Abstract
Cucumber mosaic virus (CMV) is a model virus for plant–virus protein interaction and mechanism research because of its wide distribution, high-level of replication and simple genome structure. The 2b protein is a multifunctional protein encoded by CMV that suppresses RNA silencing-based antiviral defense and contributes to CMV virulence in host plants. In this report, 12 host proteins were identified as CMV LS2b binding partners using the yeast two-hybrid screen system from the Arabidopsis thaliana cDNA library. Among the host proteins, 30S ribosomal subunit protein S11 (RPS11) was selected for further studies. The interaction between LS2b and full-length RPS11 was confirmed using the yeast two-hybrid system. Bimolecular fluorescence complementation (BIFC) assays observed by confocal laser microscopy and Glutathione S-transferase (GST) pull-down assays were used to verify the interaction between endogenous NbRPS11 and viral CMVLS2b both in vivo and in vitro. TRV-based gene silencing vector was used to knockdown NbRPS11 transcription, and immunoblot analysis revealed a decline in infectious viral RNA replication and a decrease in CMV infection in RPS11 down-regulated Nicotiana benthamiana plants. Thus, the knockdown of RPS11 likely inhibited CMV replication and accumulation. The gene silencing suppressor activity of CMV2b protein was reduced by the RPS11 knockdown. This study demonstrated that the function of viral LS2b protein was remarkably affected by the interaction with host RPS11 protein.
Highlights
Cucumber mosaic virus (CMV) has long been studied as an important plant virus model for understanding plant–virus interactions because of its broad host range and typical viral genome
Among all the host proteins identified as CMV LS2b binding partners by the yeast twohybrid screen system, ribosomal subunit protein S11 (RPS11) was selected for further studies because 30S ribosomal subunit scored the largest proportion of all positive clones
The interaction between CMV LS2b bait protein and library prey fusion proteins brought the DNA-binding domain (BD) and activating domain (AD) into proximity to activate the transcription of four independent reporter genes
Summary
Cucumber mosaic virus (CMV) has long been studied as an important plant virus model for understanding plant–virus interactions because of its broad host range and typical viral genome. The CMV genome consists of three single-stranded positive-sense RNAs, namely, RNA1, RNA2 and RNA3 (sorted by decreasing size), which encode five open-reading frames (ORFs): 1a protein by RNA1, 2a and 2b proteins by RNA2, and movement protein (MP) and capsid protein (CP) by RNA3. The subgenomic RNA4 and RNA4A encode ORFs of CP and 2b protein respectively. The 110 amino acid multifunctional 2b protein from subgroup I is among the first viral proteins identified as RNA silencing suppressors (VSR) [1]. The 2b protein was initially identified as a virulence factor [2] in a strain of CMV that lacked the 2b gene, which resulted in an asymptomatic infection. CMV 2b protein binds small RNA duplexes and longer dsRNAs [5]
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