Phosphorylation and ubiquitination synergistically promote the degradation of OsRbohB to modulate rice immunity

Abstract Plant respiratory burst oxidase homologs (Rbohs) contribute to the production of reactive oxygen species (ROS), which are crucial defense signals in plants. However, the regulation of rice (Oryza sativa) OsRboh homeostasis has remained unclear. In this study, we reported that overexpression of OsRbohB confers resistance to Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae. Mechanistically, the calcium-dependent protein kinase OsCPK4 interacts with and phosphorylates OsRbohB at Ser322 and Ser326, thereby reducing immune responses. OsRbohB phosphomimic modifications at these two sites disrupt OsRbohB-mediated disease resistance. Moreover, the RING-type E3 ubiquitin ligase OsRING142 interacts with and ubiquitinates OsRbohB at Lys266, targeting it for degradation by the 26S proteasome pathway and compromising the immune response. Overexpression of OsRbohBK266R further increased resistance compared to OsRbohB overexpression plants. Remarkably, phosphorylation at OsRbohB facilitates OsRING142-mediated ubiquitination and degradation of OsRbohB. OsRbohBK266R×S2A overexpression plants with reduced ubiquitination and phosphorylation levels of OsRbohB exhibit stronger resistance against M. oryzae. Overall, our study highlights the critical role of Rbohs in broad-spectrum resistance and demonstrates that phosphorylation and ubiquitination synergistically fine-tune Rboh protein stability and immunity.