基于RNA-Seq的稻瘟病菌Δznf1突变体的表达谱分析

Analysis of RNA-Seq-Based Expression Profiles of Δznf1 Mutants in Magnaporthe oryzae

【目的】稻瘟病菌(Magnaporthe oryzae)引起的水稻稻瘟病是威胁全球水稻生产的重要病害之一,而该菌附着胞介导的侵染又是病害循环的重要环节。在前期的研究中发现一个编码C_2H_2锌指结构的转录因子基因ZNF1,参与稻瘟病菌附着胞形成、穿透和致病过程,论文旨在从转录水平上了解受Znf1调控的基因及其调控机理,为深入研究稻瘟病菌致病分子机理提供基础数据。【方法】利用RNA-Seq技术对稻瘟病菌野生型菌株Guy11和突变体Δznf1的营养菌丝体进行表达谱测序,采用FPKM法计算基因表达量,以FDR≤0.001且log2 ratio(Δznf1/Guy11)≥1为筛选标准,获得Δznf1中差异表达基因(differentially expressed genes,DEGs);通过与Gene Ontology(GO)数据库和KEGG Pathway数据库比对,获得差异基因可能的生物学功能和参与的分子调控途径。为了更详细地研究受Znf1调控的基因,在同样的条件下,利用RNA-Seq技术对稻瘟病菌丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)编码基因PMK1的缺失突变体进行表达谱分析,通过对Δznf1和Δpmk1中的差异表达基因进行比较,筛选受Znf1和Pmk1共同调控的基因,并与前人的研究数据比较,分析获得在稻瘟病菌附着胞发育阶段上调表达但在Δznf1和Δpmk1中同时下调表达的基因。【结果】与野生型Guy11相比,Δznf1中共有709个差异表达基因,其中上调表达的有299个,下调表达的有410个;GO功能富集分析显示差异表达基因归类到生物学过程、细胞组分和分子功能上的基因数目分别有118、299和308个;KEGG Pathway富集分析显示,这些差异表达基因主要参与代谢途径、次生代谢物质生物合成、甘油磷脂代谢等。一些已知的稻瘟病菌致病相关基因,如LPP3、HOX7、PBS2、MPG1等,在Δznf1中表达水平下调。与Δpmk1中差异表达基因比较发现,Δznf1中约56%的差异表达基因同时也受Pmk1调控。其中,编码isotrichodermin C-15羟化酶的3个基因MGG_03825、MGG_02329和MGG_08498,在Δznf1和Δpmk1中的表达水平均显著下调。此外,在附着胞阶段上调表达的48个基因,在Δznf1和Δpmk1中同时下调表达,表明这些与附着胞形成可能相关的基因直接或间接受Znf1和Pmk1调控。采用q RT-PCR方法随机检测10个基因的表达情况,结果与RNA-Seq数据基本一致,说明本试验RNA-Seq数据的可靠性。【结论】RNA-Seq分析获得了受Znf1调控的基因信息和可能的生物学功能。一些与稻瘟病菌致病相关的基因受Znf1调控。此外,与Pmk1类似,一些在附着胞阶段上调表达的基因也同时受Znf1调控。结果可为进一步研究Znf1下游基因调控网络提供信息。

【Objective】Magnaporthe oryzae is the causal agent of rice blast, which is one of the most important disease threatening the production of cultivated rice worldwide. Appressorium-mediated penetration is a key step in the disease cycle of the fungus. Previously, it was reported that a C2H2 zinc finger transcription factor encoded by ZNF1 is essential for appressorium development, penetration and pathogenicity in the rice blast fungus. The objective of this study is to understand the regulatory mechanism of Znf1 and reveal the genes transcriptionally regulated by Znf1, thus providing new clues for further investigating molecular mechanism of pathogenicity in this fungus. 【Method】The transcriptome profiles of vegetative mycelia of the wild-type strain Guy11 and a Δznf1 mutant were assayed with the RNA-Seq technique. The gene expression levels were calculated using the FPKM method. The criteria of false discovery rate（FDR）≤0.001 and absolute value of log2 ratio≥1 were used to identify differentially expressed genes（DEGs）. The sequences of the DEGs were subjected to BLAST queries against the gene ontology（GO） database and KEGG pathway database to predict their biological function and pathways. In order to define in more detail about the sub-set of genes regulated by Znf1, transcriptome profiles of a mutant lacking the PMK1 MAP kinase-encoding gene was also analyzed based on the RNA-Seq technique. To identify the genes regulated by both Znf1 and Pmk1, the DEGs between Δznf1 and Δpmk1 were compared. In addition, the genes highly expressed during appressorium formation but down-regulated in either Δznf1 or Δpmk1 were obtained by comparison with the previous transcriptional profile data. 【Result】 Totally, 709 DEGs in the Δznf1 mutant, including 299 up-regulated and 410 down-regulated genes, were identified by comparison with the wild-type strain Guy11. Gene ontology enrichment analysis showed that 118, 299 and 308 DEGs were classified into cellular component, molecular function and biological process, respectively. KEGG pathway enrichment analysis revealed that the DEGs were mainly involved in metabolic pathways, biosynthesis of secondary metabolites and glycerophospholipid metabolism. Several known pathogenicity-related genes, including LPP3, HOX7, PBS2 and MPG1, were found down-regulated in Δznf1. The comparison of DEGs showed that about 56% DEGs in Δznf1 shared identical to those in Δpmk1. Three isotrichodermin C-15 hydroxylase encoding genes, MGG_03825, MGG_02329 and MGG_08498, were significantly down-regulated in both Δznf1 and Δpmk1. In addition, 48 genes up-regulated during appressorium formation were down-regulated in the two mutants, indicating that these putative appressorium-associated genes were regulated directly or indirectly by Znf1 and Pmk1. To confirm the reliability of the RNA-Seq data, 10 DEGs were randomly selected for q RT-PCR. The results showed that the expression patterns in q RT-PCR were consistent with those in RNA-Seq.【Conclusion】 The expression profiling data and predicted molecular function of Znf1-dependent DEGs were obtained by RNA-Seq technique. Several pathogenicity-associated genes were regulated by Znf1. Additionally, several genes highly expressed during appressoria formation were also regulated by Znf1 as well as Pmk1. This study provided valuable information for further research on Znf1 downstream gene regulatory network.