Probing the molecular origins of the species-selectivity of microtubule-directed fungicides

Septoria叶斑病是影响小麦和大麦生产的主要病害，特别是在北欧。Septoria目前使用抗真菌喷雾剂控制，该喷雾剂针对真菌微管，同时不影响小麦和人类微管。抑制微管可以阻止真菌细胞分裂并最终杀死它们，同时不影响小麦和人类细胞。这种方法是有效的，但阻力是一个日益严重的问题。我们迫切需要了解耐药性是如何产生的，我们需要开发新的和更好的抗真菌药物，以有效对抗Septoria真菌的耐药菌株。先正达正在努力做到这一点，并希望能够在Septoria分离的微管上测试可能的新药物，将其与小麦和人类微管的反应进行比较。理想情况下，先正达希望从耐药的Septoria细胞中纯化微管，将它们与非耐药细胞进行比较，并了解为什么耐药细胞的微管不受杀菌剂的影响。目前还不可能做到这一点——哺乳动物的微管可以很容易地纯化，但到目前为止还没有人能够纯化Septoria微管或小麦微管。目前缺乏基于纯化微管的测试，限制了先正达和其他地方设计和开发新型抗真菌药物的速度。克罗斯实验室是纯化微管蛋白的专家。例如，我们最近成功地从酵母细胞中纯化了微管蛋白。在这个项目中，我们将与先正达合作，从Septoria细胞、小麦细胞和人类细胞中分离微管蛋白，并开发小型测试，以便检查可能的抗真菌化合物，以确保它们靶向Septoria微管，同时不影响小麦和人类微管。先正达将为我们培养工业批量的Septoria细胞，我们将使用我们已经开发的纯化酵母微管蛋白的技术对它们进行加工以获得微管蛋白。我们还将从小麦幼苗和培养的人类细胞中制造微管蛋白。为了从所有这些来源中获得大量的微管蛋白，我们将把自己的方法与最新的技术相结合，进行高效的微管蛋白纯化。我们将比较不同的抗真菌剂对我们收集的Septoria、耐药Septoria、小麦和人类纯化微管的作用，使用现有的微管稳定性测量方法，也可以通过光学显微镜直接观察微管。然后我们就可以构想出杀菌剂是如何在分子水平上起作用的，以及在分子水平上，抗药性和非抗药性的Septoria有什么不同。我们将通过改造酵母微管蛋白来测试这些想法，使其对我们的抗真菌药物敏感或耐药。这是我们理解的最终基准——如果我们能够证明我们知道如何从酵母中设计微管蛋白，使其对抗真菌药物具有与Septoria蛋白相同的敏感性，那么我们就可以说我们真正了解了抗真菌药物是如何融入微管蛋白并控制其行为的。这些信息可以反馈到工作流程中，用于开发新的抗真菌药物。Septoria感染几乎影响了英国种植的所有小麦。据估计，使用杀菌剂可以提高产量约20%，因此，如果不使用杀菌剂，英国谷物种植者每年将遭受3.8亿至4.65亿英镑的经济损失（数据来自洛桑研究所的在线报告）。如果最终成功，这项研究的潜在影响将对英国经济产生重大影响。我们开发的新技术和知识将被转移到先正达，从而在项目生命周期内加速发现和开发新的mt定向抗真菌药物。

Septoria leaf blotch is a key disease affecting wheat and barley production, especially in Northern Europe. Septoria is currently controlled using antifungal sprays that target the fungal microtubules whilst leaving wheat and human microtubules unaffected. Inhibiting microtubules prevents the fungal cells from dividing and ultimately kills them, whilst leaving wheat and human cells unaffected. The approach works, but resistance is an increasing problem. We need urgently to understand how resistance arises and we need to develop new and better antifungals that are effective against the resistant strains of the Septoria fungus. Syngenta is trying to do this, and would ideally like to be able to test possible new agents on isolated microtubules from Septoria, comparing their response with that of microtubules from wheat and from humans. Ideally, Syngenta would like to have purified microtubules from resistant Septoria cells, to compare them with those from non-resistant cells and understand why the microtubules from the resistant cells are not affected by the fungicide. Nothing like this is currently possible - mammalian microtubules can be purified fairly readily, but no one has so far been able to purify Septoria microtubules, or wheat microtubules. The lack of tests based on purified microtubules at present limits the rate at which new antifungals can be designed and developed at Syngenta and elsewhere.The Cross lab is expert at purifying microtubule proteins. For example, we have recently succeeded in purifying microtubule protein from yeast cells. In this project we will collaborate with Syngenta to isolate microtubule protein from Septoria cells, wheat cells and human cells, and develop miniaturised tests so that possible antifungal compounds can be checked to make sure they target the Septoria microtubules whilst leaving wheat and human microtubules unaffected. Syngenta will grow Septoria cells for us in industrial quantities, and we will process them to obtain tubulin using the techniques we have already developed for purifying yeast tubulin. We will also make tubulin from wheat seedlings, and from cultured human cells. To obtain large quantities of microtubule protein from all these sources, we will combine our own methods with newly-available techniques for highly efficient purification of microtubule protein. We will compare the actions of different antifungals on our collection of purified microtubules from Septoria, resistant Septoria, wheat and humans, using existing measures of microtubule stability, but also by looking at the microtubules directly by light microscopy. We can then formulate ideas about how the fungicides actually work at the molecular level, and what it is that is different, at the molecular level, between the resistant and non-resistant Septoria. We will test theses ideas by engineering yeast tubulin to make it susceptible or resistant to our antifungal agents. This is the ultimate benchmark of our understanding - if we can prove that we know how to engineer the microtubule protein from yeast so that it has the same susceptibility to antifungals as the Septoria protein, than we can truly say we understand how the antifungal fits into the microtubule protein and controls its behaviour. This information can then be fed back into the workflow for the development of new antifungals. Septoria infection affects virtually all wheat grown in the UK. Application of fungicides is estimated to boost yields by ~20% and therefore without fungicide use UK cereal growers would suffer an economic loss of between £380 and £465 million each year (figures sourced from a Rothamsted on-line report). If ultimately successful, the potential impact of this research would therefore be significant for the UK economy. The new techniques and knowledge that we develop will be transferred to Syngenta, thereby accelerating the discovery and development of new MT-directed antifungals within the project lifetime.