Receptor-like Cytoplasmic Kinase 185 Mediated Chitin Perception Induced MAPK Cascade Immune Signaling In Rice

Rice(Oryza sativa)is one of the most important crops in our country.The rice blast disease which is caused by the fungi pathogen Magnaphothe oryzae,brings about devastating yield losses in crop production,threatening China food security.Chitin,the main compound of cell wall in M.oryzae,belongs to the conserved pathogen-associated molecular patterns(PAMPs).PAMPs including chitin are perceived by pattern recognition receptors(PRRs)located at the host cell membrane.The indirect recognition of chitin by the PRR with Lys M domain,Os CERK1,activates a series of biochemical and physiological reactions downstream through phosphorylation,thereby enhancing plant resistance to pathogen.Mitogen-activated protein kinase(MAPK)is a group of protein kinases that can be recognized and activated by PAMPs.It is composed of three conservative kinases,including MAPK kinase kinase(MKKK),MAPK kinase(MKK)and MAPK,these three kinases transmit upstream signals to downstream molecules through sequential phosphorylation,and form one of the important pathways in eukaryotic signaling networks.However,how plants transmit PAMPs recognition signals generated by cell membranes PRRs including Os CERK1 to downstream MAPKs,the mechanism remains elusive.Receptor-like cytoplasmic kinases(RLCKs)belong to the receptor like kinase(RLK)superfamily and they do not possess extracellular domains.It is known that PRRs can further phosphorylate RLCKs after recognition of ligands and form hetero-oligomeric complexes,thereby activating downstream intracellular signalings.Os RLCK185,one of the Os CERK1 downstream phosphorylated substrates,has a genetic phenotype mediated by the MAPK cascade activation response to chitin recognition.MAPK activation was attenuated and enhanced by chitin induction in Os RLCK185 silencing plants and overexpressing plants,respectively.Therefore,it is speculated that Os RLCK185 may play an important role in signaling transduction from chitin recognition by cell membrane receptors to the MAPK cascade pathway.In this study,MAPKKK proteins interacting with Os RLCK185 were screened by means of reverse genetics,and the role of MAPKKK interacting with Os RLCK185 in chitin mediated immunity signaling was biochemically and genetically validated.Following results were obtained:(1)Through yeast two-hybrid screening,we identified a MAPKKK protein,Os MAPKKK?,that interacts directly with Os RLCK185.Their interactions were verified by Co-IP and Pull down,and the C-terminal domain of Os MAPKKK?(aa769-1357)was demonstrated as the key domain responsible for interacting with Os RLCK185.(2)In vitro phosphorylation experiments demonstrated that Os RLCK185 can phosphorylate the serine and threonine residues within the Os MAPKKK? full-length protein and the C-terminal domain.Phosphorylation of Os RLRK185 by Os CERK1 can promote the interaction between Os RLCK185 and Os MAPKKK?,and further enhances the phosphorylation of Os MAPKKK? by Os RLRK185.Os MAPKKK? can further phosphorylate the key downstream chitin response MAPKK protein Os MKK4.(3)Genetic analysis showed that rices silencing Os MAPKKK? attenuated the activatin of MAPK response to chitin,and the resistance to M.oryzae,while the MAPK activation response to chitin increased in the full-length Os MAPKKK?-induced expression plants.MAPK and chitin reponse marker genes were constitutively activated in the Os MAPKKK? kinase domain induced expression rices,and their resistance to M.oryzae also increased.The Os MAPKKK? kinase domain overexpression Nicotiana benthamiana leaves show cell death phenotype regulated by the C-terminal domain.(4)Os RACK1 A may be involved in Os CERK1-Os RLCK185-Os MAPKKK?-mediated chitin signaling pathway as a scaffold protein.(5)Os RLCK185 interacts with Os Rac1 CA,which is the activated form of the small G protein Os Rac1,and phosphorylates it,while Os MAPKKK? interacts with the inactive form of Os Rac1;The interaction between Os Rac1 CA and Os RLCK185 may inhibit complex formation of Os RLCK185 and Os MAPKKK?.These results showed that Os Rac1 may be involved in the regulation of Os CERK1-Os RLCK185-Os MAPKKK?-mediated chitin signaling pathway.Taken together,our study clarified the molecular and biochemical mechanisms of chitin-induced plant resistance signaling transduction: After the cell membrane receptor Os CERK1 recognizes the chitin signal and phosphorylates Os RLCK185,Os RLCK185 further interacts with the C-terminal domain of the Os MAPKKK? protein.As a result,Os MAPKKK? is phosphorylated,and then the downstream Os MKK4 and Os MAPK3/6 are further phosphorylated by the MAPK cascade pathway,thereby activating the expression of disease-resistance related genes.Os RACK1 A and the small G protein Os Rac1 are also involved in the regulation of this disease resistance signaling transduction pathway.Our research has filled the gaps in the field of plant disease resistance signal transduction,promoted our understanding of the molecular mechanism of rice immunity,and provided scientific theories for the molecule breeding of disease-resistant rice through modern molecular biology techniques.