Cucumber mosaic virus suppressor 2b binds to AGO4‐related small RNAs and impairs AGO4 activities

Abstract

SUMMARYCucumber mosaic virus suppressor 2b (CMV2b) is a nuclear viral suppressor that interferes with local and systemic silencing and inhibits AGO1 slicer activity. CMV2b-mediated transgene hypomethylation and its localization in Cajal bodies suggests a role of CMV2b in RNA-directed DNA methylation (RdDM). However, its direct involvement in RdDM, or its binding with small RNAs (sRNAs) in vivo is not yet established. Here, we show that CMV2b binds both microRNAs (miRNAs) and small interfering RNAs (siRNAs) in vivo. sRNA sequencing data from the CMV2b immunocomplex revealed its preferential binding with 24-nt repeatassociated siRNAs. We provide evidence that CMV2b also has direct interaction with the AGO4 protein by recognizing its PAZ and PIWI domains. Subsequent analysis of AGO4 functions revealed that CMV2b reduced AGO4 slicer activity and the methylation of several loci, accompanied by the augmented accumulation of 24-nt siRNAs in Arabidopsis inflorescences. Intriguingly, CMV2b also regulated an AGO4-related epiallele independently of its catalytic potential, which further reinforces the repressive effects of CMV2b on AGO4 activity. Collectively, our results demonstrate that CMV2b can counteract AGO4-related functions. We propose that by adopting novel counter-host defense strategies against AGO1 and AGO4 proteins, CMV creates a favorable cellular niche for its proliferation.

Biological and Molecular Characterization of Cucumber mosaic virus Subgroup II Isolate Causing Severe Mosaic in Cucumber

“…A recent finding suggested that this suppressor protein is indirectly involved in aphid transmission [56]. A study on mechanism of 2b action showed that the suppressor specifically recruits AGO4 small RNAs and directly interacts with PIWI and PAZ domains of AGO4 affecting its slicer activity, causing hypomethylation of its loci [18]. RNA3 encodes for two proteins: movement protein (MP) located at 5 0 end and coat protein which is expressed from another ORF through subgenomic RNA 4.…”

Antiviral Silencing and Suppression of Gene Silencing in Plants

“…DCL3 has only a minor role against RNA viruses (Qu et al 2008;Raja et al 2014). The fact that silencing suppressors of RNA viruses interfere with DCL3 pathway suggests that DCL3 contributes to antiviral silencing (Azevedo et al 2010;Hamera et al 2012;Lacombe et al 2010). During antiviral silencing against DNA viruses DCL3 is essential and works presumably by inducing chromatin modifications (Akbergenov et al 2006;Blevins et al 2006;Raja et al 2014).…”

“…CMV 2b silencing suppressor protein directly interacts with AGO4 and inhibits its slicer activity and methylation and thus creates a favorable niche for CMV proliferation (Hamera et al 2012). AGO5 (besides AGO2) was shown to be required to inhibit PVX systemic infection .…”

“…Arabidopsis dcl3, drb3 and ago4 mutants fail to hypermethylate the viral genome that is required for host recovery (Raja et al 2014). Besides, AGO4 was proposed to be important in transcriptional regulation of host transcriptional response during CMV virus infection (Hamera et al 2012) or to be involved in PVX virus resistance induced by NB-LRR proteins involving AGO4-mediated translational control (Bhattacharjee et al 2009). …”

“…2b interacts both with AGO4-related siRNAs and with AGO4 protein through PAZ and PIWI domains. The interaction with 2b reduces AGO4 access to endogenous target loci and consequently modulates endogenous transcription to create a favorable niche for CMV infection (Duan et al 2012;Gonzalez et al 2010Gonzalez et al , 2012Hamera et al 2012). P38 of TCV may also suppress silencing at multiple levels.…”