The CPKs-CBP60b positive feedback loop regulates immunity in Arabidopsis

Abstract

Calmodulin-binding protein 60b (CBP60b) is a pivotal transcription factor in the transcriptional reprogramming of immune responses. Although its downstream regulatory genes and associated genetic pathways have been reported, its post-translational regulation and modifications remain unexplored. Here, we report that calcium-dependent protein kinases CPK4/5/6/11 physically interact with CBP60b and phosphorylate its serine 568 residue, thereby enhancing its transcriptional activity. The phosphodeficient variant CBP60b^S568A compromises immune responses, whereas the phosphomimetic variant CBP60b^S568D enhances resistance against pathogens, demonstrating that ser568 is essential for the function of CBP60b. Moreover, we observed that the expression of CPK4/5/6/11 is induced by pathogens, while this pathogen-induced expression of CPKs is reduced in the cbp60b mutant. Through experiments including dual luciferase (LUC) reporter assays and chromatin immunoprecipitation (ChIP) assays, we further demonstrated that CPK4, CPK5, and CPK11 are direct transcriptional targets of CBP60b. In conclusion, our results demonstrate for the first time that CBP60b is post-translationally regulated by CPKs via phosphorylation, while the pathogen-induced expression of CPKs is dependent on CBP60b. This mutual regulation establishes a positive feedback loop, which enables plants to mount a robust immune response against pathogens.