| Rice (Oryza sativa L.) is the staple food of half of the world’s human population. Rice blast disease, caused by the fungus Magnaporthe oryzae, is one of the most serious diseases of this crop. Breeding for resistance to blast is an environmental-friendly and cost-efficient way to control the disease despite the availability of effective fungicides. However, the variation of blast strains is really frequent in the fields, thus causing the loss of resistant ability of the blast-resistant rice cultivars within several years. Blast resistance is regarded primarily as partial resistance which is controlled by multiple genes. Therefore, more effort should be pushed into identifying novel blast-resistant genes in rice, and then integrating more than one blast-resistant genes into cultivated rice genotype to develop new blast-resistant rice cultivars.In this paper, CO39 (Oryza sativa L. indica) and its 4 Near-Isogenic Lines (NILs), including C101LAC、C101A51、C101PKT and C105TTP-4L23 (named CN-1, CN-2, CN-4a and CN-4b, respectively), were employed as materials. Two blast fungus race, including highly pathogenic blast fungus GUY 11 and popular blast fungus 81278ZB15 in Fujian province, were used to infect rice seedling for 24 and 48h. Genome re-sequencing, RNA-seq, two-dimension electrophoresis (2-DE) and MALDI-TOF-MS/MS were applied to identify novel blast-resistant genes in rice. The results were summaried in the following.1. Meta analysis on the blast-resistant QTLs in riceFirstly, Meta-QTL analysis were performed to integrate published blast-resistant QTLs in rice. Twenty-four Meta-QTLs were obtained with 2.7Mbp average interval distance. Meta-QTL analysis reduced the confidence interval, enhanced the accuracy of QTLs location and integrated previous research results. Secondly, these 24 Meta-QTLs were mapped to the reference genome sequence in RAP-DB by their left and right molecular markers, and then the physical location for each Meta-QTLs was acquired. According to the physical location, we found 7949 genes including 111 NBS-LRR R genes in these 24 Meta-QTLs, which were selected as candidate blast-resistant genes in rice.2. Analysis on the candidate blast-resistant genes in rice.After genome DNA re-sequencing, SOAPsnp tool was applied to detect SNPs in the 4 NILs with CO39 genome sequence as reference. Considering the different phenotype of CO39 and its 4 NILs in response to 2 kinds of fungus race infection, we found 203 candidate GUY11-resistant genes in the 24 Meta-QTLs in GUY11-resistant rice genotype, CN-4b. Whilst,202,192,74 and 289 candidate 81278ZB15-resistant genes were identified in the 4 NILs respectively, and there were 13 common genes in the 4 NILs.3. Analysis on the expression of RNA in the 24 blast-resistant Meta-QTLs in rice.RNA-seq technology was used to analyze the differentially expression of RNA in the Meta-QTLs in rice in response to blast fungus infection. We identified 201 differentially expressed genes in CN-4b in response to 24h GUY11 infection. Ten differential expressing RNA in CN-4b were the candidate GUY11-resistant genes. In these 10 genes,5 were up-regulated, among which the function of 3 genes were unknown. Meanwhile, the number of differentially expressing genes in the race-resistant rice genotype in response to 24h blast infection were higher than that under 48h blast infection, indicating that 24 hpi was an inoculation time point that overlapped the early induction of resistance gene-dependent defence reactions in blast-resistant rice genotypes.4. Analysis on the differentially expressing proteins in rice in response to blast fungus infection.Fifty common regulated proteins in the tested rice genotypes in response to blast fungus infection were identified by using 2-DE and MALDI-TOF-MS/MS. The infection of two fungus race could be classified with respect to one another on the basis of the 50 protein expression patterns, indicating that the 2 race had different infection pathway. CN-4b under 24 and 48h GUY11 infection were grouped together by the 50 proteins expression pattern, showing that the 26 up-regulated proteins in CN-4b under 24hpi of GUY11 contributed significantly to the GUY 11-resistant ability in CN-4b. The phenomenon of 12 up-regulated proteins in CN-4b was supported by the up-regulation of their corresponding mRNA. CN-4b under 24 and 48hpi of 81278ZB15 were also clustered into one node which was far from others, illustrating that CN-4b had stronger blast-resistant ability than the other rice genotypes.In summary, the results in this paper provide a new insight into the race-resistant ability in different rice genotype and mine several previously function candidate blast-resistant genes in rice. |
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