Identification Of Blast Disease Resistance-related Genes And Study On Genetic Mechanism Of OsRLK3-mediated Resistance In Rice (Oryza Sativa L.)

Rice blast,caused by the asexual spores of Magnaporthe oryzae,is one of the most devastating diseases of rice(Oryza sativa L.),and has an important effect on the yield and quality of rice.Although more than 70 rice blast defense regulatory factors have been identified,the mechanisms of rice blast resistance,such as transcriptional reprogramming,immune signaling pathways and networks,are still not well understood.In order to further explore the regulatory factors of rice blast resistance and elucidate their molecular mechanisms,transcriptome analysis of the interaction between rice and Magnaporthe oryzae was carried out,the regulatory network of rice blast resistance was revealed,and a set of core genes involved in rice immune response was identified.On this basis,five genes related to rice blast were selected for functional verification,and the mechanism of action of one receptor-like kinases(RLKs)was deeply analyzed.Therefore,this study can promote the understanding of rice immune system and provide theoretical support and gene resources for rice resistance breeding.The results of this study were as follows:1)In this study,we conducted a comprehensive transcriptome analysis and study on two Japonica rice varieties Nipponbare(Nip)and Hui 1586,which showed susceptible to and resistent to M.oryzae respectively,and obtained a high-quality and systematic transcriptome data set within 48 h.The results showed that rapid and high-range transcriptional reprogramming was important for rice defense against M.oryzae.Ribosomes and protein transaction-related genes were enriched at12 h in differentially expressed genes(DEGs)of resistant and susceptible rice varieties,suggesting that protein translation mechanism is regulated during rice immune activation.In addition,we identified a set of 321 core genes involved in rice responses to biological and abiotic stresses.2)To further verify the accuracy and reliability of our transcriptome sequencing,we performed preliminary functional analysis for some of the identified genes.The results showed that the core genes Os MT1 a and Os MT1 b positively controlled blast resistance in rice,while the core genes Perox4 negatively regulated blast resistance in rice.Among the non-core genes,we found that the expression level of Os SAMS1 gene was significantly increased after being induced by M.oryzae.Further studies showed that ossams1 mutants significantly reduced the expression of pathogen-related genes and inhibited the expression of genes related to ethylene synthesis.Os SAMS1 localized in cell membrane,cytoplasm and nucleus.Taken together,Os SAMS1 is involved in the immune response of rice and positively regulates rice blast resistance.3)Further analysis revealed that OsRLK3,the core gene encoding a RLK,was significantly induced by M.oryzae,and was involved in regulating the immune response of rice.The osrlk3 mutants were more susceptible to M.oryzae than the wild type.Overexpression of OsRLK3 enhanced resistance to M.oryzae compared with the wild type.The osrlk3 mutations inhibited the expression of defense-related genes and the accumulation of hydrogen peroxide,but did not affect the main agronomic traits such as plant height,panicle length and seed setting rate.The osrlk3 mutants also showed increased susceptibility to bacteria(Xanthomonas oryzae).These results indicated that OsRLK3 is a positive regulator of rice resistance and has a broad spectrum role in rice defense response.In addtion,in vitro kinase assay showed that OsRLK3 had kinase activity.4)In order to further elucidate the role of OsRLK3 in rice immunity,we used Immunoprecipitation-Mass Spectrometry(IP-MS)to search for proteins that interact with OsRLK3 in immunity.A total of 374 differential proteins were identified,among which LOC＿Os03g12250encodes a receptor kinase-like protein,one of the rice homolog proteins of RLK902 in Arabidopis(Arabidopsis thaliana),which was named RLK902-2 in our laboratory.To further clarify the interaction between OsRLK3 and RLK902-2,we performed split-luciferase complementation(LUC),bimolecular fluorescence complementation(Bi FC)and co-immunoprecipitation(Co-IP)assays,and confirmed the interaction between OsRLK3 and RLK902-2.In order to further determine the genetic relationship between OsRLK3 and RLK902-2,we knocked out the RLK902-2 gene in the background of osrlk3 mutant.Inoculation experiments showed that double mutants,single mutants were more susceptible compared with wild type,while osrlk3 rlk902-2 double mutants and osrlk3 single mutants showed no significant difference in disease resistant phenotype.Therefore,we hypothesized that OsRLK3 and co-receptor kinase RLK902-2 may regulate rice immune response in the same signaling pathway.In conclusion,this study identified a set of core genes involved in rice immune response,and uncovered the functions of Perox4,Os MT1 a,Os MT1 b,Os SAMS1 and OsRLK3 genes in rice blast resistance,as well as revealed the genetic mechanism of OsRLK3-regulated rice blast resistance.This study provided valuable candidate genes for the study of genetic mechanism of rice blast resistance and abundant gene resources for rice blast resistance breeding.