How do light and temperature affect lifecycle, development and pathogenicity in Verticillium?

存在于杂草和作物物种上的土传的、广泛宿主范围的维管病原体，例如大丽轮枝菌，是对全世界作物生产的重大挑战。禁止使用土壤熏蒸剂（如甲基溴）以及限制使用和即将停用现有活性物质（如氯化苦）意味着控制措施目前极为有限。在这项提案中，我们的目标是使用生物信息学和实验方法来了解病原体的生命周期和环境线索的调节，如光，温度和宿主内的位置，目的是利用这些结果来发展新的理解，这可能会导致新的控制措施。我们的研究旨在回答三个基本问题：第一，如何控制关键的发育过程，如分生孢子和微菌核形成;这些是病原体在土壤中传播和生存的机制？第二，光调控基因表达的破坏如何影响植物中大丽轮枝菌的致病性和生命周期？第三，如何以及为什么没有观察到的昼夜节律钟样行为在大丽轮枝菌（从我们的初步数据），尽管所有主要的时钟组件和时钟的主要作用，在生命周期中的相关Sordariomycete真菌，如粗糙脉孢菌？为了解决这些问题，我们将利用我们在过去三年中开发的一系列资源、技术和菌株。这些包括一套快速致病性测试，全基因组，一些高致病性大丽轮枝菌菌株的结构解析的轮枝菌基因组序列，一系列主要时钟组件的基因敲除系和RNAseq基因表达时程数据。我们将解决如何进行详细的时间过程的基因表达后，光脉冲和随后的分析差异调节基因的转录因子结合位点的关键发育过程中进行控制。将进行相同的实验，以响应野生型和其他敲除系中的温度脉冲，并进行比较，以确定参与光和温度信号传导的调控途径，以解决如何进化衍生的表型，如微菌核生产已'连接到'现有的信号网络。使用单个和多个基因敲除菌株，我们将探索已知的和其他新的温度响应转录因子如何影响植物的生命周期阶段和致病性。我们将在植物中进行感染试验和突变体的基因表达分析，以确定在感染过程中对效应子表达和生命周期变化的影响，使用荧光标记的大丽轮枝菌的共聚焦显微镜，以比较植物内病原体的发育阶段。我们还将尝试破坏病原体的生命周期在草莓和树莓植物中使用主机诱导的基因沉默的一个关键的发展，光调节基因。然后，我们将解决一个根本性的问题，即生物钟是否在轮枝菌中丢失，以及它对适应性适应的影响？我们将通过启动子交换荧光素酶读出实验测试是否由于基因frq中的启动子结合位点进化而失去了节律振荡，以及是否可以恢复大丽轮枝菌的振荡。总之，这项工作将提供基本的见解致病真菌如何响应宿主内的环境线索，以及如何有利的性状（如休息体的生产）是由涉及在其他相关模式真菌的生物钟功能的基因调节。

Soil-borne, broad host range vascular pathogens that exist on both weed and crop species, such as Verticillium dahliae, are a significant challenge to crop production across the world. The banning of soil fumigants such as methylbromide and the limited use and impending withdrawal of current actives, such as chloropicrin mean that control measures are now extremely limited. In this proposal we aim to use bioinformatic and experimental approaches to understand the lifecycle of the pathogen and regulation by environmental cues such as light, temperature and location within the host, with the intention of using the outcomes to develop new understanding which may lead to novel control measures. Our research seeks to answer three fundamental questions: First, how are key developmental processes such as coniditation and microsclerotial formation controlled; these are the mechanisms by which the pathogen can disperse and survive in the soil? Second, how does disruption of light-regulation of gene expression affect pathogenicity and lifecycle of V. dahliae in planta? Third, how and why is circadian clock-like behaviour not observed in V. dahliae (from our preliminary data) despite the conservation of all major clock components and the major role of the clock in the lifecycle of related Sordariomycete fungi such as Neurospora crassa? To address these questions, we will draw on a body of resources and techniques and strains we have developed over the past three years. These include a suite of rapid pathogenicity tests, whole genome, structurally resolved genome sequences of Verticillium for a number of highly pathogenic V. dahliae strains, a series of gene knockout lines for major clock components and RNAseq gene expression timecourse data. We will address how key developmental processes are controlled by carrying out a detailed timecourse of gene expression after a light pulse and subsequent analysis of transcription factor binding sites in differentially regulated genes. The same experiment will be carried out in response to a temperature pulse in the wildtype and other knockout lines and comparisons will be made to identify regulatory pathways involved in light and temperature signalling to address how how evolutionarily derived phenotypes, such as microsclerotial production have been 'wired into' existing signalling networks. Using single and multiple gene knockout strains we will then explore how the known and other novel temperature responsive transcription factors, affect lifecycle stages and pathogenicity in planta. We will carry out infection tests and gene expression analysis of mutants in planta to determine the effect upon effector expression and life cycle changes during infection, using confocal microscopy of fluorescently labelled V.dahliae, to compare developmental stages of the pathogen within the plant. We will also attempt to disrupt the pathogen's lifecycle in both strawberry and raspberry plants using host-induced gene silencing of a crucial developmental, light regulated gene. We will then address the fundamental question of whether the clock has been lost in Verticillium and the implications on adaptive fitness? We will test whether rhythmic oscillation has been lost due to promoter binding site evolution in the gene frq and whether oscillation can be restored to V. dahliae through promoter swap luciferase readout experiments. Taken together this work will provide fundamental insights into how pathogenic fungi respond to environmental cues within the host and how advantageous traits (such as resting body production) are regulated by genes implicated in circadian clock function in other related model fungi.