Identification And Functional Analysis Of Rice Blast Resistance-Related Genes

Rice blast caused by Magnaporthe oryzae is one of the most important diseases of rice. The major challenge in breeding blast-resistant rice cultivars is that the blast resistance controlled by the major R genes is easy to break down, due to the changes of the blast fungus strains. Utilization of durable resistance genes is desirable in the future for the breeding of blast-resistant rice cultivars. Based on the pathways of pathogen associated molecular patterns (PAMP)-triggered immunity (PTI), we aim to use transgenic technology to negatively regulate a PTI-related gene, thereby improving rice resistance to M. grisea. We have performed microarray analysis of rice genes induced by M. grisea and generated RNAi transgenic rice lines of a set of M. grisea-responsive rice genes. In this study, we focused on the screening of rice blast resistance genes by M. grisea inoculation of the RNAi transgenic rice materials, and performed bioinformatics analysis of the microarray transcriptomic data. The main results are as following:(1) For a rapid screening of transgenic rice materials resistant to M. grisea, we improved the method of spore-spraying inoculation by the use of a "micro-chamber" environment with stable temperature and humidity, young rice plants with consistent growth vigor, and paired t-tests of the difference between the disease indexes of the transgenic plants and the untransformed recipient cultivar.(2) RNAi transgenic lines of 45 M. grisea-responsive rice genes were subjected to qRT-PCR analysis. Thirty-one candidate genes showed significant RNAi effect, and their RNAi rice lines were further evaluated by M. grisea inoculation using the micro-chamber method. The RNAi lines of the candidate rice genes XQ10 and XQ24 were found to be resistant to M. grisea.(3) As indicated by the expression levels of differential expression genes (DEGs) in the Nipponbare (NPB) transformed with the rice blast resistance gene Pi9 (NPB/Pi9) and the untransformed recipient NPB, the DEGs numbers at the time point of 24 hours post inoculation (hpi) in the two genotypes were comparable to each other. And the DEGs number at the 36 hpi time point in NPB was significantly more than that in NPB/Pi9. These results suggested that the Pi’9 resistance gene had a significant effect on the transcriptome in NPB/Pi9. GO ontology analysis of the M grisea-responsive rice DEGs suggested that many of the rice chitinase genes in NPB/Pi9 were up-regulated at the 36 hpi time point, and that the up-regulation levels of most of the chitinases in NPB/Pi9 were significantly higher than those in NPB. Therefore, the rice blast resistance is closely associated with the function of chitinases.