Abstract
Using a transient plant system, it was previously found that the suppression of Cucumber mosaic virus (CMV) 2b protein relies on its double-strand (ds) RNA binding capacity, but it is independent of its interaction with ARGONAUTE (AGO) proteins. Thus, the biological meaning of the 2b-AGO interaction in the context of virus infection remains elusive. In this study, we created infectious clones of CMV mutants that expressed the 2b functional domains of dsRNA or AGO binding and tested the effect of these CMV mutants on viral pathogenicity. We found that the mutant CMV2b(1–76) expressing the 2b dsRNA-binding domain exhibited the same virulence as wild-type CMV in infection with either wild-type Arabidopsis or rdr1/6 plants with RDR1- and RDR6-deficient mutations. However, remarkably reduced viral RNA levels and increased virus (v)siRNAs were detected in CMV2b(1–76)-infected Arabidopsis in comparison to CMV infection, which demonstrated that the 2b(1–76) deleted AGO-binding domain failed to suppress the RDR1/RDR6-dependent degradation of viral RNAs. The mutant CMV2b(8–111) expressing mutant 2b, in which the N-terminal 7 amino acid (aa) was deleted, exhibited slightly reduced virulence, but not viral RNA levels, in both wild-type and rdr1/6 plants, which indicated that 2b retained the AGO-binding activity acquired the counter-RDRs degradation of viral RNAs. The deletion of the N-terminal 7 aa of 2b affected virulence due to the reduced affinity for long dsRNA. The mutant CMV2b(18–111) expressing mutant 2b lacked the N-terminal 17 aa but retained its AGO-binding activity greatly reduced virulence and viral RNA level. Together with the instability of both 2b(18–111)-EGFP and RFP-AGO4 proteins when co-expressed in Nicotiana benthamiana leaves, our data demonstrates that the effect of 2b-AGO interaction on counter-RDRs antiviral defense required the presence of 2b dsRNA-binding activity. Taken together, our findings demonstrate that the dsRNA-binding activity of the 2b was essential for virulence, whereas the 2b-AGO interaction was necessary for interference with RDR1/6-dependent antiviral silencing in Arabidopsis.
Highlights
RNA silencing (RNA interference, RNAi) is an evolutionarily conserved regulatory mechanism of gene expression in eukaryotes mediated by 20–25-nucleotides small interference RNAs
To obtain viral sources of chimeric Cucumber mosaic virus (CMV) with different 2b mutant, each of these constructs was transformed into Agrobacterium for the infiltration of N. benthamiana in the presence of 35S-RNA1 and 35S-RNA3 to examine the infectious properties of the chimeric CMV with 35S-2bx (x represents different 2b mutations shown in Figure 1A), and the related viruses were referred to as wild-type CMV, CMV 2b, CMV2b(1–76), CMV2b(8–111), and CMV2b(18–111) (Figure 1B and Supplementary Figure S1)
No major differences in the accumulation of miR173 were detected following infection with either wild-type or each mutant CMV, which supported an earlier observation that CMV infection does not alter miRNA accumulation (Diaz-Pendon et al, 2007). These results demonstrate that N terminal double-stranded RNA (dsRNA) binding activity is responsible for the induction of the virulence of CMV, which does not necessarily correlate with the accumulation of viral RNAs, and 2b-AGO binding is likely required for CMV to suppress the silencing of viral RNAs in Arabidopsis plants
Summary
RNA silencing (RNA interference, RNAi) is an evolutionarily conserved regulatory mechanism of gene expression in eukaryotes mediated by 20–25-nucleotides (nt) small interference RNAs (siRNAs; Meister and Tuschl, 2004; Baulcombe, 2005). These siRNAs are processed from doublestranded (ds) or hairpin (hp) RNA by Dicer or Dicer-like (DCL) protein. SiRNAs-guided AGO-cleaved target RNA may be recognized by RNA-dependent RNA polymerase (RDR), which amplifies the dsRNA substrate for DCLs to produce secondary siRNAs and reinforce the RNA silencing process (Peragine et al, 2004; Axtell et al, 2006; Baulcombe, 2007). Viral infection triggers the siRNA-mediated RNA silencing as a natural antiviral defense mechanism. Two of the six Arabidopsis thaliana RDRs, RDR1, and RDR6, have been implicated in defense against many viruses, including Cucumber mosaic virus (CMV; Dalmay et al, 2000; Qu et al, 2005; Schwach et al, 2005; Vaistij and Jones, 2009; Garcia-Ruiz et al, 2010; Qu, 2010; Ying et al, 2010; Li et al, 2014)
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