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Viral suppressors of RNA silencing (VSRs) are encoded by diverse viruses to counteract the RNA silencing‐mediated defence mounted by the virus‐infected host cells. In this study, we identified the NSs protein encoded by tomato zonate spot virus (TZSV) as a potent VSR, and used a potato virus X (PVX)‐based heterologous expression system to demonstrate TZSV NSs as a viral pathogenicity factor that intensified PVX symptoms in  Nicotiana benthamiana . We then used a yeast two‐hybrid screen to identify the suppressor of gene silencing 3 protein of  N. benthamiana  (NbSGS3), a known component of the plant RNA silencing pathway, as an interaction partner of TZSV NSs. We verified this interaction in plant cells with bimolecular fluorescence complementation, subcellular colocalization, and co‐immunoprecipitation. We further revealed that the NSs–NbSGS3 interaction correlated with the VSR activity of TZSV NSs. TZSV NSs reduced the concentration of NbSGS3 protein in plant cells, probably through the ubiquitination and autophagy pathways. Interestingly, TZSV infection, but not NSs overexpression, significantly up‐regulated the  NbSGS3  transcript levels. Our data indicate that TZSV NSs suppresses RNA silencing of the host plant and enhances TZSV pathogenicity through its interaction with NbSGS3. This study reveals a novel molecular mechanism of NSs‐mediated suppression of plant host antiviral defence.

The nonstructural protein NSs encoded by tomato zonate spot virus suppresses RNA silencing by interacting with NbSGS3