Barley stripe mosaic virus infection requires PKA-mediated phosphorylation of γb for suppression of both RNA silencing and the host cell death response

The Barley stripe mosaic virus (BSMV) cb 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 gamma b. Here, we demonstrate that gamma b 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 gamma b phosphorylation spread similarly to the wild-type virus. Furthermore, the S96A mutant had reduced local and systemic cb 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 gamma b phosphorylation is functionally distinct from its RNA silencing suppressor activities. These results provide new insights into the function of gamma b 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.