The Function Study Of Calcium Dependent Protein Kinase OsCPK4 In Rice Immunity

Plants develop multiple layers of innate immunity during long-term coevolution with microorganisms to defend against attacks from phytopathogenic organisms.The immune responses triggered by the pattern recognition receptors(PRRs)on the plant cell surface constitute the first layer of plant immunity.Cytosolic calcium increase is a typical early defense response induced by pathogen-associated molecular patterns(PAMPs).Ca2+-binding proteins can sense elevated intracellular Ca2+concentration and therefore induce downstream signal transduction.Calcium-dependent protein kinases(CDPKs)are a family of typical Ca2+-binding proteins involved in plant growth,development,responses to various biotic and abiotic stresses,and hormone-regulated cellular processes.Previous studies demonstrated that OsCPK4 plays important roles in salt and drought tolerance as well as plant growth and development in rice.However,little is known on the molecular mechanisms underlying OsCPK4 function in rice immunity.This study illustrated the function of OsCPK4 in rice immunity and further explored its interacting proteins and underlying molecular mechanisms.Initially,a series of assays were performed to investigate OsCPK4 function in innate immunity.OsCPK4 expression was demonstrated to be rapidly induced in rice suspension cells and seedlings upon chitin and flg22 stimulation,respectively.The results indicate that OsCPK4 is an early PAMP-responsive gene.ROS burst induced by PAMPs was significantly more robust in the oscpk4 cells than that in the wild-type cells.Meanwhile,the basal levels of PR gene expression and ROS generation in the leaves of two independent oscpk4 mutants were also enhanced compared with that in the wild type.In addition,inoculation assays demonstrated that the oscpk4 mutant lines possessed enhanced resistance against bacterial blight and fungal blast.These results indicate that OsCPK4 negatively regulates innate immunity in rice.To further explore how OsCPK4 functions in rice immunity,we demonstrated that a known positive immune regulator,the receptor-like cytoplasmic kinase OsRLCK176,interacted with OsCPK4 specifically through bimolecular fluorescence complementation(BiFC)and co-immunoprecipitation(Co-IP)assays.The protein stability assay in N.benthamiana and cell-free degradation assays suggest that OsCPK4 promotes the degradation of OsRLCK176 to negatively regulate innate immunity.Besides,it was revealed that OsCPK4 and OsRLCK176 transiently expressed in rice protoplast and in N.benthamiana were both activated after PAMP stimulation.The two proteins were mutually phosphorylated in vitro,and the in-gel kinase assays suggest that OsCPK4 can phosphorylate OsRLCK176 in vivo and such phosphorylation is enhanced upon PAMP treatment.Interestingly,the activation of OsCPK4-OsRLCK176 circuit can remit OsRLCK176 degradation.Furthermore,this research demonstrated that OsCPK4 interacts with a negative regulator OsMPK3 in vitro.OsCPK4,OsRLCK176 and OsMPK3 interact with each other in vivo which imply they may function as a complex in plant immunity.OsCPK4 was able to phosphorylate OsMPK3,OsMPK4 and OsMPK7 as well as become phosphorylated by the three compounds.In-gel kinase assays suggested that the overall OsMPK activity is significantly reduced in oscpk4 plants in comparison with that in wild-type plants when treated with flg22.Such results imply that OsCPK4 also mediates plant immunity by impacting OsMPK pathway.Collectively,the findings in this study suggest that OsCPK4 promotes the degradation of OsRLCK176 and keeps the defense surveillance system in an inactivated state,thus promoting plant growth and development during resting state without pathogen attacks.After challenging with pathogens and PAMP stimulation,OsCPK4 is activated and forms a phosphorylation feedback with OsRLCK176,this phosphorylation circuit prevents OsRLCK176 degradation and positively contributes to plant immunity.Besides,OsCPK4 regulate plant immunity by impacting OsMPK signal cascades.The study proposes a novel defense buffering mechanism and further deepens the understanding of molecular mechanisms underlying rice innate immunity.