Background

Rice blast, caused by the ascomycete fungus M. oryzae, is one of the most important diseases of rice. Although many blast resistance (R) genes have been identified and deployed in rice varieties, the molecular mechanisms responsible for the R gene-mediated defense responses are yet not fully understood.

Results

In this study, we used comparative transcriptomic analysis to explore the molecular mechanism involved in Piz-t-mediated resistance in a transgenic line containing Piz-t (NPB-Piz-t) compared to Nipponbare (NPB). Clustering and principal component analysis (PCA) revealed that the time-point at 24-h post inoculation (hpi) was the most important factor distinguishing the four time-points, which consisted of four genes of mitogen-activated protein kinases (MAPKs) signaling pathway, one gene related to WRKY DNA-binding domain containing protein, five pathogenesis-related protein (OsPR1s) genes, and three genes of R proteins involving in the most significant protein-protein interaction (PPI) pathway. Using weighted gene co-expression network analysis (WGCNA) to investigate RNA-seq data across 0, 24, 48, and 72 hpi, nine modules with similar patterns expression pattern (SEP) and three modules with differential expression pattern (DEP) between NPB-Piz-t and NPB across 0, 24, 48, and 72 hpi with KJ201 (referred to as Piz-t-KJ201 and NPB-KJ201) were identified. Among these the most representative SEP green-yellow module is associated with photosynthesis, and DEP pink module comprised of two specific expressed nucleotide-binding domain and leucine-rich repeat (NLR) genes of LOC_Os06g17900 and LOC_Os06g17920 of Pi2/9 homologous, three NLR genes of LOC_Os11g11810, LOC_Os11g11770, and LOC_Os11g11920 which are putatively associated with important agronomic traits, and a B3 DNA binding domain containing protein related genes (LOC_Os10g39190). Knockout of LOC_Os10g39190 via CRISPR-Cas9 resulted in plant death at the seedling stage.

Conclusions

The research suggested that Piz-t and multiple NLR network might play important roles in the regulation of the resistance response in the Piz-t-KJ201 interaction system. The identified genes provide an NLR repository to study the rice-M. oryzae interaction system and facilitate the breeding of blast-resistant cultivars in the future.

中文翻译：

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发病过程中加权基因共表达网络与关键时间点分析相结合，预测稻瘟病抗性的分子机制

背景

稻瘟病是由子囊真菌米曲霉引起的，是稻米中最重要的疾病之一。尽管许多抗稻瘟病（[R ）的基因已被确定并部署在水稻品种，负责的分子机制[R基因介导的防御反应尚未尚不完全清楚。

结果

在这项研究中，我们使用了比较转录组学分析方法，探索了与日本晴（NPB）相比，含有Piz-t（NPB-Piz-t）的转基因品系中Piz-t介导的抗性的分子机制。聚类和主成分分析（PCA）显示，接种后24小时（hpi）的时间点是区分这四个时间点的最重要因素，这四个时间点由有丝分裂原激活的蛋白激酶（MAPKs）信号转导的四个基因组成途径，与含有WRKY DNA结合域的蛋白质相关的一个基因，五个与发病相关的蛋白质（OsPR1s）基因和三个R基因涉及最重要的蛋白质-蛋白质相互作用（PPI）途径的蛋白质。使用加权基因共表达网络分析（WGCNA）研究0、24、48和72 hpi的RNA-seq数据，NPB之间的9个具有相似模式表达模式（SEP）的模块和3个具有差异表达模式（DEP）的模块用KJ201（称为Piz-t-KJ201和NPB-KJ201）鉴定了0、24、48和72 hpi的-Piz-t和NPB。其中最有代表性的SEP绿黄色模块与光合作用相关，DEP粉红模块由与Pi2 / 9同源的LOC_Os06g17900和LOC_Os06g17920的两个特异性表达的核苷酸结合域和富含亮氨酸的重复（NLR）基因组成，三个NLR可能与重要农艺性状相关的LOC_Os11g11810，LOC_Os11g11770和LOC_Os11g11920基因，以及含有蛋白质相关基因的B3 DNA结合域（LOC_Os10g39190）。通过CRISPR-Cas9敲除LOC_Os10g39190导致幼苗期植物死亡。

结论

研究表明，Piz-t和多重NLR网络可能在调节Piz-t-KJ201相互作用系统的抗性反应中起重要作用。鉴定出的基因提供了一个NLR信息库，用于研究水稻与米曲霉的相互作用系统，并在将来促进了抗病育种的育种。