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. 2023 Mar 3;35(6):2391–2412. doi: 10.1093/plcell/koad064

Figure 2.

Figure 2.

OsMKK10-2 activates phosphorylation of OsMPKs and OsWRKY31. A) The recombinant proteins were expressed with GST plus 3×Myc or 3×Flag tag. GST-WRKY31-3flag with appropriate combination of kinase(s) were subjected to phosphorylation at 30 °C for 2 h. Proteins were separated on Phos-tag gels and blotted with αFlag antibody. The retarded bands indicated the phosphorylated GST-WRKY31-3flag. OsMKK10-2DD: mutations of Arg203 and Ser209 to Asp for constitutive activation. B) Seven-day-old seedlings cultured hydroponically were treated with 10 µM dexamethasone (Dex) or DMSO as the mock. Proteins extracted were separated on 10% SDS-PAGE gels and blotted with αpTEpY, αOsMPK6, or αW31pS6 (generated for detection of OsWRKY31 S6 phosphorylation) antibody. Representative data from four independent experiments. C) Ubi:fW31h#2 mpk3ko and Ubi:fW31h#2 seedlings were grown hydroponically for 7 d, and then treated with 200 µg mL−1 chitin. The proteins extracted were analyzed by immunoblots with αpTEpY, αW31pS6, and αFlag. CBB, Coomassie brilliant blue staining for loading control. Representative data from three independent experiments. Relative intensity (numbers below the gels) of bands was analyzed using ImageJ software. The values below indicate the relative gray density of three experiments. The value marked with * indicates statistically significant difference between Dex treatment B) and chitin C), analyzed by the Student's t-test (*, P < 0.05). Dex:MKK10-2 for Dex:MKK10-2-myc; mpk3ko for OsMPK3 knockout; Ubi:fW31h for Ubi:Flag-WRKY31-6×His.