Impact of mutations in the target-encoding CYP51 gene in Mycosphaerella graminicola populations developing resistance to triazole fungicides

抗真菌化合物耐药性的出现对农业和医学真菌疾病的管理是一个日益严重的问题。最重要的杀菌剂之一是三唑类，用于治疗农作物和人类的真菌感染。不幸的是，当真菌暴露于这些化学物质时，治疗方法可能会选择对化学物质不太敏感的真菌菌株。随着时间的推移，真菌的抗性菌株可以发展，化学物质不再控制疾病。人类致病性念珠菌就发生过这种情况，现在有证据表明，一些植物致病性真菌对三唑也变得不那么敏感了。该项目的目的是了解导致小麦分枝杆菌（Mycosphaerella graminicola）对三唑敏感性变化的机制，分枝杆菌是英国最重要的小麦叶面病——Septoria leaf blotch的致病因子。这种病原体已经适应了许多用来控制它的杀菌剂。2002年首次发现的对strobilurin杀菌剂的耐药性，现在在英国和欧洲的禾草杆菌种群中广泛存在，因此控制这种疾病现在严重依赖于三唑类药物。在过去的十年中，真菌对这些杀菌剂的敏感性逐渐发生了变化，因此现在需要更高的剂量来实现疾病控制。在农业和农用化学工业中，越来越多的人担心，三唑敏感性的进一步变化将降低我们管理这一重要疾病的能力。很少有新的Septoria杀菌剂可用，而用于Septoria控制的旧化学品对环境的破坏更大。已知有几种机制有助于对三唑类药物产生耐药性。这些变化包括靶蛋白的变化，以及其他蛋白质的参与，这些蛋白质被称为转运蛋白，能够将杀菌剂从真菌中泵出。因此，为了了解靶蛋白的变化如何影响三唑的敏感性，必须对蛋白的改变形式进行分离表达和研究。拟议的项目旨在确定三唑靶蛋白突变对敏感性的影响，该蛋白是一种称为CYP51的细胞色素P450，参与固醇14a-去甲基化。这项研究将利用几种方法评估这些突变如何影响杀菌剂和蛋白质之间的相互作用，以及酶本身的活性。突变蛋白将在另一种真菌——酿酒酵母中表达，以观察对不同三唑的敏感性是如何受到影响的，并制作蛋白质的纯样品来测量酶的活性、改变的性质和抑制作用。特别是，该项目将集中研究最近在暴露于三唑类杀菌剂的禾状芽孢杆菌种群中发生的几种突变。我们还打算通过靶向突变引入念珠菌中鉴定的蛋白质的进一步变化，以评估其对敏感性和蛋白质功能的影响。该项目将提供靶点突变在田间对M. graminicola azole敏感性现状的贡献以及未来耐药性发展潜力的关键信息。从这些研究中获得的信息将用于设计新的分子诊断方法，以检测目标位点的变化并监测真菌田间种群中这种变化的发生。这将显示真菌是如何对不同的三氮唑处理作出反应而进化的，并有助于制定控制策略，以保持这组化学品对graminicola的有效性。研究结果也有助于设计更有效的三唑类化合物。此外，确定目标位点变化、杀菌剂敏感性和酶活性之间的关系将有助于了解植物病原真菌在选择反应中CYP51蛋白的分子进化。

The emergence of resistance to antifungal compounds is an increasing problem for the management of fungal diseases in agriculture and medicine. One of the most important groups of fungicides is the triazoles, used for the treatment of fungal infections of both crop plants and humans. Unfortunately, when fungi are exposed to these chemicals, the treatment can select for strains of the fungus that are less sensitive to the chemical. Over time, resistant strains of the fungus can develop and the chemical no longer controls the disease. This has happened with the human pathogenic yeast Candida, and there is now evidence that some plant pathogenic fungi are also becoming less sensitive to triazoles. The aim of this project is to understand the mechanisms responsible for these changes in sensitivity to triazoles in Mycosphaerella graminicola, the causal agent of Septoria leaf blotch, the most important foliar disease of wheat in the UK. This pathogen has already adapted to many of the fungicides used to control it. Resistance to the strobilurin fungicides, first detected in 2002, is now widespread in UK and European M. graminicola populations, so control of the disease now relies heavily on triazoles. Over the past decade there has been a gradual change in the sensitivity of the fungus to these fungicides so that higher doses are now required to achieve disease control. There is a growing concern, amongst both the agricultural and agrochemical industries, that further shifts in triazole sensitivity will reduce our ability to manage this important disease. There are few new Septoria fungicides available, and the older chemicals used for Septoria control are more damaging to the environment. Several mechanisms are known to contribute to resistance to triazoles. These include changes in the target site protein, as well as participation of other proteins, known as transporters, that are able to pump the fungicide out of the fungus. Therefore, to understand how changes in the target protein may affect triazole sensitivity, the altered forms of the protein must be expressed and studied in isolation. The proposed project aims to determine the effect on sensitivity of mutations in the triazole target protein, a cytochrome P450 called CYP51 involved in sterol 14a-demethylation. The research will assess how such mutations affect interactions between the fungicide and the protein, as well as the activity of the enzyme itself, using several approaches. The mutant proteins will be expressed in another fungus, the yeast Saccharomyces cerevisiae, to see how sensitivity to different triazoles is affected, and to make pure samples of the protein to measure enzyme activity, altered properties and inhibition. In particular the project will concentrate on several mutations that have occurred quite recently in M. graminicola populations exposed to triazole fungicides. We also intend to introduce further changes identified in Candida to the protein by targeted mutation to assess their effects on sensitivity and protein function. The project will provide key information on the contribution of target site mutations to the current status of M. graminicola azole sensitivity in the field and the potential for future resistance development. Information gained from these studies will be used to design novel molecular diagnostics to detect target site changes and monitor the occurrence of such changes in field populations of the fungus. This will show how the fungus evolves in response to treatment by different triazoles and help to devise control strategies to maintain the effectiveness of this group of chemicals against M. graminicola. Results should also aid the design of more effective triazole compounds. Furthermore, determining the relationship between target site changes, fungicide sensitivity and enzyme activity will improve understanding of the molecular evolution of the CYP51 protein in plant pathogenic fungi in response to selection.