Table_1_The fungicide pyraclostrobin affects gene expression by altering the DNA methylation pattern in Magnaporthe oryzae.docx

IntroductionRice blast disease caused by Magnaporthe oryzae has long been the main cause of rice (Oryza sativa L.) yield reduction worldwide. The quinone external inhibitor pyraclostrobin is widely used as a fungicide to effectively control the spread of pathogenic fungi, including M. oryzae. However, M. oryzae can develop resistance through multiple levels of mutation, such as target protein cytb mutation G143A/S, leading to a decrease in the effectiveness of the biocide after a period of application. Therefore, uncovering the possible mutational mechanisms from multiple perspectives will further provide feasible targets for drug development.MethodsIn this work, we determined the gene expression changes in M. oryzae in response to pyraclostrobin stress and their relationship with DNA methylation by transcriptome and methylome.ResultsThe results showed that under pyraclostrobin treatment, endoplasmic reticulum (ER)-associated and ubiquitin-mediated proteolysis were enhanced, suggesting that more aberrant proteins may be generated that need to be cleared. DNA replication and repair processes were inhibited. Glutathione metabolism was enhanced, while lipid metabolism was impaired. The number of alternative splicing events increased. These changes may be related to the elevated methylation levels of cytosine and adenine in gene bodies. Both hypermethylation and hypomethylation of differentially methylated genes (DMGs) mainly occurred in exons and promoters. Some DMGs and differentially expressed genes (DEGs) were annotated to the same pathways by GO and KEGG, including protein processing in the ER, ubiquitin-mediated proteolysis, RNA transport and glutathione metabolism, suggesting that pyraclostrobin may affect gene expression by altering the methylation patterns of cytosine and adenine.DiscussionOur results revealed that 5mC and 6mA in the gene body are associated with gene expression and contribute to adversity adaptation in M. oryzae. This enriched the understanding for potential mechanism of quinone inhibitor resistance, which will facilitate the development of feasible strategies for maintaining the high efficacy of this kind of fungicide.

稻瘟病，由稻瘟菌（Magnaporthe oryzae）引起，长久以来一直是全球范围内水稻（Oryza sativa L.）产量降低的主要病因。吡嘧磺隆（pyraclostrobin）作为一种喹啉类外源抑制剂，广泛用作杀菌剂，以有效控制病原真菌的传播，包括稻瘟菌。然而，稻瘟菌可以通过多个层次的突变发展出抗药性，例如靶蛋白细胞色素c（cytb）的突变G143A/S，导致在一段时间应用后，这种生物杀灭剂的有效性降低。因此，从多个角度揭示可能的突变机制，将进一步为药物开发提供可行的靶点。

在本研究中，我们通过转录组学和甲基化组学确定了稻瘟菌对吡嘧磺隆胁迫的基因表达变化及其与DNA甲基化的关系。结果发现，在吡嘧磺隆处理下，内质网（ER）相关和泛素介导的蛋白质降解增强，这表明可能产生了更多需要清除的异常蛋白。DNA复制和修复过程受到抑制。谷胱甘肽代谢增强，而脂质代谢受损。可变剪接事件的数量增加。这些变化可能与基因体内胞嘧啶和腺嘌呤的高甲基化水平有关。差异甲基化基因（DMGs）的过甲基化和低甲基化主要发生在外显子和启动子区域。一些DMGs和差异表达基因（DEGs）通过GO和KEGG注释到相同的通路中，包括内质网中的蛋白质加工、泛素介导的蛋白质降解、RNA转运和谷胱甘肽代谢，这表明吡嘧磺隆可能通过改变胞嘧啶和腺嘌呤的甲基化模式来影响基因表达。

我们的结果表明，基因体内的5mC和6mA与基因表达相关，并有助于稻瘟菌的逆境适应。这丰富了我们对喹啉类抑制剂抗药性潜在机制的认知，将有助于开发维持此类杀菌剂高效性的可行策略。