Fungicide mode of action and resistance development in crop pathogenic fungi

世界人口的持续增长伴随着对粮食需求的增加。因此，农业实践得到了加强。玉米、小麦等重要农作物的大面积单一栽培，为植物病原真菌提供了丰富的食物来源。事实上，真菌是我们粮食安全面临的最大挑战。其中最具破坏性的作物病原菌是玉米黑粉菌(Ustilago Maydis)和小麦斑点病菌(SepVictoria Tritici/Mycosphaerella graminicola)。我们的农民通过喷洒抗真菌化学药剂来对抗这些真菌，即所谓的杀菌剂。这些药物通常针对真菌细胞，而对作物几乎没有毒性。为了了解杀菌剂是如何起作用的，需要在抗真菌化学物质存在的情况下详细研究真菌细胞的生理学。其结果是了解杀菌剂对真菌的影响，这描述了“作用模式”(MOA)。在以前，这样的研究受到技术限制的限制。因此，许多杀菌剂的MOA要么未知，要么我们的知识支离破碎。最近，针对真菌病原体的活细胞成像技术和工具的发展使得在杀菌剂存在的情况下对真菌细胞进行可视化。PI的实验室在真菌病原体的活细胞成像方面处于世界领先地位。该项目旨在使用这种现代方法来监测杀菌剂对玉米黑粉菌和小麦纹枯病菌细胞的诱导变化。在初步研究中，我们提供了用于控制苹果真菌病害的杀菌剂多丁的原理研究证据。这揭示了多丁的一种新的MOA，并说明了这种方法的力量。在项目的第一部分，我们将使用细胞标记和活细胞成像来调查12种主要杀菌剂的MOAS，这些杀菌剂涵盖了全球使用的最重要的杀菌剂类别。真菌具有适应杀菌剂的能力。与细菌类似，它们也会产生抗药性，这具有很高的经济重要性，因为它会使各自的杀菌剂失效。抗药性可以通过修改杀菌剂结合和抑制的蛋白质来实现。或者，它可以通过其他鲜为人知的方法来实现，包括增加细胞泵的活性，将杀菌剂从真菌细胞中移除。我们对真菌产生抗药性的机制的了解仅限于病原体遗传信息的局部变化。然而，快速对抗药性真菌的整个基因组信息进行测序的能力打开了寻找所有变化的机会，伴随着杀菌剂抗药性的出现。我们已经在实验室开发了产生抗杀菌剂真菌的技术(到目前为止，大约有150个真菌细胞系(=菌株)对12种主要杀菌剂中的大多数都具有抗药性)。我们已经对这些真菌菌株中的15个菌株的基因组信息进行了测序，并已经发现了一个意想不到的和新的机制在玉米粉虱中产生抗药性的强烈迹象。我们的目标是增加耐药菌株的数量，并扩大无偏见的方法，对耐药真菌的整个基因组进行测序。这一点，以及随后对突变基因的分析，为真菌病原体对杀菌剂的分子适应提供了新的见解。控制小麦病原菌Z.tritici的侵染变得越来越困难。这是由于出现了对主要杀菌剂类别的抗药性菌株。据推测，对杀菌剂的适应能力是由于存在8条“可有可无”的染色体。它们对病原体的生存并不是必需的，因此在细胞分裂过程中可能会丢失。在该项目的这一部分中，我们将产生完全相同的小黑麦菌株，但它们缺乏单独的可选染色体。我们将把它们暴露在杀菌剂下，并分析它们对抗真菌药物产生抗药性的能力。

Continuous growth of the world population comes with increasing demand for food. As a consequence, agricultural practises have intensified. The large monocultures of important crop plant, such as maize and wheat, provide a rich food source for plant pathogenic fungi. In fact, fungi are the biggest challenge for our food security. Amongst the most devastating crop pathogens are the corn smut fungus (Ustilago maydis) and the Septoria tritici wheat blotch fungus (Zymoseptoria tritici/Mycosphaerella graminicola). Our farmers fight these fungi by spraying anti-fungal chemistries, so-called fungicides. These usually target the fungal cell, whilst showing little toxicity to the crop. To understand how a fungicide acts, detailed studies of the physiology of the fungal cell in the presence of the antifungal chemistry are required. The outcome is an understanding of the impact of the fungicide on the fungus, which describes is "mode of action" (MoA). In previous times, such studies were restricted by technical limitations. Consequently, the MoA of many fungicides is either not known or our knowledge is fragmentary. The recent development of live cell imaging techniques and tools for fungal pathogens allows visualisation of fungal cell in the presence of a fungicide. The PI's laboratory is world-leading in live cell imaging of fungal pathogens. The project aims to use this modern approach to monitor fungicide-induced changes in cells of U. maydis and Z. tritici. In a preliminary study, we provide a proof of principle study with the fungicide dodine, used to control fungal disease on apples. This revealed a novel MoA for dodine and illustrates the power of this approach. In the first part of the project, we will use the cell markers and live cell imaging to investigate the MoAs of 12 major fungicides that cover the most economically important fungicide groups in global use. Fungi have the ability to adapt to fungicides. Similar to bacteria, they can develop resistance, which is of high economic importance, as it renders the respective fungicide useless. Resistance can be achieved by modification of the protein that the fungicide binds to and inhibits. Alternatively, it can be achieved by other, much less understood ways, including an increased activity of cellular pumps that remove the fungicide from the fungal cell. Our understanding of the mechanism by which fungi develop resistance is limited to local changes in the genetic information of the pathogen. However, the ability to quickly sequence the entire genomic information of a resistant fungus opens the opportunity to look for all changes, accompanied by the appearance of fungicide resistance. We have developed techniques to generate fungicide resistant fungi in our laboratory (so far ~150 fungal cell lines (=strains), resistant against most of the 12 major fungicides). We have sequenced the genomic information of 15 of these fungal strains and already found strong indication for an unexpected and new mechanism conferring resistance in U. maydis. We aim to increase the number of resistant strains and extend the unbiased approach of sequencing entire genomes of resistant fungi. This, and the subsequent analysis of mutated genes, promises novel insight into the molecular adaptation of fungal pathogens to fungicides. It is increasingly difficult to control infections by the wheat pathogen Z. tritici. This is due to the appearance of resistance strains against the major fungicide classes. It was speculated that the ability to adapt to fungicides is due to the presence of 8 "dispensable" chromosomes. These are not essential for survival of the pathogen and, therefore, can be lost during cell division. In this part of the project, we will generate Z. tritici strains that are identical, but which lack individual dispensable chromosomes. We will expose these to fungicides and analyse the ability to develop resistance against the anti-fungal chemistries.