Molecular Mechanism Of OsCDPK19 In Regulation Of Innate Immunity In Rice

Rice(Oryza sativa L.)is one of the most important cereal crops in the world,and plays a decisive role in the maintenance of society.However,blast caused by fungi(Magnaporthe oryzae,M.oryzae)is a major disease in rice,which seriously threatens the yield safety of rice.Avr-Pita/Pi-ta is a classic pathosystem to study the mechanisms of molecular interaction between blast fungi and rice,while the intrinsic function of Avr-Pita and in vivo recognition processes of Avr-Pita and Pi-ta are still largely unknown.In order to understand the molecular basis for the interaction of rice and pathogen,two Avr-Pita interacting factors,OsCDPK19 and OsCOX11,were studied using protein interaction assays,gene expression analyses,subcellular localization analyses,gene knockout technology.The main results are as follows:(1)An Avr-Pita interacting OsCDPK19 protein was fished out by co-immunoprecipitation assays.(2)Transcription analysis showed that OsCDPK19 has two transcripts,OsCDPK19-X1 and OsCDPK19-X2.The OsCDPK19-X2 interacts with Avr-Pita in Y2 H,meanwhile the truncated protein containing phosphokinase domain of OsCDPK19 interacts with Avr-Pita and Pi-ta.(3)qRT-PCR analyses showed that OsCDPK19 expresses in the root,stem,leaf and panicle.The expression of OsCDPK19 is high in the leaf,but is not induced by pathogen M.oryzae.Subcellular localization analysis indicates that OsCDPK19 protein is localized in the cytoplasm and nucleus.(4)Multiple oscdpk19 allelic mutants were obtained from CRISPR/Cas9 gene editing.(5)Punch inoculation with compatible strain 13219(without Avr-Pita)indicated that OsCDPK19 is a negative regulator of basal resistance in rice.(6)The punch inoculation with incompatible strain 08T13(with Avr-Pita)indicated that OsCDPK19 is a positive regulator of Pi-ta mediated resistance in rice.(7)Pull-down and yeast two-hybrid assays demonstrated that OsCOX11 interacts with Avr-Pita and OsCOX19,suggesting that OsCOX11 is involved in the assembly of COX(Cytochrome c oxidase)complex.(8)qRT-PCR analysis showed that OsCOX11,OsCOX17 and OsCOX19 are induced by Chitin,but the induction of OsCOX11 is suppressed by overexpression of Avr-Pita.(9)Inoculation of OsCOX11-OE plant by M.oryzae(without Avr-Pita gene)indicated that OsCOX11 is a negative regulator of rice basic immune response.(10)The Avr-Pita positively regulates the activity of COX and inhibits the production of ROS via association with OsCOX11 and OsCOX19.In conclusion,we study a calcium dependent protein kinase OsCDPK19 and demonstrate that OsCDPK19 plays a positive role in Pita-mediated ETI and plays a negative role together with Avr-Pita in suppressing rice PTI innate immunity by eliminating ROS via association with OsCOX11 and OsCOX19 by enhancing COX activity.