Fig. 4: XopC2 interacts with and phosphorylates OSK1.
From: A bacterial kinase phosphorylates OSK1 to suppress stomatal immunity in rice
a OSK1 was phosphorylated by XopC2 in vitro. His6-XopC2 was incubated with His6-TF-OsCullin1a, His6-OSK1, His6-OsRBX1, His6-OsJAZ9, and His6-TF-OsCOI1b individually in in vitro kinase assays. Protein phosphorylation was detected by autoradiography. Protein loading was indicated by CBB staining. The experiment was repeated 3 times with similar results. b In vivo OSK1 phosphorylation was enhanced during Xoc infection. The OSK1-FLAG-OE-11 transgenic line overexpressing OSK1-FLAG was inoculated with 10 mM MgCl2 (mock), Xoc RS105 (Xoc), ΔxopC2, C-ΔxopC2, and C-ΔxopC2D391A strains. OSK1-FLAG was immunoprecipitated from total protein extracts of inoculated leaves at 2 dpi. Phosphorylated and total OSK1-FLAG proteins were detected with anti-phosphoserine and anti-FLAG antibodies, respectively. Data are shown as means ± SE (n = 3 independent experiments). The chart shows Ser phosphorylation intensities relative to total OSK protein evaluated by Photoshop with three independent repeats. The letters (a, b) indicate a statistically significant difference in OSK1 phosphorylation after inoculation of different strains (one-way ANOVA, Duncan’s multiple range test). c OSK1 was strongly phosphorylated by XopC2 in a semi-in vitro kinase assay. XopC2-FLAG and its kinase-defective variant XopC2D391A/N396A-FLAG were expressed in rice protoplasts and immunoprecipitated with anti-FLAG M2 agarose beads, which were incubated with His6-OSK1 in a kinase assay. Protein phosphorylation was detected by autoradiography. The protein loading was indicated by CBB staining. FLAG-tagged proteins bound to the beads were detected by immunoblotting with an anti-FLAG-HRP antibody. The experiment was repeated 3 times with similar results. d In vitro GST-pulldown assay to detect the His6-OSK1 and GST-XopC2 interaction. His6-OSK1 was co-expressed with GST, GST-XopC2, and GST-XopC2D391A in E. coli. GST-XopC2- and GST-precipitated complexes were detected before (Input) and after affinity purification (pulldown) by immunoblotting with anti-His and anti-GST antibodies. The experiment was repeated 3 times with similar results. e Luciferase complementation imaging to detect in vivo interaction of OSK1 and XopC2. A strong luminescence signal was recorded in N. benthamiana leaves co-expressing OSK1-NLuc and CLuc-XopC2 at 2 days post agroinfiltration. Little or no luminescence was present in negative controls co-expressing NLuc and CLuc, OSK1-NLuc and CLuc-AvrBs2, and pKIWI502-NLuc and CLuc-XopC2, respectively.
