication or due to secondary or tertiary structure of target viral RNA, through a Dicer-like enzyme and thus keep pace with the initial replication and accumulation of viral RNAs. However, an immediate antiviral systemic response requires RDR6 to recognize the systemic signal that precedes or

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accompanies the transported target viral RNA, to yield a dsRNA substrate for Dicer. Most viral silencing suppressors tend to operate in a counter-defense manner through defeating siRNAsrelated steps of RNA silencing by blocking siRNA production, or interfere with siRNA function and/or preventing secondary siRNA synthesis. The capacity of a suppressor in preventing the onset of mobile systemic signal and the concomitant blockage of secondary VIGS, will lead to effective viral infection. On the other hand, strategies devoted to escape rather than suppress RNA silencing activity appears to be also effective tools to protect viral RNA inside the cell and facilitate viral infection. Acknowledgements We thank Dr. Juan Antonio Garcia and Ms. Yangsun Chan for critical reading of the manuscript. H.-S.G. was supported by National Basic Research Priorities Program (Grant No. 2002CCA03000) and National Natural Science Foundation of China (Grant No. 30525004). Q.X. was supported by National Natural Science Foundation of China (Grant No. 30325030). References Baulcombe, D., 2004. RNA silencing in plants. Nature 431, 356–363. Baulcombe, D., 2005. RNA silencing. Trends Biochem. Sci. 30, 290–293. Bayne, E.H., Rakitina, D.V., Morozov, S.Y., Baulcombe, D.C., 2005. Cellto-cell movement of Potato Potexvirus X is dependent on suppression of RNA silencing. Plant J. 44, 471–482. Bennasser, Y., Le, S.Y., Benkirane, M., Jeang, K.T., 2005. Evidence that HIV-1 encodes an siRNA and a suppressor of RNA silencing. Immunity 22, 607–619. Cao, X., Zhou, P., Zhang, X., Zhu, S., Zhong, X., Xiao, Q., Ding, B., Li, Y., 2005. Identification of an RNA silencing suppressor from a plant double-stranded RNA virus. J. Virol. 79, 13018–13027. Chapman, E.J., Prokhnevsky, A.I., Gopinath, K., Dolja, V.V., Carrington, J.C., 2004. Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev. 18, 1179–1186. Chellappan, P., Vanitharani, R., Fauquet, C.M., 2004. Short interfering RNA accumulation correlates with host recovery in DNA virus-infected hosts, and gene silencing targets specific viral sequences. J. Virol. 78, 7465–7477. Chen, J., Li, W.X., Xie, D., Peng, J.R., Ding, S.W., 2004. Viral virulence protein suppresses RNA silencing-mediated defense but upregulates the role of microrna in host gene expression. Plant Cell 16, 1302–1313. Covey, S.N., Al-Kaff, N.S., Langara, A., Turner, D.S., 1997. Plants combat infection by gene silencing. Nature 385, 781–782. Dalmay, T., Hamilton, A., Rudd, S., Angell, S., Baulcombe, D.C., 2000. An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101, 543–553. Ding, S.W., Li, H., Lu, R., Li, F., Li, W.X., 2004a. RNA silencing: a conserved antiviral immunity of plants and animals. Virus Res. 102, 109–115. Ding, X.S., Liu, J., Cheng, N.H., Folimonov, A., Hou, Y.M., Bao, Y., Katagi, C., Carter, S.A., Nelson, R.S., 2004b. The Tobacco mosaic virus 126-kDa protein associated with virus replication and movement suppresses RNA silencing. Mol. Plant Microbe Interact. 17, 583–592. Dunoyer, P., Himber, C., Voinnet, O., 2005. DICER-like 4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal. Nat. Genet. 37, 1356–1360. Dunoyer, P., Lecellier, C.H., Parizotto, E.A., Himber, C., Voinnet, O., 2004. Probing the microRNA and small interfering RNA pathways with virusencoded suppressors of RNA silencing. Plant Cell 16, 1235–1250.

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