![]() ![]() Values at the bottom of this panel refer to the averages of the relative hybridization signal intensity of RNA1, 2, and 3 in three independent experiments with the accumulation levels of CMV-2aTΔ2b in wild-type plants (left panel) or sgs3 plants (right panel) set as 1. 25S rRNA was used as the loading control. ![]() (C) Accumulation of CMV-Δ2b and CMV-2aTΔ2b genomic RNAs (RNAs 1-3) and the subgenomic RNA (RNA4) in wild-type and mutant plants 3 weeks after inoculation detected by a probe specific for the 3′-untranslated region conserved in all of the four CMV RNAs. Infected plants were photographed 3 weeks after inoculation. (B) Pathogenetic responses of CMV-2aTΔ2b and CMV-Δ2b in wild-type (wt) and mutant plants. By contrast, CMV-Δ2b contained point mutations that inactivated the expression of 2b but did not alter coding in the 2a reading frame. The 295-nucleotide deletion introduced into RNA2 of CMV-2aTΔ2b abolished expression of the 2b protein and also resulted in the truncation of the 2a protein. Wild-type RNA2 encodes the viral RdRp 2a protein and the 2b protein in overlapping reading frames. (A) Genome organization of RNA2 from wild-type and mutant CMV isolates. RNA Silencing of Distinct CMV Mutants by Single and Double RDR Pathways. Possible mechanisms for the observed qualitative difference in RNA silencing between 21- and 22-nucleotide secondary siRNAs are discussed. Notably, although DCL2 is able to produce abundant viral secondary siRNAs in the absence of DCL4, the resultant 22-nucleotide viral siRNAs alone do not guide efficient silencing of CMV-Δ2b. Our findings also illustrate that dicing of the viral RNA precursors of primary and secondary siRNA is insufficient to confer virus resistance. Examination of 25 single, double, and triple mutants impaired in nine ARGONAUTE (AGO) genes combined with coimmunoprecipitation and deep sequencing identifies an essential function for AGO1 and AGO2 in defense against CMV-Δ2b, which act downstream the biogenesis of viral secondary siRNAs in a nonredundant and cooperative manner. We show that production of the viral secondary siRNAs targeting CMV-Δ2b requires SUPPRESSOR OF GENE SILENCING3 and DICER-LIKE4 (DCL4) in addition to RDR6. Here, we describe a mutant of Cucumber mosaic virus (CMV-Δ2b) that is silenced predominantly by the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent viral secondary siRNA pathway. However, little is known about the biogenesis pathway and effector mechanism of viral secondary siRNAs. Arabidopsis thaliana defense against distinct positive-strand RNA viruses requires production of virus-derived secondary small interfering RNAs (siRNAs) by multiple RNA-dependent RNA polymerases. ![]()
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