Calcium-dependent Protein Kinase 5/6 Phosphorylate Chitin Receptor To Regulate Innate Immune Response In Arabidopsis

Plant diseases are serious challenge for human beings,which seriously affect food production and human health.Using the resistance genes from plants to fight against phytopathogen can not only protect the plants themselves,but also reduce the use of pesticides so as to protect the environment.Chitin,a major component of fungalcell wall,is a typical microbe associated molecular pattern?MAMP?that is recognized by two plasma membrane located Lys M receptors,CERK1 and LYK5,to trigger immune response in plants.Previous studies suggested that the role of LYK5 is mainly to perceive chitin,while CERK1 is mainly to transduce the immune signal.Although LYK5 is a pseudokinase,the cytoplasmic domain of LYK5 is required for the chitin-induced immune response.To study the role of the cytoplasmic domain of LYK5 in chitin-induced immune response,we conducted the following studies:1. We transformed the construct of LYK5-Myc into lyk5 background and selected a transgenic line high expression level under 35S promotor control,and purified LYK5-Myc receptor complex from the small seedlings before and after chitin treatment.The samples were subjected to mass spectrum?MS?analysis and identified 48 protein candidates.2. Of which,CPK5 and CPK6 are two components of the candidates.Using the bimolecular fluorescence complement?Bi FC?assay and coimmunoprecipitation assay,we found LYK5 interact with CPK5 and chitin enhanced LYK5 interact with CPK5 in planta.Furthermore,we confirmed that CPK6 and CPK5 interact with CERK1 and LYK4 by Bi FC assay.3. A range of physiological and biochemical characteristics of plant defense were detected after chitin treatment in the cpk5 single mutant and cpk5/6 double mutant plants compared to wildtype Col-0,it displayed reduced immune response,including ROS production,WRKY33 and NHL10 expression,MAPK cascade activation.These results clearly showed CPK5 and CPK6 are involved in chitin induced innate response.4. CPK5/6 and chitin receptors,CERK1 and LYK5 are protein kinase.We performed in vitro kinase assay to explore the trans-phosphorylation between these protein kinases.We found CPK5 and CPK6 phosphorylate the cytoplasmic domain of LYK4 and LYK5,but not CERK1 by in vitro kinase assay.The phosphorylated LYK5 cytoplasmic domain was subjected to liquid chromatography-tandem mass spectrometry?LC-MS?analysis,and identified Ser-323 and Ser-542 of LYK5 are important phosphorylation residues of LYK5.In the in vitro kinase assay,it displayed a reduced radiative signal in the LYK5^S542A and LYK5^S323A samples compared to wildtype LYK5.These results indicated LYK5^S542A and LYK5^S323Aare main phosphor-sites by CPK5.5. We made substitution from Ser to Ala at 542 amino acid of LYK5 by PCR-based site-direct mutagenesis method and transformed the construct to lyk5 mutant under its native promoter control.The LYK5^S542A plant displayed reduced MAPK cascade signaling transduction and WRKY53 expression after chitin treatment compared to Col-0.6. We overexpressed CPK5 in wildtype Col-0 background and explore the chitin induced defense response.We found chitin induce CERK1 to degrade,but the protein level of CERK1 in CPK5 overexpression lines is higher than that in wildtype Col-0.Also,we found CERK1 is excessive induced in the CPK5 overexpression lines compared to wildtype Col-0.These results clearly indicated CPK5 play positive function in regulation of CERK1 protein level.Taken together,these results indicated that chitin promote CPK5 interact with LYK5,and CPK5 can increase CERK1 protein level.The above results provide significant contribution to the molecular mechanism of chitin receptors to deliver immune signals,and provide theoretical guidance for the improvement of plant resistance to pathogens.