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

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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.