The role of two component signalling in the regulation of stress responses and virulence in the fungal phytopathogen, Zymoseptoria tritici

小麦黑斑病（STB）是小麦生产和全球粮食安全的主要威胁，真菌病原体小麦黑斑病酵母（Zymoseptoria tritici）是黑斑病的病原体。近年来，一系列小麦曲霉分子工具的发展为破译其致病性调控途径和提供新的控制策略提供了机会。由于抗杀菌剂菌株的发病率日益增加，威胁到可持续农业和粮食生产，因此确定这种战略很重要。对宿主施加的环境压力进行有效反应的能力已被证明是多种植物和人类真菌病原体的重要毒力属性。这些反应的核心是进化上保守的应激激活MAP激酶（SAPK）途径。在小麦中，Hog1 SAP激酶的缺失导致对一系列环境胁迫的敏感性和毒力的丧失。然而，SAPK通路所控制的基因及其调控机制尚不清楚。重要的是，真菌是不同的，因为它们使用“双组分”磷中继系统来感知和传输特定的应激信号到SAPK模块。典型的真菌双组分系统是在出芽酵母中发现的，它由组氨酸激酶Sln1、磷酸化接力蛋白Ypd1和真菌特异性反应调节蛋白Ssk1组成。小麦小麦的基因组序列分析表明，编码组氨酸激酶的基因有多个，但只有Ypd1和Ssk1的同源基因。因此，本项目的目的是确定ZtYpd1和ZtSsk1在小麦小偃麦草的胁迫反应和毒力中的作用。使用实验室中已经建立的方法，将构建携带这些基因缺失的菌株。根据对模型真菌的分析，可以预测ZtYpd1的缺失会导致SAPK的组成性激活，而ZtSsk1的缺失会阻止SAPK对特定信号的激活。因此，这些敲除菌株将允许确定干扰双组分信号对抗逆性的影响。将监测SAPK通路激活（ZtHog1磷酸化），以确定哪些胁迫信号通过双组分系统传递，转录谱分析将用于揭示ZtYpd1和ZtSsk1对胁迫应答基因表达的影响。最终，利用敲除菌株进行小麦侵染试验，将用于评估两组分系统对小麦小麦瘟病弧菌毒力的贡献。

The fungal pathogen Zymoseptoria tritici is the causative agent of Septoria tritici blotch (STB), a disease that represents a major threat to wheat production and food security worldwide. Recently, the development of a range of molecular tools for Z. tritici has provided an opportunity to decipher the pathways that regulate its pathogenicity and inform new strategies for its control. The identification of such strategies is important due to the increasing incidence of fungicide resistant strains which threaten sustainable agriculture and food production. The ability to mount effective responses to host-imposed environmental stresses has been shown to be an important virulence attribute in a variety of plant and human fungal pathogens. Central to these responses are evolutionarily conserved stress activated MAP kinase (SAPK) pathways. In Z. tritici deletion of the Hog1 SAP kinase results in sensitivity to a range of environmental stresses and loss of virulence. However, the genes controlled by the SAPK pathway and the mechanisms by which is regulated have yet to be determined. Importantly, fungi are distinct as they use 'two component' phosphorelay systems to sense and transmit specific stress signals to SAPK modules. The prototypical fungal two component system was characterised in the budding yeast, S. cerevisiae and consists of a histidine kinase Sln1, a phosphorelay protein Ypd1 and a fungal-specific response regulator protein Ssk1. Analysis of the Z. tritici genome sequence has revealed that there are multiple genes encoding histidine kinases but single homologues of Ypd1 and Ssk1. Therefore, the aim of this project is to determine the role of ZtYpd1 and ZtSsk1 in the stress response and virulence of Z. tritici. Using methodology that is already established in the lab, strains carrying deletions in these genes will be constructed. Based on analyses of model fungi, it would be predicted that loss of ZtYpd1 would result in constitutive SAPK activation whereas loss of ZtSsk1 would prevent SAPK activation in response to specific signals. Therefore, these knockout strains will allow the effect of perturbing two component signalling on stress resistance to be determined. SAPK pathway activation (ZtHog1 phosphorylation) will monitored in order to identify which stress signals are relayed via the two component system and transcript profiling will be employed to reveal the impact of ZtYpd1 and ZtSsk1 on stress-responsive gene expression. Ultimately, wheat infection assays using the knockout strains will be used to assess the contribution of the two component system to the virulence of Z. tritici.