Abstract
ABSTRACTMany viral suppressors (VSRs) counteract antiviral RNA silencing, a central component of the plant’s immune response by sequestration of virus-derived antiviral small interfering RNAs (siRNAs). Here, we addressed how VSRs affect the activities of cellular microRNAs (miRNAs) during a viral infection by characterizing the interactions of two unrelated VSRs, the Tombusvirus p19 and the Cucumovirus 2b, with miRNA 162 (miR162), miR168, and miR403. These miRNAs regulate the expression of the important silencing factors Dicer-like protein 1 (DCL1) and Argonaute proteins 1 and 2 (AGO1 and AGO2), respectively. Interestingly, while the two VSRs showed similar binding profiles, the miRNAs were bound with significantly different affinities, for example, with the affinity of miR162 greatly exceeding that of miR168. In vitro silencing experiments revealed that p19 and 2b affect miRNA-mediated silencing of the DCL1, AGO1, and AGO2 mRNAs in strict accordance with the VSR’s miRNA-binding profiles. In Tombusvirus-infected plants, the miRNA-binding behavior of p19 closely corresponded to that in vitro. Most importantly, in contrast to controls with a Δp19 virus, infections with wild-type (wt) virus led to changes of the levels of the miRNA-targeted mRNAs, and these changes correlated with the miRNA-binding preferences of p19. This was observed exclusively in the early stage of infection when viral genomes are proposed to be susceptible to silencing and viral siRNA (vsiRNA) concentrations are low. Accordingly, our study suggests that differential binding of miRNAs by VSRs is a widespread viral mechanism to coordinately modulate cellular gene expression and the antiviral immune response during infection initiation.
Highlights
Many viral suppressors (VSRs) counteract antiviral RNA silencing, a central component of the plant’s immune response by sequestration of virus-derived antiviral small interfering RNAs
Several miRNAs (10 examples shown in Fig. 1A), among them an isoform of miRNA 162 (miR162), were found to be enriched in the precipitate compared to input extract
DISCUSSION miRNAs regulate a wide variety of biological processes, including the measures taken by the plant to counteract and clear pathogen infections [54]
Summary
Many viral suppressors (VSRs) counteract antiviral RNA silencing, a central component of the plant’s immune response by sequestration of virus-derived antiviral small interfering RNAs (siRNAs). Experiments in plants showed that in the early infection phase, the Tombusvirus VSR p19 modulates the activity of these miRNAs on their target mRNAs very differently and that this differential regulation strictly correlates with the binding affinities of p19 for the respective miRNAs. Our data suggest that VSRs may control plant gene expression and the early immune response by differential sequestration of miRNAs. RNA silencing regulates gene expression on the transcriptional or posttranscriptional levels and decisively affects multiple biological processes in eukaryotic cells. P19-dependent sequestration of siRNAs prevents RISC assembly [24,25,26] and interferes with the systemic spread of silencing Another example is the Cucumovirus 2b protein, which suppresses RNA silencing through the binding of dsRNAs of various sizes [27, 28]. As with most siRNA-binding VSRs of different virus species, the primary protein structures of p19 and 2b are entirely unrelated [20]
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