Fine-tuning OsCPK18/OsCPK4 activity via genome editing of phosphorylation motif improves rice yield and immunity

Abstract

Plants have evolved complex signalling networks to regulate growth and defence responses under an ever-changing environment. However, the molecular mechanisms underlying the growth-defence tradeoff are largely unclear. We previously reported that rice CALCIUM-DEPENDENT PROTEIN KINASE 18 (OsCPK18) and MITOGEN-ACTIVATED PROTEIN KINASE 5 (OsMPK5) mutually phosphorylate each other and that OsCPK18 phosphorylates and positively regulates OsMPK5 to suppress rice immunity. In this study, we found that OsCPK18 and its paralog OsCPK4 positively regulate plant height and yield-related traits. Further analysis reveals that OsCPK18 and OsMPK5 synergistically regulate defence-related genes but differentially regulate development-related genes. In vitro and in vivo kinase assays demonstrated that OsMPK5 phosphorylates C-terminal threonine (T505) and serine (S512) residues of OsCPK18 and OsCPK4, respectively. The kinase activity of OsCPK18^T505D , in which T505 was replaced by aspartic acid to mimic T505 phosphorylation, displayed less calcium sensitivity than that of wild-type OsCPK18. Interestingly, editing the MAPK phosphorylation motif in OsCPK18 and its paralog OsCPK4, which deprives OsMPK5-mediated phosphorylation but retains calcium-dependent activation of kinase activity, simultaneously increases rice yields and immunity. This editing event also changed the last seven amino acid residues of OsCPK18 and attenuated its binding with OsMPK5. This study presents a new regulatory circuit that fine tunes the growth-defence tradeoff by modulating OsCPK18/4 activity and suggests that CRISPR/Cas9-mediated engineering phosphorylation pathways could simultaneously improve crop yield and immunity.

Keywords: CDPK; MAPK; genome editing; growth; immunity; rice.