Shuai Wang, Shuying Han, Xiangui Zhou, Changjiang Zhao, Lina Guo, Junqi Zhang, Fei Liu, Qixin Huo, Wensheng Zhao, Zejian Guo, Xujun Chen

Plant Cell; 2023 May 29; 35(6):2391-2412. doi: 10.1093/plcell/koad064.

Abstract

Mitogen-activated protein kinase (MPK) cascades play vital roles in plant innate immunity, growth, and development. Here, we report that the rice (Oryza sativa) transcription factor gene OsWRKY31 is a key component in a MPK signaling pathway involved in plant disease resistance in rice. We found that the activation of OsMKK10-2 enhances resistance against the rice blast pathogen Magnaporthe oryzae and suppresses growth through an increase in jasmonic acid and salicylic acid accumulation and a decrease of indole-3-acetic acid levels. Knockout of OsWRKY31 compromises the defense responses mediated by OsMKK10-2. OsMKK10-2 and OsWRKY31 physically interact, and OsWRKY31 is phosphorylated by OsMPK3, OsMPK4, and OsMPK6. Phosphomimetic OsWRKY31 has elevated DNA-binding activity and confers enhanced resistance to M. oryzae. In addition, OsWRKY31 stability is regulated by phosphorylation and ubiquitination via RING-finger E3 ubiquitin ligases interacting with WRKY 1 (OsREIW1). Taken together, our findings indicate that modification of OsWRKY31 by phosphorylation and ubiquitination functions in the OsMKK10-2-mediated defense signaling pathway.

See https://pubmed.ncbi.nlm.nih.gov/36869655/

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. OsMKK10-2^DD: 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.