Catalytically inactive subgroup VIII receptor-like cytoplasmic kinases regulate the immune-triggered oxidative burst in <i>Arabidopsis thaliana</i>

Protein kinases are key components of multiple cell signaling pathways. Several receptor-like cytoplasmic kinases (RLCKs) have demonstrated roles in immune and developmental signaling across various plant species, making them of interest in the study of phosphorylation-based signal relay. Here, we present our investigation of a subgroup of RLCKs in Arabidopsis thaliana. Specifically, we focus on subgroup VIII RLCKs: MAZ and its paralog CARK6, as well as CARK7 and its paralog CARK9. We found that both MAZ and CARK7 associate with the calcium-dependent protein kinase CPK28 in planta and, furthermore, that CPK28 phosphorylates both MAZ and CARK7 on multiple residues in areas that are known to be critical for protein kinase activation. Genetic analysis suggested redundant roles for MAZ and CARK6 as negative regulators of the immune-triggered oxidative burst. We provide evidence that supports homo- and heterodimerization between CARK7 and MAZ, which may be a general feature of this subgroup. Multiple biochemical experiments indicated that neither MAZ nor CARK7 demonstrate catalytic protein kinase activity in vitro. Interestingly, we found that a mutant variant of MAZ incapable of protein kinase activity can complement maz-1 mutants, suggesting non-catalytic roles of MAZ in planta. Overall, our study identifies subgroup VIII RLCKs as new players in Arabidopsis immune signaling and highlights the importance of non-catalytic functions of protein kinases.