The phagocytosis oxidase/Bem1p domain-containing protein PB1CP negatively regulates the NADPH oxidase RBOHD in plant immunity

Perception of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors activates RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) through direct phosphorylation by BOTRYTIS-INDUCED KINASE 1 (BIK1) and induces the production of reactive oxygen species (ROS). RBOHD activity must be tightly controlled to avoid the detrimental effects of ROS, but little is known about RBOHD downregulation. To understand the regulation of RBOHD, we used co-immunoprecipitation of RBOHD with mass spectrometry analysis and identified PHAGOCYTOSIS OXIDASE/BEM1P (PB1) DOMAIN-CONTAINING PROTEIN (PB1CP). PB1CP negatively regulates RBOHD and the resistance against the fungal pathogen Colletotrichum higginsianum. PB1CP competes with BIK1 for binding to RBOHD in vitro. Furthermore, PAMP treatment enhances the PB1CP-RBOHD interaction, thereby leading to the dissociation of phosphorylated BIK1 from RBOHD in vivo. PB1CP localizes at the cell periphery and PAMP treatment induces relocalization of PB1CP and RBOHD to the same small endomembrane compartments. Additionally, overexpression of PB1CP in Arabidopsis leads to a reduction in the abundance of RBOHD protein, suggesting the possible involvement of PB1CP in RBOHD endocytosis. We found PB1CP, a novel negative regulator of RBOHD, and revealed its possible regulatory mechanisms involving the removal of phosphorylated BIK1 from RBOHD and the promotion of RBOHD endocytosis.