ARGONAUTE2 Mediates RNA-Silencing Antiviral Defenses against Potato virus X in Arabidopsis 1[W][OA] Marianne Jaubert, Saikat Bhattacharjee, Alexandre F.S. Mello, Keith L. Perry, and Peter Moffett* Boyce Thompson Institute for Plant Research, Ithaca, New York 14853 (M.J., S.B., P.M.); Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, New York 14853 (A.F.S.M., K.L.P.); and Centre de Recherche en Ame ´lioration Ve ´ge ´tale, De ´partement de Biologie, Universite ´ de Sherbrooke, Sherbrooke, Quebec, Canada J1K 2R1 (P.M.) RNA-silencing mechanisms control many aspects of gene regulation including the detection and degradation of viral RNA through the action of, among others, Dicer-like and Argonaute (AGO) proteins. However, the extent to which RNA silencing restricts virus host range has been difficult to separate from other factors that can affect virus-plant compatibility. Here we show that Potato virus X (PVX) can infect Arabidopsis (Arabidopsis thaliana), which is normally a nonhost for PVX, if coinfected with a second virus, Pepper ringspot virus. Here we show that the pepper ringspot virus 12K protein functions as a suppressor of silencing that appears to enable PVX to infect Arabidopsis. We also show that PVX is able to infect Arabidopsis Dicer-like mutants, indicating that RNA silencing is responsible for Arabidopsis nonhost resistance to PVX. Furthermore, we find that restriction of PVX on Arabidopsis also depends on AGO2, suggesting that this AGO protein has evolved to specialize in antiviral defenses. The outcome of a plant-virus interaction is deter- mined by the balance between the ability of the virus to exploit host cellular mechanisms, and the multiple defense mechanisms employed against it by the host. As obligate parasites, viruses are unique in that their genetic material is transcribed, translated, and repli- cated entirely inside the host cell. As such, the inability of a virus to infect a given plant may be due to an incompatibility between viral proteins and the host translational machinery (Robaglia and Caranta, 2006), inhibition of viral replication (Ishibashi et al., 2007, 2009) and movement (Chisholm et al., 2000; Whitham et al., 2000; Cosson et al., 2010), or by detection and elimina- tion by disease resistance (R) proteins (Moffett, 2009). A major defense mechanism targeting foreign nucleic acids is based on RNA silencing (Ding and Voinnet, 2007). RNA silencing refers to a number of related cellular processes that employ small RNAs to regulate the expression of genetic material in a sequence-specific manner. Small RNAs are generated from dsRNA precursors by DICER-LIKE (DCL) RNase III-related enzymes and subsequently incorporated into RNA- induced silencing complexes (RISCs). The functions of RISCs are carried out in large part by the activity of RNAse H-like Argonaute (AGO) proteins. AGO pro- teins bind small RNAs, using them as guides to interact with homologous RNA molecules, and subsequently effect endonuclease activity, translational repression of mRNAs, or DNA methylation (Voinnet, 2009). The highly structured or dsRNA of viruses is targeted by DCL proteins to generate virus-derived small interfer- ing RNAs (vsiRNAs). These vsiRNAs can be incorpo- rated into virus-induced RISC complexes that target viral RNAs, thus making RNA silencing a doubly ef- fective antiviral mechanism (Ding and Voinnet, 2007; Omarov et al., 2007). Furthermore, small RNAs can serve as primers for host RNA-dependent RNA poly- merases to generate additional dsRNA targets for DCL enzymes to amplify the silencing signal (Voinnet, 2008; Vaistij and Jones, 2009; Garcia-Ruiz et al., 2010). In response to the pressure exerted by RNA silenc- ing, plant viruses have evolved viral suppressors of RNA silencing (VSRs; Diaz-Pendon and Ding, 2008; Alvarado and Scholthof, 2009). Mixed infection by two viruses can extend the host range, increase virus accu- mulation, and exacerbate viral symptoms (Latham and Wilson, 2008). In certain cases, this synergistic effect has been shown to be due to the action, in trans, of the VSR of one of the coinfecting viruses, likely because the VSRs of different viruses can target dif- ferent RNA-silencing components (Wu et al., 2010). An example of such a synergism is illustrated by the helper component proteinase from potyviruses that enhances the accumulation and systemic spreading of Potato virus X (PVX) in tobacco (Nicotiana tabacum) and sweet potato (Ipomoea batatas; Pruss et al., 1997; Sonoda et al., 2000). 1 This work was supported by the National Science Foundation (grant no. IOB–0343327 to P.M.). * Corresponding author; e-mail peter.moffett@usherbrooke.ca. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Peter Moffett (peter.moffett@usherbrooke.ca). [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a sub- scription. www.plantphysiol.org/cgi/doi/10.1104/pp.111.178012 1556 Plant Physiology Ò , July 2011, Vol. 156, pp. 1556–1564, www.plantphysiol.org Ó 2011 American Society of Plant Biologists