The Functional Study Of OsAGO2 And OsAGO18 In Rice Defense Responses Against Magnaporthe Oryzae And AR156-mediated Plant Disease Resistance

Rice is one of major food crops around the world,and is the second food crop in China.Rice blasts is one of severest rice diseases,resulting in different level rice production reduction and even crop failure in some years.Magnaporthe oryzae infects plants through spores spreading,as spores can spread to surrounding healthy rice fields by air and finally cause disease.Rice defense mechanisms against M.oryzae are generally divided into two categories:(PAMP-Triggered Immunity,PTI)and(Effector-Triggered Immunity,ETI).PTI is the first layer of defense in rice,and which can be triggered by the process of PRRs(plasma membrane receptors,PRRs)recognizing PAMPs,during which the accumulation of ROS,callose deposition happen.ETI occurs when rice R genes recognize specific avirulence genes secreted by M.oryzae,and which is regarded as the second layer of defense in rice.There are about one hundred R genes found and more than twenty genes cloned.The functions of five pairs of genes and their respective avirulence genes have been fully explored,which include:Avr-Pita and Pita,Avr-Pik and Pik,AvrPiz-t and Piz-t,Avr-Pia and Pia,Avr-CO39 and Pi-CO39.Researches on rice blast mostly focused on two aspects:on one hand is the study of the pathogenesis of the M.oryzae,on the other hand is the study of the rice resistance.In this study,we mainly focused on rice defense response against the M.oryzae.RNA silencing is a conserved immune process in plants,in which defense-related gene expression can be regulated by endogenous plant miRNAs.RNA silencing depends on AGOs protein family,AGOs protein and miRNA specifically recognize each other to form RISC,RISC degrade or inhibit target mRNA translation.AGO2 proteins in Arabidopsis can specifically bind miR393*,which is involved in Arabidopsis defense response against bacteria.AGO2 is closely related to reproduction and development of rice.AGO18 in rice can be associated by miR168,thus prevent the reductive expression of AGO1 and enhance the rice antivirus capabilities,it is projected that AGO18 is involved in the antiviral responses in rice.Therefore,with the combination of our preliminary laboratory results,we propose that OsAGO2 and OsAG(O18 participate in the defense responses to M.oryzae.In this study,we used T-DNA insertion lines and identified nine single insertion homozygote mutants which have correct insertion point.The mutants are as follows,osagold,osago2,osago3,osagoll,osago12,osago14,osago15,osago17,osago18.These mutants were inoculated with M.oryzae strains(Guy11,JS153,98-06)causing different disease symptoms.We compared phenotype with wild type,and found that when inoculated with Guy11,osago2 and osago18 showed significant resistance compared to the wild type plants(Oryza sativa L.japaonica.cv.Nipponbare);when inoculated with JS153,obvious disease phenotype observed.Furthermore,the rice leaves sheathes experiments validated that compared to NPB,the hyphae extended suppressively in osago2 when it is Guy11 infected,and extended widely in osago2 when it is JS153 infected.Thus,we speculated that OsAGO2 and OsAGO18 may be involved in the recognition of avirulent effectors.When rice are infected by Guy11,compare 0 h to 144 h,OsLOX,OsPAD4,OsWRKY53 genes expression are up-regulated remarkably in osago2.The results indicate that OsAG02 may induce denfense against Guy11 through SA,JA signaling pathway and PTI.With the infection of JS153,compare 0 h to 144 h,OsAOS2,OsLOX,OsWRKY53 genes expression are down-regulated markedly in osago2,which means that OsAGO2 may induce denfense against JS153 through JA signaling pathway and PTI.With the infection of Guy11,compare 0 h to 144 h,OsRGA4 gene expression is down-regulated observably in osago18.The results conclude that OsAG018 may induce denfense against Guy11 through ETI.When rice are infected by JS153,compare 0 h to 144 h,OsLOX and OsRGA4 genes expression are down-regulated markedly in osago18.We suspect that OsAGO18 may induce denfense against JS153 through JA signaling pathway and ETI.In addition,we found that AR156 could activate systemic acquired resistance(SAR)to enhance the resistance to bacteria.We used some plants to determine AR156 play a role in different signaling pathways.Arabidopsis transgenic NahG and mutant nprl can not happen SAR.Compared to the control,leaves from jarl and ein2 mutants which have treated by AR156 had similarly moderate symptom.In contrast,NahG and nprl showed no significant difference between the control and AR156-treated plants.Determination of bacterial growth,according to the results,we found that Pst(EV)accumulation decresase significantly in jarl and ein2 mutants which have treated by AR156,and in transgenic NahG and mutant nprl there was no evident difference Pst(EV)accumulation between the control-and AR156-treated plants.Therefore,we conclude that AR156 can induce SAR to enhance resistance of Aradidopsis against Pst DC3000,and AR156-mediated SAR is dependent on the SA signaling pathway and NPR1.We further investigated whether AR156 may participate in the defense of rice against blast.Therefore,we infected rice which are the control and AR156-pretreated plants by two different fungus(Guyll and JS153),and observed phenotype between osago2,osago18 and NPB.When compared with plants pre-treated with control,the results show that AR156 may improve resistance to Guyll in NPB and osago18.And AR156 may improve resistance to JS153 in osagol8.The signaling pathways employed by OsAGO2 and OsAGO18 remain unresolved.