Fig. 7: OsVIRK1 phosphorylates RSV NS3 to dampen its VSR function.
a OsVIRK1C phosphorylates NS3. Purified NS3-GST or CP-GST was incubated with purified MBP-OsVIRK1C and subjected to an in vitro kinase activity assay. b Phos-tag SDS-PAGE coupled immunoblotting assay showing that OsVIRK1-phosphorylated NS3 in RSV-infected WT rice, and this phosphorylation was diminished by the addition of λPP. c Phos-tag SDS-PAGE coupled immunoblotting assay showing that OsVIRK1 is required for NS3 phosphorylation in RSV-infected rice. The numbers represent the normalized intensities of the phosphorylated NS3 signals. d The VSR function of NS3 was drastically reduced when co-expressed with OsVIRK1-MYC. The VSR activity of NS3 was assessed by transient co-expression of 35S::GFP and FLAG-HA-NS3 with or without OsVIRK1-MYC in 16c leaves. GFP fluorescence was imaged 5 days post agroinfiltration. EV, empty vector. e OsVIRK1 reduces NS3 accumulation. Western blot analysis of the accumulation of GFP, FLAG-HA-NS3, and OsVIRK1-MYC in the materials described in d. f The phosphomimic NS35D lost VSR function as assessed by transient co-expression of 35S::GFP with FLAG-HA-NS3 or FLAG-HA-NS35D in 16c leaves. GFP fluorescence was imaged 5 days post agroinfiltration. g Compared to NS3, the phosphomimic NS35D showed reduced protein accumulation. Western blot analysis of the accumulation of GFP, FLAG-HA-NS3, and FLAG-HA-NS35D in the materials described in f. h, i Subcellular localization of NS3-GFP and NS35D-GFP proteins in N. benthamiana leaves; NS3-GFP and NS35D-GFP were expressed in different zones of one leaf. All experiments were repeated two or three times with similar results. j A model illustrating that OsVIRK1 recognizes CP and NS3 and inhibits RSV infection. After infection, RSV produces the viral proteins CP and NS3 and viral RNAs in plant cells. Plant antiviral RNA silencing mediates cleavage of the viral RNAs and inhibits infection, and this is counteracted by the viral suppressor NS3. OsVIRK1 expression is induced by RSV infection. OsVIRK1 protein accumulates through autophosphorylation and redox regulation and localizes to the TGN/EE, where it recognizes CP and NS3. Recognition of CP by OsVIRK1 activates the expression of antiviral defense genes. OsVIRK1 directly phosphorylates NS3, promoting and reducing NS3 accumulation in the nucleus and cytoplasm, respectively, thereby inhibiting its VSR activity and releasing antiviral RNA silencing.
