Early Interaction Between Rice And Rhizoctonia Solani Regulated By OsSBR2 Gene

Rice sheath blight(Rhizoctonia solani;R.solani),an important fungal disease of rice,exists in almost all rice growing areas in the world.Rice sheath blight resistance is controlled by QTL loci,and so far,no rice germplasm with major-gene resistance to R.solani has been identified.Rice varieties with QTL-mediated disease resistance can be developed to control rice sheath blight.In previous studies,comparative transcriptome analysis of R.solani-resistant rice genotypes was performed,differentially expressed candidate genes were functionally characterized in RNAi-transgenic rice,the rice sheath blight resistance-related gene OsSBR2 was obtained.CRISPR/CAS9 edited OsSBR1-mutated rice lines,OsSBR1-RNAi and OsSBR1 overexpression lines were generated.In this study,we continued to characterize the biological function of OsSBR2 by R.solani inoculation to the field rice plants of the above-mentioned rice lines,transcriptome analysis of OsSBR2 overexpression lines,and proteomic analysis of ossbr1 mutant lines.The results were as follows.(1)At the late tillering stage of rice plants,OsSBR2 rice genetic materials were inoculated by R.solani inoculation of rice sheath.The average disease grades of the ossbr2 mutant line and the wild-type rice cultivar XD were 5.41 and 7.65,respectively,of which the difference was at significant level(p=0.05).The OsSBR2 overexpression line,showing an average disease grade of 8.33,was more susceptible than the wild-type XD,and the difference of two genotypes was significant(p=0.05).The average disease grades of the OsSBR2-RNAi rice line and the wild-type TJ394 were5.20 and 7.35,respectively,of which the difference was significant(p=0.05).These data validated the results in previous studies obtained by in vitro leaf inoculation that the OsSBR2 mutant and RNAi lines were more resistant than the wildtype rice cultivars,and the OsSBR2 overexpression line was more susceptible than the wildtype.Therefore,OsSBR2 negatively regulates rice sheath sheath resistance.(2)Comparative transcriptome analysis was performed on the susceptible wildtype XD and the more susceptible OsSBR2 overexpression rice line.There were173 rice differentially expressed genes(DEGs)between the uninoculated XD and overexpression line,and 263 rice DEGs were identified in the two rice genotypes infected by R.solani for 24 h.Rice DEGs of the two treatments were significantly enriched among the GO items of "oxidoreductase activity" and "metal ion transport activity".The 173 DEGs of uninoculation treatment were functionally enriched in the GO items of "extracellular monooxygenase activity",and the 263 DEGs of the uninoculation treatment were enriched among the GO items of "chlorophyll binding","xylan 1,4-β-xyloside activity" and "amylase".R.solani transcripts were detected in the R.solani infected rice samples,and 92 R.solani DEGs were identified between the inoculated and uninoculation samples.The R.solani DEGs were the gene families of glucoside hydrolase and biobiose hydrolase(AG1IA＿07877,AG1IA＿01834,AG1IA＿00256,AG1IA＿00667,AG1IA＿09613,AG1IA＿09169),which suggested that R.solani infection depends on cell wall degradation enzymes and other differentially expressed molecules.(3)Comparative proteomic analysis was performed on the resistant genotype ossbr2 mutant line and the susceptible wildtype XD.A total of 148 differentially expressed proteins(DEPs),including peroxidase precursor,glutathione S-transferase GSTU6,glycosylhydrolase and chitinase,were identified from the uninoculated samples of the two genotypes.These results indicated that ossbr2 mutation resulted in the preexisting differential expression of metal ion transport activity and oxidoreductase activity.The inoculated samples of the two rice genotypes had 127 rice DEPs,including peroxidase,oxidoreductase,glutathione S-transferase,glycosyl hydrolases,chitinase,phenylalanine deaminase,suggesting that OsSBR2 can affect the activities of oxidoreductase,metal ion transport,and some key enzymes related to secondary metabolism and further regulate rice sheath blight resistance.In addition,56 R.solani DEPs were identified from the inoculation samples of the susceptible and resistant rice genotypes.The DEPs were cellulose and fibrinose degrading enzymes,glycoside hydrolyases,oxidoreductase and virulence related protein ATM1.These results indicated that OsSBR2 indirectly regulates the cell wall degrading enzymes,oxidoreductases and virulence related proteins of R.solani.In conclusion,during the process of rice and R.solani interaction,OsSBR2 regulated the activities of metal ion transport,oxidoreductase and key enzymes of secondary metabolism in rice,and affected rice sheath blight resistance by indirectly regulating the activities of cell wall degradation enzymes and virulence related proteins in R.solani.The results of transcriptome and proteome analysis obtained in this study are of important significance for breeding rice varieties resistant to the rice sheath blight.