Functional Analysis Of Differential Expression Genes Induced By Rhizoctonia Solani

Rice sheath blight (SB) caused by Rhizoctonia solani (R. solani) is one of the most serious diseases worldwide. Rice resistance to R. solani is controlled by multiple genes. As the difficulty of phenotype identification and the small effects of quantitative resistance genes, the reports of rice sheath blight resistance genes are rare, which significantly limits the study of molecular mechanism of rice resistance to sheath blight. By using the rice whole genome gene chip (microarray), a series of genes response to R. solani infection were identified from susceptible cultivar Lemont and the resistant cultivar YSBR1 based on the fold changes of gene expression at 10 hours and 20 hours after inoculation, respectively. According to the position of sheath blight resistance QTL on chromosome, and the differential expression genes specifically identified on YSBR1 and the geens associated with disease resistance, nine differential genes were further investigated in the present study.The main results were as follows:1. According to the rice gene annotation database, we found that the 9 genes encode powdery mildew resistance protein PM3F(hereafter named OsPM3F), powdery mildew locus O like protein 1 (OsMLO), plasma membrane intrinsic protein 2c(OsPIP), cellulose synthase-like A6(OsCslA), expansin-related protein 2 precursor(OsEXPR), immunophilin FK506 binding protein(OsFKBP65), RNA-binding region RNP-1 domain containing protein(OsRNP), gigantea homologue (OsGI), and guanylate kinase virescent-2(OsV2), respectively.2. Through qRT-PCR, we investigated the temporal and spatial expression patterns of 7 genes except for OsGI and OsV2. The OsRNP was found strongtly expressed at seedling stage, while the remaining 6 genes showed high expression at or after tillering stage. For different organs, we found that OsCslA highly expressed in spike, while the other 6 genes strongly expressed in either leaf or leaf sheath. Further, we found that all 7 genes response to the inoculation of Rhizoctonia solani and Xanthomonas oryzae, although they showed different expression profiles at different timepoints after inoculation between two different varieties, and between two pathogens. These results indicate that all 9 geens are disease related genes and involve in rice resistance response to pathogen.3. In order to further confirm the role of the 9 genes, we generated RNA interference construct for each of 9 genes. Through agrobacterium-mediated transformation method, each RNAi constuct was introduced into YSBR1. A series of positive transformants were obtained. Through PCR detection in subsequently self-pollination generations of each transformant, we obtained some transgenetic homozygote lines for each of 9 genes. Expression level of target gene in each homozygous transgenetic line were analyzed and showed that most genes were significantly down-regulated.4. At least two homozygous lines for each of 8 genes (not include OsEXPR) were inoculated with sheath blight fungus using detached tillers in growth champer. We found that decreased the OsV2 expression significantly reduce the resistant level of YSBR1 to sheath blight, indicating the important role of OsV2 involved in regulation of rice resistance to sheath blight. Some homozygous lines for transgenes,OsPM3F-RNAi、OsMLO-RNAi and OsRNP-RNAi, were also found significnatly reduced resistance to sheath blight compared with the control YSBR1, suggesting that they probably regulate rice sheath blightr resistance either. For the remaining genes, the phenotyping results were not consistence which apparently needs more transgenic lines to confirm the phenotype.5. The XOO was also used to inoculate the homozygous transgenic lines (not include OsEXPR, OsV2 and OsGI) for testing their phenotype to blight disease resistance. Transgenic lines for 4 transgenes, OsPIP-RNAi, OsRNP-RNAi, OsFKBP65-RNAi and OsCslA-RNAi, were found with significantly longer lesion length than that of control YSBR1. This demonstrates that down regulate of the 4 genes expression is able to reduce rice resistance to blight disease. For OsMLO, we did not found its transgenic lines showing significant difference on lesion length compared to the control YSBR1. The phenotyping results for two homozygous lines for transgene OsPM3F-RNAi were not consistent, which needs further test.In general, the results above showed temporal and spacial expression patterns of 9 disease-related genes and their expression profiles in response to R. solani and Xoo. In addition, we developed RNAi lines for each of 9 geens, and preliminarly found some of them involved in regulation of rice resistance to either sheath blight or blight. These results provide a strong foundation for further investigating their molecular mechanism, and elucidating the molecular basis of resistant variety YSBR1 to sheath blight.