UC Riverside

Small interfering RNAs (siRNAs) are processed from virus-specific dsRNA to direct antiviral RNA interference (RNAi) in diverse eukaryotic hosts. We have recently performed a sensitized genetic screen in Arabidopsis (Arabidopsis thaliana) and identified two related phospholipid flippases required for antiviral RNAi and the amplification of virus-derived siRNAs by plant RNA-dependent RNA polymerase1 (RDR1) and RDR6. Here we report the identification and cloning of ANTIVIRAL RNAI-DEFECTIVE2 (AVI2) from the same genetic screen. AVI2 encodes a multispan transmembrane protein broadly conserved in plants and animals with two homologous human proteins known as magnesium transporters. We show that avi2 mutant plants display no developmental defects and develop severe disease symptoms after infection with a mutant Cucumber mosaic virus (CMV) defective in RNAi suppression. AVI2 is induced by CMV infection, particularly in veins, and is required for antiviral RNAi and RDR6-dependent biogenesis of viral siRNAs. AVI2 is also necessary for Dicer-like2-mediated amplification of 22-nucleotide viral siRNAs induced in dcl4 mutant plants by infection, but dispensable for RDR6-dependent biogenesis of endogenous transacting siRNAs. Further genetic studies illustrate that AVI2 plays a partially redundant role with AVI2H, the most closely related member in the AVI2 gene family, in RDR1-dependent biogenesis of viral siRNAs and the endogenous virus-activated siRNAs (vasi-RNAs). Interestingly, we discovered a specific genetic interaction of AVI2 with AVI1 flippase that is critical for plant development. We propose that AVI1 and AVI2 participate in the virus-induced formation of the RDR1/RDR6-specific, membrane-bound RNA synthesis compartment, essential for the biogenesis of highly abundant viral siRNAs and vasi-RNAs.