Abstract
BackgroundRNA silencing has an important role mediating sequence-specific virus resistance in plants. The complex interaction of viruses with RNA silencing involves the loading of viral small interfering RNAs (vsiRNAs) into its host ARGONAUTE (AGO) proteins. As a side effect of their antiviral activity, vsiRNAs loading into AGO proteins can also mediate the silencing of endogenous genes. Here, we analyze at the genome-wide level both aspects of the interference of cucumber mosaic virus (CMV) with the RNA silencing machinery of Arabidopsis thaliana.ResultsWe observe CMV-derived vsiRNAs affect the levels of endogenous sRNA classes. Furthermore, we analyze the incorporation of vsiRNAs into AGO proteins with a described antiviral role and the viral suppressor of RNA silencing (VSR) 2b, by combining protein immunoprecipitation with sRNA high-throughput sequencing. Interestingly, vsiRNAs represent a substantial percentage of AGO-loaded sRNAs and displace other endogenous sRNAs. As a countermeasure, the VSR 2b loaded vsiRNAs and mRNA-derived siRNAs, which affect the expression of the genes they derive from. Additionally, we analyze how vsiRNAs incorporate into the endogenous RNA silencing pathways by exploring their target mRNAs using parallel analysis of RNA end (PARE) sequencing, which allow us to identify vsiRNA-targeted genes genome-wide.ConclusionsThis work exemplifies the complex relationship of RNA viruses with the endogenous RNA silencing machinery and the multiple aspects of virus resistance and virulence that this interaction induces.
Highlights
RNA silencing has an important role mediating sequence-specific virus resistance in plants
This considerable population of viral small interfering RNAs (vsiRNAs) was mainly constituted by vsiRNAs that were 21-nucleotide long (70.2%), followed by 22-nt (18.14%) and 20nt vsiRNAs (7.5%), as it was previously reported for RNA viruses in different studies [29,30,31] (Fig. 1D)
VsiRNAs were produced from the three genomic RNAs that constitute the cucumber mosaic virus (CMV) genome, predominantly from RNA 2 (42.94%) and RNA 3 (41.94%), with a lower proportion coming from RNA 1 (15.11%) (Additional file 2: Fig. S1A-B)
Summary
RNA silencing has an important role mediating sequence-specific virus resistance in plants. One of the genetic tools that helps plants to cope with stress is an adaptive and highly specific mechanism to regulate gene expression, termed RNA interference (RNAi) or RNA silencing [4, 5] This mechanism is triggered by the presence of a double-stranded RNA (dsRNA), which is cleaved by Dicer-like (DCL) proteins to produce small interfering RNAs (siRNAs), typically of 21- to 24-nt long. 2b can inhibit PTGS and TGS by interfering with the function of AGOs by blocking AGO1 cleavage activity and, inhibit miRNA pathways [21], and by directly interacting with AGO4 and interfering with the RNA-directed DNA methylation pathway [20] These proteins exemplify the duality of the interaction of viruses with the RNA silencing machinery, which is used by the host to control viral accumulation, but can be used by the virus to regulate host processes
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