<i>Arabidopsis</i> DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1

TLDR

Plant RNA silencing involves DCLs, AGOs, DRBs, and RDRs, and while several endogenous pathways are well characterized, the antiviral pathway remains incompletely understood. The study investigates how specific DCLs, AGOs, DRB4, and RDR6 affect viral RNA accumulation in Arabidopsis using a suppressor‑deficient virus. They assessed viral RNA levels in Arabidopsis mutants lacking DCLs, AGOs, DRB4, or RDR6, infected with a virus that cannot suppress silencing. All four DCLs, DRB4, AGO1, AGO7, and RDR6 participate in antiviral silencing, with DCL1 acting as a negative regulator of DCL4 and DCL3, AGO1 preferentially targeting compact viral RNAs while AGO7 and RDR6 favor less structured RNAs, thereby clarifying key steps in the pathway.

Abstract

Plant RNA silencing machinery enlists four primary classes of proteins to achieve sequence-specific regulation of gene expression and mount an antiviral defense. These include Dicer-like ribonucleases (DCLs), Argonaute proteins (AGOs), dsRNA-binding proteins (DRBs), and RNA-dependent RNA polymerases (RDRs). Although at least four distinct endogenous RNA silencing pathways have been thoroughly characterized, a detailed understanding of the antiviral RNA silencing pathway is just emerging. In this report, we have examined the role of four DCLs, two AGOs, one DRB, and one RDR in controlling viral RNA accumulation in infected Arabidopsis plants by using a mutant virus lacking its silencing suppressor. Our results show that all four DCLs contribute to antiviral RNA silencing. We confirm previous reports implicating both DCL4 and DCL2 in this process and establish a minor role for DCL3. Surprisingly, we found that DCL1 represses antiviral RNA silencing through negatively regulating the expression of DCL4 and DCL3. We also implicate DRB4 in antiviral RNA silencing. Finally, we show that both AGO1 and AGO7 function to ensure efficient clearance of viral RNAs and establish that AGO1 is capable of targeting viral RNAs with more compact structures, whereas AGO7 and RDR6 favor less structured RNA targets. Our results resolve several key steps in the antiviral RNA silencing pathway and provide a basis for further in-depth analysis.