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

Plant innate immune response must be a well-balanced process with positive and negative regulations for the plants to survive. Calcium signalling is essential for pathogen-associated molecular pattern (PAMP)-driven Respiratory burst oxidase homolog D (RbohD)-mediated reactive oxygen species (ROS) burst. We show 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. Plasma membrane localization of CBL1 and CBL9 and CIPK6 kinase activity were associated with the ROS production and immune response. CBL1/9-CIPK6 module interacts with RbohD at the plasma membrane and phosphorylates it’s N-terminal cytoplasmic domain at a non-conserved (Ser ³³ ) and a conserve (Ser ³⁹ ) Serine residue. While Ser ³⁹ phosphorylation increased RbohD activity, Ser ³³ phosphorylation drastically reduced it and superseded the effect of Ser ³⁹ phosphorylation. Replacement of Ser ³³ with alanine or aspartic acid made RbohD a super-active or low-active enzyme, respectively. Our study reports a direct mechanism of negative regulation of ROS production and plant immune response by a Calcium-signalling module in Arabidopsis . Overall, this study provides a novel insight into how calcium signaling integrates with immune regulation to prevent excessive ROS accumulation, ensuring a balanced plant immune response.