Identification of a plasma membrane complex that interacts with phyB to regulate ROS production

Reactive oxygen species (ROS) regulate plant growth, development, and responses to the environment. ROS production by the RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN D (RBOHD) protein is regulated by PHYTOCHROME B (phyB), and phyB is phosphorylated by FERONIA, highlighting the possibility that these 3 proteins interact to regulate ROS levels during stress. We used immunoprecipitation and proximity labeling, followed by split-luciferase and functional validation assays, to study interactions among FERONIA, phyB, and RBOHD under excess light (EL) stress in Arabidopsis (Arabidopsis thaliana). We found that phyB, RBOHD, and FERONIA interact, that phosphorylation of phyB by FERONIA, as well as the kinase activity of FERONIA, are required for RBOHD-driven ROS production in response to EL stress, and that CYSTEINE-RICH RECEPTOR LIKE KINASE 10 (CRK10) and PLASMA MEMBRANE INTRINSIC PROTEIN 2;6 (PIP2;6) interact with RBOHD and phyB and are also required for EL-driven RBOHD ROS production. Our findings uncover a putative plasma membrane complex among FERONIA, RBOHD, CRK10, and PIP2;6 that interacts with phyB to regulate ROS production in Arabidopsis in response to stress. This complex could play a canonical role in the integration and regulation of multiple signaling pathways in plants.