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P0 protein of some polerovirus members can target ARGONAUTE1 (AGO1) to suppress RNA silencing. Although P0 harbors an F-box-like motif reported to be essential for interaction with S phase kinase-associated protein 1 (SKP1) and RNA silencing suppression, it is the autophagy pathway that was shown to contribute to AGO1 degradation. Therefore, the role of P0-SKP1 interaction in silencing suppression remains unclear. We conducted global mutagenesis and comparative functional analysis of P0 encoded by Brassica yellows virus (BrYV) (P0Br ). We found that several residues within P0Br are required for local and systemic silencing suppression activities. Remarkably, the F-box-like motif mutant of P0Br , which failed to interact with SKP1, is destabilized in vivo. Both the 26S proteasome system and autophagy pathway play a role in destabilization of the mutant protein. Furthermore, silencing of a Nicotiana benthamiana SKP1 ortholog leads to the destabilization of P0Br . Genetic analyses indicated that the P0Br -SKP1 interaction is not directly required for silencing suppression activity of P0Br , but it facilitates stability of P0Br to ensure efficient RNA silencing suppression. Consistent with these findings, efficient systemic infection of BrYV requires P0Br . Our results reveal a novel strategy used by BrYV for facilitating viral suppressors of RNA silencing stability against degradation by plant cells.

Summary: The Barley stripe mosaic virus (BSMV) γb protein is a viral suppressor of RNA silencing (VSR) and symptom determinant. However, it is unclear how post‐translational modification affects the different functions of γb. Here, we demonstrate that γb is phosphorylated at Ser‐96 by a PKA‐like kinase in vivo and in vitro. Mutant viruses containing a nonphosphorylatable substitution (BSMVS96A or BSMVS96R) exhibited reduced viral accumulation in Nicotiana benthamiana due to transient induction of the cell death response that constrained the virus to necrotic areas. By contrast, a BSMVS96D mutant virus that mimics γb phosphorylation spread similarly to the wild‐type virus. Furthermore, the S96A mutant had reduced local and systemic γb VSR activity due to having compromised its binding activity to 21‐bp dsRNA. However, overexpression of other VSRs in trans or in cis failed to rescue the necrosis induced by BSMVS96A, demonstrating that suppression of cell death by γb phosphorylation is functionally distinct from its RNA silencing suppressor activities. These results provide new insights into the function of γb phosphorylation in regulating RNA silencing and the BSMV‐induced host cell death response, and contribute to our understanding of how the virus optimizes the balance between viral replication and virus survival in the host plants during virus infection. [ABSTRACT FROM AUTHOR]