We investigated the genetic pathway in Arabidopsis thaliana targeted during infection by cucumber mosaic virus (CMV) 2b protein, known to suppress non-cell-autonomous transgene silencing and salicylic acid (SA)-mediated virus resistance. We show that 2b expressed from the CMV genome drastically reduced the accumulation of 21-, 22-, and 24-nucleotide classes of viral small interfering RNAs (siRNAs) produced by Dicer-like4 (DCL4), DCL2, and DCL3, respectively. The defect of a CMV 2b-deletion mutant (CMV-D2b) in plant infection was efficiently rescued in Arabidopsis mutants producing neither 21-nor 22-nucleotide viral siRNAs. Since genetic analysis further identifies a unique antiviral role for DCL3 upstream of DCL4, our data indicate that inhibition of the accumulation of distinct viral siRNAs plays a key role in 2b suppression of antiviral silencing. Strikingly, disease symptoms caused by CMV-D2b in Arabidopsis mutants defective in antiviral silencing were as severe as those caused by CMV, demonstrating an indirect role for the silencing suppressor activity in virus virulence. We found that production of CMV siRNAs without 2b interference depended largely on RNA-dependent RNA polymerase 1 (RDR1) inducible by SA. Given the known role of RDR6-dependent transgene siRNAs in non-cell-autonomous silencing, our results suggest a model in which 2b inhibits the production of RDR1-dependent viral siRNAs that confer SA-dependent virus resistance by directing non-cell-autonomous antiviral silencing.

Plant and animal viruses overcome host antiviral silencing by encoding diverse viral suppressors of RNA silencing (VSRs). Prior to the identification and characterization of their silencing suppression activities mostly in transgene silencing assays, plant VSRs were known to enhance virus accumulation in the inoculated protoplasts, promote cell-to-cell virus movement in the inoculated leaves, facilitate the phloem-dependent long-distance virus spread, and/or intensify disease symptoms in systemically infected tissues. Here we discuss how the various silencing suppression activities of VSRs may facilitate these distinct steps during plant infection and why VSRs may not play a direct role in eliciting disease symptoms by general impairments of host endogenous small RNA pathways. We also highlight many of the key questions still to be addressed on the role of viral suppression of antiviral silencing in plant infection.