CBL1/9-CIPK6 complex negatively regulates Respiratory burst oxidase homolog D in Arabidopsis thaliana

Plant innate immune response is a well-balanced process with positive and negative regulations for the plants to survive. Calcium signaling is essential for pathogen-associated molecular pattern (PAMP)-driven respiratory burst oxidase homolog D (RBOHD)-mediated reactive oxygen species (ROS) burst. We show that calcium sensors calcineurin B like protein 1 (CBL1) and CBL9 and their interacting protein kinase CIPK6 negatively regulate RBOHD activity and immune response in Arabidopsis thaliana. Arabidopsis mutant cbl1cbl9, like cipk6, exhibited enhanced resistance and ROS production when infected with the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). CBL1 and CBL9 enhanced kinase activity of CIPK6. CBL1/9-CIPK6 module interacts with RBOHD at the plasma membrane. CIPK6 along with CBL1 reduces RBOHD activity in planta. CIPK6 phosphorylates the N-terminal cytoplasmic domain of RBOHD at a non-conserved (S³³) and a conserved (S³⁹) serine residue. While S³⁹ phosphorylation increased RBOHD activity, S³³ phosphorylation drastically reduced it and superseded the effect of S³⁹ phosphorylation. We propose a model that CIPK6 phosphorylates RBOHD at S³³ to suppress its activity to balance ROS generation in post-PTI situation in Arabidopsis. Our study reports a direct mechanism of negative regulation of ROS production and plant immune response by a calcium-signaling module in Arabidopsis thaliana.