Functional Identification And Omics Analysis Of Rice Sheath Blight Resistance-related Gene OsSBR1

Rice sheath blight(Rhizoctonia solani)is one of the important diseases of rice.Rice sheath blight resistance is controlled by multiple quantitative trait inheritance loci.So far,rice germplasms with high-level resistance or immunity to sheath blight have not been identified.Studying the molecular mechanism of sheath blight resistance in rice is of great significance for breeding resistant rice varieties and controlling the disease.In this laboratory,a preliminary study was performed on the rice sheath blight resistance-related gene OsSBR1,and the OsSBR1 RNAi and over-expressing transgenic rice lines showed resistance and susceptibility to R.solani,respectively.To further characterize the biological function of OsSBR1 and the mechanism of OsSBR1 regulating disease resistance,this study focused on the development of OsSBR1 transgenic rice lines amd their evaluation of sheath blight resistance,subcellular localization of the OsSBR1 protein,transcriptome analysis of OsSBR1-RNAi rice lines and OsSBR1 gene-edited mutant lines,as well as the changes in the content of plant hormones and differential metabolites in the OsSBR1-RNAi rice lines.The research results are as follows:(1)Detached leaf tissues of OsSBR1 over-expression rice lines and gene-edited mutant lines were inoculated with R.solani.Based on the size of infection area on leaf tissues,the OsSBR1 overexpression lines were susceptible to sheath blight and the OsSBR1 knockout lines showed resistance,which verified that OsSBR1 negatively regulates sheath blight resistance in rice.The vector expressing OsSBR1-GFP fusion protein and the vector expressing an endoplasmic-reticulum protein fused with RFP were co-transformed into rice protoplasts and the GFP and RFP fluorescence signals completely overlapped,thereby localizeing the OsSBR1 protein on the rice endoplasmic reticulum.(2)R.solani transcripts were detected in the rice tissues of the resistant genotype(OsSBR1-RNAi rice line)and the susceptible genotype(TJ394)that were infected with R.solani.165 and 536 differentially-expressed genes(DEGs)of R.solani were identified,respectively,from the 24 hpi and 48 hpi tissues of both rice genotypes by comparative transcriptome analysis.266 differentially expressed genes of R.solani were identified from the rice tissues of the OsSBR1 knock-out rice line(resistant genotype)and the susceptible cultivar XUDAO 3 that were infected with R.solani for 24 h.According to the gene annotation information,a number of R.solani DEGs are related to pathogenicity.The results that sheath blight-resistant and-susceptible rice genotypes differentiated R.solani gene expression have implication that OsSBR1 negatively regulates the sheath blight resistance in rice.(3)Comparative transcriptome analysis was performed on the OsSBR1 gene-edited mutant line and the susceptible cultivar XUDAO 3 24 h post R.solani inoculation.851 and 2341 DEGs between the two rice genotypes were identified in mock and R.solani inoculation,respectively,and 83 transcription factors including WRKY and MYB were differentially expressed upon R.solani inoculation.GO and KEGG enrichment results showed that DEGs between the two rice genotypes were significantly enriched in photosynthesis,redox process,signal transduction pathway,metabolic pathway,secondary metabolic biosynthesis and other pathways.Rice genes related to jasmonate signal pathway and ethylene biosynthesis pathway and ethylene transcription factors were differentially expressed in the two rice genotypes,indicating that OsSBR1 regulates both jasmonate and ethylene signaling pathways.(4)Comparative transcriptome analysis was performed on the OsSBR1-RNAi resistant rice line and the susceptible TJ394 cultivar at the 24 hpi infection time-ponit.The data as above were integrated with the transcriptome data of the same rice genotypes at the 48 hpi,and the transcriptome dynamics of rice at different inoculation time points were analyzed comprehensively.The results of GO classification enrichment analysis showed that under the conditions of inoculation and non-inoculation,DEGs beween the two genotypes were significantly enriched in redox process,photosynthesis,ion binding and other items,which suggested that there was "pre-existing expression" of disease resistance related genes in the OsSBR1-RNAi rice line.Compared with that in the susceptible TJ394 cultivar,the jasmonate biosynthesis related genes in the OsSBR1-RNAi rice line were significantly down regulated,and the ethylene transcription factors and ethylene biosynthesis related genes were differentially expressed,indicating that OsSBR1 may regulate jasmonate signal transduction pathway and ethylene signal transduction pathway.(5)The rice tussues of OsSBR1-RNAi line and the susceptible TJ394 cultivar 12 h,24 h and 48 h post R.solani inoculation were assayed for measurement of the contents of jasmonic acid(JA)and its derivatives JA-Ile and SA.There was no significant difference between the the hormone contents of both genotypes in mock inoculation condition.Compared with the mock inoculation samples,the JA and JA-Ile contents in both rice genotypes inoculated with R.solani were induced,and the changes of JA and JA-Ile contents showed sinilar trends.The JA and JA-Ile contents of 12 hpi samples of the OsSBR1-RNAi rice line was slightly higher than those in TJ394,while the JA and JA-Ile contents in TJ394 samples at 24 hpi and 48 hpi were significantly higher than those of the OsSBR1-RNAi line.SA contents in the rice samples were basically opposite to those of JA,indicating that SA and JA were antagonistic to each other.The metabolomic analysis of OsSBR1-RNAi resistant strains and susceptible control varieties showed that SA was significantly upregulated in OsSBR1-RNAi resistant strains,suggesting that the sheath blight resistance in OsSBR1-RNAi was achieved by the JA antagonism against SA.This study has laid a foundation for analyzing the molecular mechanism of rice sheath blight resistance and the molecular interaction mechanism between rice and sheath blight.The gene-edited R.solani-resistant rice lines provide elite rice germplasms for rice breeding.