The Weak Small RNA-Binding Activity of the 2b Proteins of Subgroup II Cucumber Mosaic Virus Strains Is Insufficient for RNA Silencing Suppression.

Yingying Gao,Xiaobei Zhang,Zhiyou Du,Zhouhang Gu,Aizhong Chen,Jinrui Yang

doi:10.3389/fmicb.2021.760937

Abstract

The 2b proteins encoded by cucumber mosaic virus (CMV) subgroup I strains suppress RNA silencing primarily by competitively binding small RNAs (sRNAs) in the host cell cytoplasm. Interestingly, 2b proteins encoded by CMV subgroup II strains accumulate predominantly in nuclei. Here we determined that whereas the 2b protein (Fny2b) of subgroup IA strain Fny-CMV is highly effective in suppressing both sense RNA-induced and inverted repeat-induced posttranscriptional gene silencing, the 2b protein (LS2b) of the subgroup II strain LS-CMV was not as effective. Reducing nuclear accumulation of LS2b by mutating a residue in its nuclear localization sequence had no effect on RNA silencing suppressor activity, while attenuated viral symptoms. Electrophoretic mobility shift assays showed that the sRNA binding of LS2b was weaker and more selective than that of Fny2b. The domain determining the differential sRNA-binding ability was delimited to the putative helix α1 region. Moreover, LS2b mutants that completely lost suppressor activity still retained their weak sRNA-binding ability, suggesting that sRNA binding is not sufficient for LS2b to suppress RNA silencing. Considering the subgroup I strain-encoded 2b proteins that require sRNA-binding ability for the suppression of RNA silencing, we suggest that in addition to binding sRNA, the 2b proteins of subgroup II CMV strains would require extra biological activities to achieve RNA silencing inhibition.

Highlights

RNA silencing is a conserved mechanism in eukaryotes in which RNA accumulation is regulated by sequence-specific interactions with small RNAs
To better understand viral suppressors of RNA silencing (VSRs) activity of 2b proteins encoded by cucumber mosaic virus (CMV) subgroup II strains, we compared LS2b with Fny2b in suppression of local green flurorescent protein (GFP) silencing in N. benthamiana via agroinfiltration, using GUS expression as a negative control
We used two plasmid constructs to induce GFP silencing, 35S-FP and 35S-dsFP (Xiong et al, 2009; Li et al, 2014). 35S-FP expresses an aberrant sense RNA (FP) corresponding to the 3'-terminal 400 nt of GFP that triggers sense RNA-mediated posttranscriptional gene silencing (PTGS) (S-PTGS), which is dependent on host RDRs to synthesize double-stranded FP molecules as substrates for generation of GFP short-interfering RNAs (siRNAs) by DCLs. 35S-dsFP expresses an inverted repeat (IR) RNA of the FP fragment, which can be diced directly by DCLs to produce primary siRNAs conferring IR-PTGS

Summary

Introduction

RNA silencing is a conserved mechanism in eukaryotes in which RNA accumulation is regulated by sequence-specific interactions with small RNAs (sRNAs; Palukaitis, 2011). Most plant viruses express one or more viral suppressors of RNA silencing (VSRs) to counteract siRNAmediated antiviral defense (Roth et al, 2004; Li and Ding, 2006; Csorba et al, 2015; Li and Wang, 2019). Certain VSRs inhibit vsiRNA biogenesis by interfering with the activities of double-RNA-binding protein 4, DCLs, the host RNA-dependent RNA polymerase 6 (RDR6), or the suppressor of gene silencing 3 (SGS3). Other VSRs compromise slicing activity or stability of AGO proteins, while many VSRs sequestering vsiRNAs inhibits their incorporation into RISCs (Roth et al, 2004; Li and Ding, 2006; Csorba et al, 2015; Li and Wang, 2019). VSRs can influence viral pathogenicity in manners that are dependent or independent of their VSR activities (Diaz-Pendon and Ding, 2008; Csorba et al, 2015)

Methods

Results

Conclusion