Fig. 2. VLN3 is phosphorylated by MPK3/6 during plant innate immunity.

a The C terminus of VLN3 is phosphorylated upon flg22 activation. HA-tagged N-VLN3 and C-VLN3 were expressed in Arabidopsis protoplasts. Following treatment with mock or 100 nM flg22 for 10 min, total protein was separated in a phos-tag gel and VLN3 fragments were detected using anti-HA antibodies. Rubisco was used as a loading control. b-c MPK3/MPK6 interact with VLN3 in vivo. The indicated constructs were transiently expressed in N. benthamiana. Luciferase complementation imaging assay (b) and coIP assay (c) was performed. Nluc N-terminal fragment of firefly luciferase, Cluc C-terminal fragment of firefly luciferase. d Activated MPK3 and MPK6 phosphorylate VLN3 in vitro. VLN3 proteins were incubated with MPK3/6 activated by MKK5^DD or a constitutively active form of MPK4 (MPK4^CA)⁵⁰ in an in vitro kinase assay. VLN3 phosphorylation was detected by autoradiography after gel electrophoresis. e–f Ser779 is required for VLN3 phosphorylation in vitro (e) and in vivo (f). e Phosphorylation of wild-type and mutated VLN3 was assessed using an in vitro kinase assay. VLN3 phosphorylation was detected by autoradiography after gel electrophoresis and the band intensity was quantified by densitometric analyses. f Protoplast expressing C-VLN3 and C-VLN3^S779A tagged with HA were treated with flg22. Total protein was subject to phos-tag gel analyses. g MPK3/MPK6 are required for VLN3 phosphorylation in vivo. The HA-C-VLN3 was expressed in MPK6SR protoplasts. Prior to 10-min flg22 (100 nM) treatment, the MPK6SR protoplasts were treated with mock or NAPP1 (2.5 µM) for 12 h. The phosphorylation of C-VLN3 was detected by phos-tag gel analyses. h Flg22 induces Ser779 phosphorylation in a MPK3/6-dependent manner. HA-VLN3 was expressed in protoplasts of MPK6SR rosette leaves. The MPK6SR protoplasts were treated with mock or NAPP1 (2.5 µM) for 12 h before treatment with 100 nM flg22 for 10 min. HA-VLN3 protein was affinity purified with anti-HA antibodies, and Ser779 phosphorylation was detected by immunoblotting with anti-pSer779 antibodies. Total HA-VLN3 protein was detected by anti-HA immunoblot. Values in (e) are means ± SD. n = 4. Different letters indicate significant differences at P < 0.05, as determined by two-way ANOVA with Tukey’s multiple comparisons test. The exact p values are provided in the Source Data file. The experiments in (a, c, d, f–h) were repeated three times with similar results.