Identification of a New Host Factor Required for Antiviral RNAi and Amplification of Viral siRNAs

Abstract

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 () 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 - () from the same genetic screen. encodes a multispan transmembrane protein broadly conserved in plants and animals with two homologous human proteins known as magnesium transporters. We show that mutant plants display no developmental defects and develop severe disease symptoms after infection with a mutant (CMV) defective in RNAi suppression. 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 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 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.

CMV2b-Dependent Regulation of Host Defense Pathways in the Context of Viral Infection

“…Transcriptome and RT-qPCR analyses revealed that CMV infection induced the expression of a number of R genes ( Figure 5 ), as well as SA biosynthesis and signaling ( Figure 7 ), which required the presence of the 2b protein, consistent with recent reports that CMV infection induced host factors with antiviral effects and these effects relied on the function of 2b [ 47 , 48 ]. Although only a few of these genes were found to be related to the direct effect of 2b-mediated demethylation on their induction ( Figure 3 C), a large amount of 2b-dependent Hypo-DMR loci detected in CMV infection would also imply a defensive effect ( Figure 4 ).…”

A putative nuclear copper chaperone promotes plant immunity in Arabidopsis

“…GUS staining assays were performed as described previously ( Guo et al , 2018 ). Briefly, plant tissues were treated with X-Gluc solution (3 mM 5-bromo-4-chloro-3-indolyl-β- d -glucuronide, 10 mM potassium ferricyanide, 0.1% Triton X-100, 10 mM potassium ferrocyanide, 50 mM NaH 2 PO 4 buffer, pH 7.0) at 37 °C overnight, washed in an ethanol series, and photographed using a digital camera.…”

Sterol isomerase HYDRA1 interacts with RNA silencing suppressor P1b and restricts potyviral infection

“…Moreover, AGO1 association to a membrane compartment, through a yet unknown mechanism that depends on the mevalonate (MVA)-isoprenoid pathway, is required to sustain a functional RNA silencing activity (Brodersen et al, 2012). Host factors presumably participating in the formation of membrane-bound compartments and specifically involved in antiviral RNA silencing have been recently identified (Guo et al, 2017;Guo et al, 2018;Zhu et al, 2017). Viral RNA silencing suppressor (RSS) proteins aim to arrest the silencing mechanism to protect the viral genome from degradation (Csorba, Kontra, & Burgyán, 2015;Nakahara & Masuta, 2014).…”

“…Moreover, AGO1 association to a membrane compartment, through a yet unknown mechanism that depends on the mevalonate (MVA)‐isoprenoid pathway, is required to sustain a functional RNA silencing activity (Brodersen et al, ). Host factors presumably participating in the formation of membrane‐bound compartments and specifically involved in antiviral RNA silencing have been recently identified (Guo et al, ; Guo et al, ; Zhu et al, ).…”