RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA-and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation.

In plants, microRNAs (miRNAs) comprise one of three classes of small RNAs regulating gene expression at the post-transcriptional level. Many plant miRNAs are conserved, and play a role in development, abiotic stress responses or pathogen responses. However, some miRNAs have only been found in certain species. Here, we use deep-sequencing, computational and molecular methods to identify, profile, and describe conserved and non-conserved miRNAs in four grapevine (Vitis vinifera) tissues. A total of 24 conserved miRNA families were identified in all four tissues, and 26 known but non-conserved miRNAs were also found. In addition to known miRNAs, we also found 21 new grapevine-specific miRNAs together with their star strands. We have also shown that almost all of them originated from single genes. Furthermore, 21 other plausible miRNA candidates have been described. We have found that many known and new miRNAs showed tissue-specific expression. Finally, 112 target mRNAs of known and 44 target mRNAs of new grapevine-specific miRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs.

Symptoms on virus-infected plants are often very specific to the given virus. The molecular mechanisms involved in viral symptom induction have been extensively studied, but are still poorly understood. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies the typical symptom induced by CMV in specific hosts; Y-sat causes a bright yellow mosaic on its natural host Nicotiana tabacum. The Y-sat-induced yellow mosaic failed to develop in the infected Arabidopsis and tomato plants suggesting a very specific interaction between Y-sat and its host. In this study, we revealed that Y-sat produces specific short interfering RNAs (siRNAs), which interfere with a host gene, thus inducing the specific symptom. We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I (ChlI, the key gene involved in chlorophyll synthesis) had a 22-nt sequence that was complementary to the Y-sat sequence, including four G-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plant downregulate the mRNA of ChlI by targeting the complementary sequence. ChlI mRNA was also downregulated in the transgenic lines that express Y-sat inverted repeats. Strikingly, modifying the Y-sat sequence in order to restore the 22-nt complementarity to Arabidopsis and tomato ChlI mRNA resulted in yellowing symptoms in Y-sat-infected Arabidopsis and tomato, respectively. In 5′-RACE experiments, the ChlI transcript was cleaved at the expected middle position of the 22-nt complementary sequence. In GFP sensor experiments using agroinfiltration, we further demonstrated that Y-sat specifically targeted the sensor mRNA containing the 22-nt complementary sequence of ChlI. Our findings provide direct evidence that the identified siRNAs derived from viral satellite RNA directly modulate the viral disease symptom by RNA silencing-based regulation of a host gene.

RNA silencing is conserved in a broad range of eukaryotes and operates in the development and maintenance of genome integrity in many organisms. Plants have adapted this system for antiviral defense, and plant viruses have in turn developed mechanisms to suppress RNA silencing. RNA silencing-related RNA inactivation is likely based on target RNA cleavage or translational arrest. Although it is widely assumed that virus-induced gene silencing (VIGS) promotes the endonucleolytic cleavage of the viral RNA genome, this popular assumption has never been tested experimentally. Here we analyzed the viral RNA targeting by VIGS in tombusvirusinfected plants, and we show evidence that antiviral response of VIGS is based on viral RNA cleavage by RNA-induced silencing effector complex (RISC) programmed by virus-specific small interfering RNAs (siRNAs). In addition, we found that the RISC-mediated cleavages do not occur randomly on the viral genome. Indeed, sequence analysis of cloned cleavage products identified hot spots for target RNA cleavage, and the regions of specific RISC-mediated cleavages are asymmetrically distributed along the positive-and negativesense viral RNA strands. In addition, we identified viral siRNAs containing high-molecular-mass protein complexes purified from the recovery leaves of the silencing suppressor mutant virus-infected plants. Strikingly, these large nucleoproteins cofractionated with microRNA-containing complexes, suggesting that these nucleoproteins are silencing related effector complexes.RNA silencing is an ancient self-defense mechanism induced by double-stranded RNA (dsRNA) leading to a homology-dependent degradation of a target RNA. RNA silencing is conserved across kingdoms and is known as quelling in fungi, RNA interference in animals, and posttranscriptional gene silencing in plants.The unifying feature of RNA silencing relies on a set of highly conserved reactions that are triggered by dsRNA, which is processed into 21-to 24-nucleotide (nt) small interfering RNAs (siRNAs) (13,14,33) by the RNase III enzyme Dicer and its homologues (4, 30). These siRNAs program the multisubunit RNA-induced silencing complex (RISC) (15) and guide the complex to degrade cellular RNA molecules having perfect or near perfect base pairing between mRNA and the guide siRNA, while they mediate translation repression in case of partial complementarity (2,8,11,18).Replicating plant viruses are strong inducers as well as targets of virus-induced RNA silencing (VIGS). Indeed, the accumulation of virus-derived siRNAs were reported in local and systemic tissues of virus-infected plants (14, 41) demonstrating the activation of VIGS, which resulted in lowering the accumulation of the invading virus and the recovery phenotype in upper noninoculated leaves (41). Thus, VIGS emerged as an RNA-mediated defense response to protect plants against viral infection (27,46,47,49). The finding that many plant viruses have evolved proteins that suppress at different stages of the silencing pathway (20,23,39,40,48) provides th...