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

对抗真菌化合物的抗药性的出现是农业和医药中真菌疾病管理的一个日益严重的问题。三唑类杀菌剂是最重要的杀菌剂之一，用于治疗农作物和人类的真菌感染。不幸的是，当真菌接触到这些化学物质时，治疗方法可能会选择对化学物质不那么敏感的真菌菌株。随着时间的推移，这种真菌会产生抗药性菌株，这种化学物质不再能控制疾病。这已经发生在人类致病酵母菌身上，现在有证据表明，一些植物病原真菌对三氮唑也变得不那么敏感了。本项目的目的是了解引起小麦叶斑病(英国最重要的叶部病害)的病原菌--禾谷分枝孢霉对三唑类药物敏感性变化的机制。这种病原体已经适应了许多用于控制它的杀菌剂。对2002年首次发现的斯特比林杀菌剂的抗药性，现在在英国和欧洲的麦草分枝杆菌种群中很普遍，所以现在疾病的控制在很大程度上依赖于三唑类药物。在过去的十年里，这种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。农业和农化行业越来越担心，三氮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。几乎没有新的杀菌剂可供使用，而用于控制褐腐病的旧化学物质对环境的破坏更大。已知有几种机制导致对三氮唑类药物的耐药性。这些变化包括靶部位蛋白质的变化，以及其他蛋白质的参与，这些蛋白质被称为转运蛋白，能够将杀菌剂泵出真菌。因此，为了了解靶蛋白的变化如何影响三氮唑的敏感性，必须在分离的情况下表达和研究蛋白的变化形式。这项拟议的项目旨在确定三唑靶蛋白突变对敏感性的影响。三唑靶蛋白是一种名为细胞色素P450的细胞色素P450，与甾醇14a去甲基化有关。这项研究将使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，以了解对不同三氮唑的敏感性是如何受到影响的，并制作蛋白质的纯样本，以测量酶活性、改变的性质和抑制作用。特别是，该项目将集中于最近在暴露于三唑杀菌剂的禾谷分枝杆菌种群中发生的几个突变。我们还打算通过定向突变将念珠菌中发现的进一步变化引入蛋白质，以评估它们对敏感性和蛋白质功能的影响。该项目将提供有关目标位点突变对现场格拉米尼科拉唑敏感度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被用于设计新的分子诊断方法，以检测目标位置的变化，并监测这种真菌田间种群的这种变化的发生。这将显示真菌如何在不同的三氮唑处理下进化，并有助于制定控制策略，以保持这组化学物质对禾谷分支杆菌的有效性。研究结果也将有助于设计更有效的三氮唑化合物。此外，确定靶位变化、杀菌剂敏感性和酶活性之间的关系将有助于理解植物病原真菌中CYP51蛋白对选择的响应的分子进化。

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 ofr the molecular evolution of the CYP51 protein in plant pathogenic fungi in response to selection.