OsCPK17- and OsMPK6-mediated dual phosphorylation of OsPUB12 maintains OsPUB2-regulated OsPUB12 homeostasis and balances rice immunity

Numerous E3 ubiquitin ligases regulate plant immunity through mediating protein homeostasis, yet how homeostasis of E3 ligases is finely regulated per se remains poorly understood. Here, we reveal that the ubiquitin E3 ligase plant U-box 2 (OsPUB2) specifically interacts with OsPUB12 and targets it for ubiquitin-mediated degradation. In contrast to the immunity-suppressing role of OsPUB12, OsPUB2 positively regulates resistance to rice bacterial diseases and contributes to pathogen-associated molecular pattern-induced reactive oxygen burst and defense gene expression. Upon pathogen challenge, the calcium-dependent kinase OsCPK17 and mitogen-activated protein kinase OsMPK6 phosphorylate OsPUB12 at Thr154 and Ser460 in a temporally sequential manner. These two phosphorylation events oppositely regulate OsPUB12’s interaction with OsPUB2 and its subsequent ubiquitination, leading to divergent regulation of OsPUB12 stability and rice immunity. Our findings indicate that distinct phosphorylation of OsPUB12 by immune-related kinases OsCPK17 and OsMPK6 synergistically maintains OsPUB2-mediated OsPUB12 homeostasis, serving as a core mechanism to fine-tune rice immunity. OsPUB2 degrades OsPUB12 to promote rice immunity. Phosphorylation of OsPUB12 by OsCPK17 and OsMPK6 at two sites fine-tunes its stability and the plant’s defense response.