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性的出现是农业和医学中真菌疾病管理的一个日益严重的问题。杀真菌剂中最重要的一类是三唑类，用于治疗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，治疗可以选择对化学物质不太敏感的真菌菌株。随着时间的推移，真菌的耐药性菌株可以发展，化学物质不再控制疾病。这种情况已经发生在人类致病酵母假丝酵母上，现在有证据表明，一些植物病原真菌对三唑也变得不那么敏感。该项目的目的是了解在禾生球腔菌，壳针孢叶斑病，在英国最重要的小麦叶部疾病的病原体，对三唑类敏感性的这些变化的机制。这种病原菌已经适应了许多用于控制它的杀菌剂，对嗜球果伞素杀菌剂的抗药性于2002年首次发现，现在在英国和欧洲的M。因此，现在对该疾病的控制主要依赖于三唑类。在过去的十年中，真菌对这些杀真菌剂的敏感性逐渐发生变化，因此现在需要更高的剂量来控制疾病。在农业和农业化学工业中，人们越来越担心三唑敏感性的进一步变化将降低我们管理这种重要疾病的能力。有几个新的壳针孢杀真菌剂可用，和旧的化学品用于壳针孢控制更破坏环境。已知几种机制有助于对三唑类的耐药性。这些变化包括靶点蛋白的变化，以及其他蛋白质的参与，称为转运蛋白，能够将杀真菌剂泵出真菌。因此，为了了解靶蛋白的变化如何影响三唑敏感性，必须单独表达和研究蛋白质的改变形式。该项目旨在确定三唑靶蛋白突变对敏感性的影响，该靶蛋白是一种称为CYP 51的细胞色素P450，参与甾醇14 a-脱甲基化。该研究将使用几种方法评估这些突变如何影响杀真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酵母酿酒酵母中表达，以观察对不同三唑的敏感性如何受到影响，并制备蛋白质的纯样品以测量酶活性，改变的性质和抑制作用。特别地，该项目将集中于最近在M.暴露于三唑类杀真菌剂的禾生草群。我们还打算通过靶向突变将念珠菌中鉴定的进一步变化引入蛋白质，以评估其对敏感性和蛋白质功能的影响。该项目将提供关于靶位点突变对M的当前状态的贡献的关键信息。禾生菌对唑的田间敏感性和未来抗性发展的潜力。从这些研究中获得的信息将用于设计新的分子诊断方法，以检测靶位点的变化，并监测真菌田间种群中此类变化的发生。这将显示真菌如何对不同三唑类药物的处理作出反应，并有助于制定控制策略，以保持这组化学品对M的有效性。禾生菌研究结果还有助于设计更有效的三唑化合物。此外，确定目标位点的变化，杀菌剂的敏感性和酶活性之间的关系，将提高理解的CYP 51蛋白在植物病原真菌的分子进化，以响应选择。

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.