Plants employ post-transcriptional gene silencing (PTGS) as an antiviral defense response. In this mechanism, viral-derived small RNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. ARGONAUTE1 (AGO1) is a key component of RISC: it carries the RNA slicer activity. As a counter-defense, viruses have evolved various proteins that suppress PTGS. Recently, we showed that the Polerovirus P0 protein carries an F box motif required to form an SCF-like complex, which is also essential for P0's silencing suppressor function. Here, we investigate the molecular mechanism by which P0 impairs PTGS. First we show that P0's expression does not affect the biogenesis of primary siRNAs in an inverted repeat-PTGS assay, but it does affect their activity. Moreover, P0's expression in transformed Arabidopsis plants leads to various developmental abnormalities reminiscent of mutants affected in miRNA pathways, which is accompanied by enhanced levels of several miRNA-target transcripts, suggesting that P0 acts at the level of RISC. Interestingly, ectopic expression of P0 triggered AGO1 protein decay in planta. Finally, we provide evidence that P0 physically interacts with AGO1. Based on these results, we propose that P0 hijacks the host SCF machinery to modulate gene silencing by destabilizing AGO1.

To counteract plant defence mechanisms, plant viruses have evolved to encode RNA silencing suppressor (RSS) proteins. These proteins can be identified by a range of silencing suppressor assays. Here, we describe a simple method using beet necrotic yellow vein virus (BNYVV) that allows a rapid screening of RSS activity. The viral inoculum consisted of BNYVV RNA1, which encodes proteins involved in viral replication, and two BNYVV-derived replicons: rep3-P30, which expresses the movement protein P30 of tobacco mosaic virus, and rep5-X, which allows the expression of a putative RSS (X). This approach has been validated through the use of several known RSSs. Two potential candidates have been tested and we show that, in our system, the P13 protein of burdock mottle virus displays RSS activity while the P0 protein of cereal yellow dwarf virus-RPV does not.RNA silencing suppressor (RSS) proteins are pathogenicity determinants widely expressed by plant and animal viruses (Li & Ding, 2006;Moissiard & Voinnet, 2004) that have recently also been identified amongst bacterial effectors (Navarro et al., 2008). Although these proteins are often multifunctional (Diaz-Pendon & Ding, 2008), the activity that will concern us here is their ability to block or attenuate plant host defence mechanisms, particularly post-transcriptional gene silencing. A large number of RSSs have been identified using procedures described in recent reviews (Li & Ding, 2006;Moissiard & Voinnet, 2004;Qu & Morris, 2005). The most popular assay is the 'patch' technique developed by Voinnet et al. (1998) based on the infiltration of Agrobacterium tumefaciens cultures harbouring the putative RSS and a reporter gene [usually the gene for green fluorescent protein (GFP)] on Nicotiana benthamiana. Viral vectors such as potato virus X have also proven to be useful to express and characterize RSS in a silencing reversal assay (Voinnet et al., 1999). More recently, alternative technologies based on functional complementation of defective viral mutants have been developed (Chiba et al., 2006;Powers et al., 2008). Here, we describe another simple experimental approach to screen for RSS activity based on a viral system derived from beet necrotic yellow vein virus (BNYVV). This test was used to assess the RSS activity of two uncharacterized viral proteins.For successful amplification on a plant host, most viruses need to fulfil three main functions: (i) replication, (ii) movement from the initial point of infection and (iii) suppression of the host RNA silencing mechanism. If infection is successful, symptoms can be observed and progeny RNA is detected. BNYVV has intrinsic properties that lend themselves to use in an assay for RNA silencing suppression activity. First, BNYVV RNA1, which encodes the viral RNA-dependent RNA polymerase, can replicate autonomously (Bouzoubaa & Scheidecker, 1990; Gilmer et al., 1992). Second, functional replicons have been constructed from genomic RNA3 and 5, viral RNAs which are not required for viral multiplication on leaves but are ne...