| Rice (Oryza sativa) is one of the most important food crops in the world. However, rice sheath blight, caused by Rhizoctonia solani, is one of the three major rice diseases, leading to severe losses in rice production. Therefore, the development of sheath blast-resistant rice cultivars is considered to be an important approach to maintain the rice yield. The resistance to sheath blight in rice belongs to a quantitative genetic trait, and rice germplasm resources with major resistance genes against sheath blight are not available so far. Thus, the rice breeding for sheath blight resistance is quite difficult. In this work, we performed a comprehensive transcriptome profiling analysis of the japonica rice variety Taijing 394 infected with Rhizoctonia strain YN-7 to identify the differentially expressed genes in response to sheath blight. Moreover, in order to increase the efficiency of characterizing the sheath blight resistance in rice and to facilitate large-scale screening of rice germplasms, we improved the detached-leaf inoculation method for determination of rice resistance to sheath blight by optimizing the inoculation conditions. The main results are as follows:(1) RNA isolated from leaves of the japonica rice cultivar Taijing 394, 10h and 20h post inoculation (hpi) with R. solani strain YN-7 or post mock inoculation, was subject to transcriptomic analysis using RNA sequencing technology (RNA-Seq). Two biological replicates were performed for each time point and each condition. In total, we obtained 9,683,448 high quality clean reads. With mapped to the rice genome sequence, it is found that genes with 70.63%-90.17% of their sequence covered by reads were generated in each library. Of the total clean reads, there were 67.31%-85.27% and 3.32%-5.10% matching to unique genomic locations and multiple genomic locations, respectively. Through the analysis of the correlation among groups of the sequencing data, it is found that at P≤0.05 probability, the Pearson correlation coefficients of the biological replicates among 10h control group, 10h treatment group,20h control group and 20h treatment group were 0.96,0.97,0.96 and 0.99, respectively, indicating that the RNA-Seq data showed high repeatability and reliability.(2) The gene expression amount was calculated by RPKM method, and a total of 34496 rice expression genes were detected in the eight libraries. The differential expression genes (DEG) were screened by NOIseq method. Compared with the control group, a total of 361 DEGs was identified between 10hpi and 20hpi treatments. Using GO functional classification analysis, we categorized the 361 DEGs according to the secondary classification of GO terms.136,205, and 170 functional groups were in the cellular component, molecular function, and biological process category, respectively. A set of PR1 genes, chitinase-related genes, terpenoid compounds biosynthesis pathway related genes, as well as genes involved in JA signaling pathway were induced in Taijing 394 at 20 hpi, indicating that these genes and pathways may be involved in the response of rice against R. solani infection.(3) In order to improve the efficiency of screening and characterizing the sheath blight-resistant rice germplasm, we also improved the detached-leaf inoculation method for determination of rice resistance to sheath blight. Rice sheath blight strains YN-7 and MH12 were used for inoculation of rice cultivar YSBR1, Lemont and Taijing 394. The inoculation conditions for the detached-leaf method were precisely controlled, and a quantitative protocol for the evaluation of disease severity was developed. The detached leaf tissues inoculated with R. solani were subject to quantitative reverse-transcription qRT-PCR analysis. The rice pathogenesis related genes showed induced expression, which verified the infection of the pathogen into rice. Furthermore, a sheath blight resistant rice cultivar and a sensitive cultivar were tested using the detached-leaf method, the field inoculation method for rice adult plants, and the micro-chamber inoculation method for seedlings. The sheath blight scores of the resistant and sensitive cultivars were significantly different based on statistical tests, and the three methods showed similar results. Taken together, the improved detached-leaf inoculation method has the advantages of easy operation and good repeatability, and can be used for large-scale screening of sheath blight-resistant rice germplasm. |
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