Satellite RNA-Derived Small Interfering RNA satsiR-12 Targeting the 3′ Untranslated Region of Cucumber Mosaic Virus Triggers Viral RNAs for Degradation

Abstract

RNA silencing provides protection against RNA viruses by targeting both the helper virus and its satellite RNA (satRNA). Virus-derived small interfering RNAs (vsiRNAs) bound with Argonaute (AGO) proteins are presumed participants in the silencing process. Here, we show that a vsiRNA targeted to virus RNAs triggers the host RNA-dependent RNA polymerase 6 (RDR6)-mediated degradation of viral RNAs. We confirmed that satRNA-derived small interfering RNAs (satsiRNAs) could be associated with different AGO proteins in planta. Diverse RNA silencing processes exist in plants, including silencing by microRNAs (miRNAs) and several classes of endogenous small interfering RNAs (siRNAs) involved in pathways with multiple Dicer-like proteins (DCLs) and Argonaute proteins (AGOs) (3, 4). There are four specialized DCLs (DCL1 to DCL4) that produce different kinds of small RNAs (sRNAs; 21 to 25 nucleotides [nt] in length) from doublestranded RNAs or stem-loop precursors in Arabidopsis thaliana. The resulting sRNAs bind to an AGO protein to form effector complexes, termed RNA-induced silencing complexes (RISCs), to target the cleavage or translational suppression of cRNA based on sequence complementarity (1). There are three clades of AGOs, including 10 subfamily members in Arabidopsis (50). Recent studies reveal distinct binding affinities of sRNAs with different 5Ј-terminal nucleotides targeted to AGO proteins (31). AGO1 displays a strong bias for sRNAs with a 5Ј-terminal uridine, while AGO2 and AGO4 are associated mainly with sRNA sequences that are initiated with a 5Ј adenosine, and AGO5 is highly enriched for sRNA sequences that are initiated with a 5Ј cytosine (31).RNA silencing also provides protection against diverse RNA viruses in many eukaryotic organisms (28,38,52). Two classes of viral siRNAs are generated in virus infections: the primary siRNAs, derived from the DCL-mediated cleavage of a viral RNA precursor, and the secondary siRNAs, whose biogenesis requires host RNA-dependent RNA polymerases (RDRs) (12,16,20,52) in a process likely resembling the production of secondary siRNAs in plants, which is triggered by an AGO1-containing 22-nt miRNA or siRNA (7,9,42). To counter or escape from host antiviral silencing, many viruses have evolved viral suppressors of RNA silencing (VSR) to interfere with host RNA silencing at different stages (11,14,27). For example, the 2b protein, a VSR encoded by a Cucumber mosaic virus (CMV) (5, 21, 29), perturbs RNA silencing by directly interacting with AGO1, which results in a block of the loading of sRNAs into the AGO1 RISC and, thereby, interferes with the Slicer activity of AGO1 (55).CMV is one of the best-characterized tripartite positivesense single-stranded RNA viruses, with genes encoding five proteins distributed on three genomic and two subgenomic RNAs. The 1a and 2a proteins encoded by genomic RNA1 and RNA2, respectively, are RDRs involved in viral replication (24,35,36). Genomic RNA3 encodes the 3a protein, or movement

The role of virus-derived small interfering RNAs in RNA silencing in plants

“…Moreover, using CMV-Δ2b-infected transgenic plants expressing asatsiR-12, we found that asatsiR-12 was able to reduce the accumulation of CMV RNAs. Also, we verified that asatsiR-12-mediated reduction of viral RNAs in the presence of RDR6 was inhibited by the 2b suppressor [48]. A high concentration of replicating satRNAs could reduce the accumulation of viral RNAs in N. benthamiana on later infection by CMV-2b [49].…”

“…By introducing mutations into the region generating satsiR-12, we confirmed that satsiR-12 was indeed involved in the satRNA-mediated reduction in CMV RNAs. Our findings provided the first demonstration that viral satellite RNA-derived siRNAs could mediate silencing against their helper virus [48].…”

“…Among these satsiRNAs, satsiR-12 mediated the host RDR6-dependent antiviral silencing by targeting the 3′ UTR of CMV RNA. Moreover, the satsiR-12-mediated RDR6-dependent antiviral defense response was inhibited by the suppressor 2b [48]. On the other hand, the abundant replication of satRNA affected the accumulation of 2b coding RNA [49].…”

“…Our results indicate that these satsiRNAs can combine with AGO proteins in host plants. The most frequently cloned satsiRNA, named satsiR-12 that targets the 3′ UTR of CMV RNA, triggers the RDR6-dependent antiviral silencing pathway [48]. Firstly, using a plant transient expression system, we found the expression of artificial satsiR-12 (asatsiR-12) could mediate the cleavage of a GFP-sensor harboring the satsiR-12 target site.…”

Plant subviral RNAs as a long noncoding RNA (lncRNA): Analogy with animal lncRNAs in host–virus interactions

“…There are many examples of animal lncRNAs that control chromatin modifications. mechanism may still be true, Zhu et al (2011) demonstrated another possibility, that siRNAs derived from CMV satRNAs could target the 3 untranslated region in CMV genomic RNAs for cleavage of CMV RNAs, which then triggers RDR6-dependent secondary siRNA amplification, resulting in a decrease of CMV accumulation. Their results suggest that satRNA can attack the helper virus through the plant's major defense system, RNA silencing, and thus strengthen the defense response against viruses in plant cells.…”