Fig. 3: Autophosphorylation and redox regulation promote the accumulation of OsVIRK1.
a Autophosphorylation analysis of OsVIRK1 in vitro. The MBP-fused C-terminal region of OsVIRK1 (MBP-OsVIRK1C) and 391 Lys-mutated OsVIRK1C (MBP-OsVIRK1CK391E) were purified and subjected to an in vitro kinase activity assay. b Autophosphorylation analysis of OsVIRK1 in vivo. OsVIRK1-GFP, OsVIRK1K391E-GFP, and the control vector were transiently expressed in rice protoplasts. Total proteins were extracted for anti-GFP immunoprecipitation and subsequent anti-pThr and anti-pSer immunoblotting. c Anti-HA western blot assay showing the accumulation of OsVIRK1-FLAG-HA, OsVIRK1K391E-FLAG-HA, and OsVIRK1C4A-FLAG-HA proteins in the corresponding transgenic rice lines. d Subcellular localization of OsVIRK1-GFP, OsVIRK1K391E-GFP, and OsVIRK1C4A-GFP proteins in rice protoplasts. Arrows indicate representative fluorescence signals of puncta. Scale bars, 10 μm. e Analysis of the redox regulation of OsVIRK1 in N. benthamiana. OsVIRK1-GFP, OsVIRK1K391E-GFP, or OsVIRK1C4A-GFP was transiently expressed in N. benthamiana leaves. f Analysis of the redox regulation of OsVIRK1 in rice. Transgenic rice constitutively expressing OsVIRK1-FLAG-HA, OsVIRK1K391E-FLAG-HA, or OsVIRK1C4A-FLAG-HA was inoculated with RSV or mock. In e, f, total protein was extracted with or without DTT (50 mM) and separated by non-reducing SDS-PAGE, followed by western blot analysis with anti-GFP, anti-HA, or anti-CP antibodies. In a–c, f, the Rubisco large subunit bands were visualized by Coomassie Brilliant Blue (CBB) staining and served as a loading control. In e, actin was used as a loading control. All experiments were repeated two or three times with similar results.
