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

土壤传播的、广泛寄主的血管病原体存在于杂草和作物物种中，如大丽花黄萎病，对世界各地的作物生产构成重大挑战。甲基溴等土壤熏蒸剂的禁用以及氯丁等现有活性物质的有限使用和即将撤出意味着控制措施现在极为有限。在本提案中，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（如光、温度和宿主位置）的调节，目的是利用这些结果来发展新的认识，从而可能导致新的控制措施。我们的研究旨在回答三个基本问题：第一，如何控制条件和微硬化形成等关键发育过程；这些是病原体在土壤中传播和生存的机制吗？第二，基因表达的光调控中断如何影响植物中大丽花弧菌的致病性和生命周期？第三，尽管所有主要时钟成分都保存完好，并且时钟在相关索达菌真菌（如神经孢子虫）的生命周期中发挥了主要作用，但在大丽花中如何以及为什么没有观察到类似生物钟的行为（从我们的初步数据来看）？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，对一些高致病性V. dahliae菌株的黄萎病菌的全基因组，结构分解的基因组序列，一系列主要时钟成分的基因敲除系和RNAseq基因表达时序数据。我们将通过在光脉冲和随后的转录因子结合位点的差异调节基因中进行详细的基因表达时间序列分析来解决关键的发育过程是如何被控制的。同样的实验将在野生型和其他基因敲除系中对温度脉冲进行响应，并将进行比较，以确定涉及光和温度信号的调节途径，以解决进化衍生的表型（如微硬化产生）如何“连接”到现有的信号网络中。利用单基因和多基因敲除菌株，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。我们将对植物中的突变体进行感染试验和基因表达分析，以确定感染期间对效应体表达和生命周期变化的影响，使用荧光标记的大丽花共聚焦显微镜，比较植物内病原体的发育阶段。我们还将尝试利用宿主诱导的一个关键发育、光调节基因的基因沉默来破坏草莓和覆盆子植物中病原体的生命周期。然后，我们将解决一个基本问题，即黄萎病菌是否失去了生物钟，以及它对适应性的影响？我们将测试frq基因中启动子结合位点的进化是否导致了节律性振荡的丧失，以及是否可以通过启动子交换荧光素酶读出实验恢复大丽花的振荡。总之，这项工作将为致病真菌如何对宿主内的环境线索作出反应以及其他相关模型真菌中与生物钟功能有关的基因如何调节优势性状（如静息体生产）提供基本见解。

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.