Abstract:

【Objective】The purpose of the study is to analyze the mechanism behind Pi9 gene-mediated rice blast resistance at the transcriptional level, and to lay a theoretical basis for breeding disease-resistant rice cultivars. 【Method】Rice cultivar Nipponbare (NPB) and NPB-transgenic line carrying the Pi9 rice blast resistance gene (NPB/Pi9) were inoculated with Magnaporthe oryzae. Leaf tissues were sampled at 0 h, 12 h, 24 h, and 36 h after inoculation, respectively. Gene-chip transcriptome analysis of 12503 rice genes was performed using the rice samples, and the microarray data were verified by qRT-PCR of a set of differentially expressed genes (DEGs). 【Result】7754 DEGs were identified by comparing the gene expression levels in the resistant NPB/Pi9 line at 12 h, 24 h and 36 h after inoculation, respectively, to that at 0 h after inoculation. Accordingly, 7385 DEGs were identified in the susceptible rice NPB at the same time points. At 36 h after inoculation, the DEG number of NPB/Pi9 was significantly higher than that of NPB. 4065 DEGs were identified by comparing the gene expression levels in NPB/Pi9 and NPB at the same time points; there were significantly more DEGs at 36 h after inoculation than at 0 h, 12 h or 24 h after inoculation. Therefore, NPB/Pi9 exhibited more intensive defense responses to Magnaporthe oryzae. Gene Ontology (GO) and KEGG analyses were carried out on the DEGs between NPB/Pi9 and NPB at the same time points. The GO terms classifications related to extracellular regions, plant response to stimuli, transcriptional regulation, redox activity, ion binding activity, secondary metabolism and plant hormones were significantly enriched for the time points post inoculation. The KEGG pathways of phenylalanine metabolism, flavonoid biosynthesis and plant hormone signaling were enriched significantly at the time points post inoculation. Distinctive gene expression patterns were observed between NPB/Pi9 and NPB at the same time points for the genes of the effector-triggered immunity (ETI) related salicylic acid signaling pathway and chitinase, and the PAMP-triggered immunity (PTI) related extracellular regions, response to stimuli, and lignin biosynthesis. Moreover, distinctive gene expression patterns were observed between NPB/Pi9 and NPB at the same time points for the genes of WRKY transcription factors, MAPK kinases, jasmonate and ethylene signaling pathways that were utilized by both PTI and ETI. Based on all the results, the differential expression patterns between NPB/Pi9 and NPB are related to ETI and PTI, which are involved with each other and play important roles in the Pi9-mediated rice blast rice resistance. 【Conclusion】 Compared with NPB, the resistant genotype NPB/Pi9 showed more intensive defense responses against Magnaporthe oryzae. Transcription factors, kinases, NBS-LRR genes, chitinases, salicylic acid-, jasmonic acid-, and ethylene-signaling pathways, and plant secondary metabolism play important roles in Pi9 gene-mediated rice blast resistance.

Key words: rice blast, transcriptome, resistance genes, plant hormones, secondary metabolism

摘要：

【目的】在转录水平上解析Pi9基因介导的稻瘟病抗性调控机理,为培育抗病水稻品种提供理论依据。【方法】向水稻品种日本晴（NPB）及其转Pi9抗稻瘟病基因株系(NPB/Pi9)接种稻瘟菌。分别于接种后0 h、12 h、24 h、36 h提取叶组织样品,选取12 503个水稻基因定制基因芯片,进行水稻基因转录组分析,并通过qRT-PCR对部分差异表达基因进行验证。【结果】NPB/Pi9在接种后12 h、24 h和36 h的基因表达量分别与其接种0 h表达量比较,共检测到7 754个差异表达基因;相应地,感病水稻NPB在以上时间点共检测到7 385个差异表达基因;在接种后36 h,NPB/Pi9的差异表达基因数目显著多于NPB。比较NPB/Pi9和NPB相同时间点的基因表达量,共获得4 065个差异表达基因,其中接种后36 h的差异表达基因显著多于接种后0 h、12 h或24 h。因此,NPB/Pi9的稻瘟病防御反应更强烈。对NPB/Pi9与NPB相同时间点的差异表达基因进行GO和KEGG分析,细胞外区域、植物对刺激应答、转录调控、氧化还原、离子结合、次生代谢和植物激素相关的GO分类在接种后呈显著富集,苯丙氨酸代谢、类黄酮生物合成和植物激素信号途径的KEGG通路在接种后显著富集。与效应分子触发的免疫反应(ETI)相关的水杨酸信号途径、几丁质酶,以及与病原相关分子模式触发的免疫反应(PTI)相关的胞外区域、对刺激的应答、木质素合成等,均在抗感水稻之间差异表达。而且PTI/ETI共有的WRKY转录因子、MAPK激酶、茉莉酸和乙烯信号途径等发生差异表达。综上所述,NPB/Pi9和NPB的差异表达模式与ETI和PTI相关,两者相互联系并在Pi9介导的稻瘟病抗性中发挥作用。【结论】与日本晴比较,抗病基因型NPB/Pi9对稻瘟病防御反应更强烈。转录因子、激酶、NBS-LRR基因、几丁质酶、水杨酸、茉莉酸和乙烯信号途径,以及植物次生代谢在Pi9介导的稻瘟病抗病反应中发挥重要作用。

关键词: 水稻稻瘟病, 转录组, 抗病基因, 植物激素, 次生代谢