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

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.

The project seeks to understand how the fungal pathogen Verticillium dahliae (Vd) responds to environmental signals such as light and temperature using knowledge gained through study of the model fungus Neurospora crassa. The molecular components that have been described as playing a central role in the circadian clock in Nc appear to be key to light and temperature signalling in Vd. The combination of RNAseq in fluctuating environmental conditions over time in wild type and mutant backgrounds along with bioinformatic prediction of co-regulated gene sets with common promoter motifs, will be used to generate hypotheses about the regulatory pathways controlling conidiation and microsclerotial production in response to light and temperature. Genetic analysis by single and double knockouts and promoter binding assays will lead to elucidation of these regulatory networks in Vd.
Following this, pathogenicity screens with mutants, confocal microscopy and gene expression profiling of plant-specific stages of fungal growth (using cell sorting) will be used to assess the role of 'clock' components in infection and development within plants, leading to a greater understanding of how the fungus responds to the environment in the roots and xylem. Using a hairpin WC-1 construct transformed into strawberry and raspberry we will test whether inhibition of condidial and microsclerotial production is possible using host-induced gene silencing. In order to assess the role of the clock in planta and to understand the regulatory changes that have led to the apparent loss of clock-like behaviour in Vd, promoter and gene swap experiments will be carried out and LUC-PEST constructs will be used to read the status of rhythmic oscillations. Taken together this will lead to novel information that could be used as the basis for novel disease control through disruption of environmentally modulated developmental transitions important for pathogenicity.

While this work is of a fundamental nature, there are several routes to achieving impact. In the UK alone the UK strawberry industry is worth £1bn per annum (up from £500m in 2012) and the raspberry market around £150M. Large amounts of production have moved out of soil and into expensive and sometimes hard to source substrates. In many regions there is a desire to return to the soil as costs are lower. The primary problem in soil-borne production is Verticillium, which in the UK is also a problem on potatoes and globally on lettuce, hops and tomato. In time, this is a potential area in which this research could have a meaningful impact.

Direct beneficiaries:
Breeding companies- the use of HIGS against core fungal developmental genes.
Many studies have now shown that trans-kingdom RNAi-mediated gene silencing is possible. The trialling of HIGS in two important soft fruit crops is important, not only for the control of Verticillium, but also as a tool in its own right.

Chemical and biocontrol companies- developing new inhibitors of light/environmental sensing pathways. Just as chemical disruption of mating has been successful in insect pests, it could be possible to disrupt the lifecycle of soil borne pathogens, by disrupting responses to light and temperature. This may lead to the development of new control strategies, based upon the findings from this research. While actively studied in humans and a large area of medical research, chronobiology is in its infancy in fungal/crop sectors and merits further research.

Indirect beneficiaries
Growers and farmers- The likely complete withdrawal of the main soil fumigant in the UK could ultimately lead to annual crop losses between £60-125M with the existing susceptible varieties if chloropicrin is not re-approved. This could be avoided if resistant varieties are developed and even if chloropicrin remains available in the short term, the fumigation costs of £4,000 per hectare could be saved, representing a national saving of c. £10M.

Government, public and policy benefits

Food security- Novel, technology-driven approaches to addressing agricultural problems are a key component in the drive to improve food security and self sufficiency. Research at this level into novel technologies and areas are important, as the form the first part of the value chain that eventually ends up in the hands first of industry, then ultimately the consumer

Less toxic chemistry leading to greater environmental conservation- The wider environmental impacts of alternative control strategies that remove the need for traditional chemical control are also central to improving the environment, quality of soil, water and produce. Chloropicrin is a necessary but harmful chemical- if alternatives could be found the environmental benefits would be large.