Do G-protein coupled receptors regulate pathogenesis and mycotoxin biosynthesis in filamentous phytopathogenic fungi?

DO G-PROTEIN COUPLED RECEPTORS REGULATE PATHOGENESIS AND MYCOTOXIN BIOSYNTHESIS IN FILAMENTOUS PHYTOPATHOGENIC FUNGI?
Fungal pathogens constantly attack crop plants and cause major diseases. In order to cause disease, some fungal pathogens secrete chemicals that are toxic to both plants and humans. Therefore, fungal diseases reduce a farmer's crop production and profit, while threatening human health.

When a fungal pathogen lands on a plant, it senses the surrounding and decides whether this location is suitable for infection. How fungi sense the "touch and taste" of their environment through external receptors is largely unknown. These receptors, and their sensing of the environment, are thought to control how a fungal pathogen causes disease and whether it produces toxins. Therefore, we plan to identify fungal receptors that are required for disease in the cereal pathogen, and toxin producer, Fusarium graminearum.

At Rothamsted Research, we will create a collection of mutated fungi missing different receptors. Within specialised containment facilities, we will test if the loss of these receptors reduces the ability of F. graminearum to cause disease and produce toxins in wheat. What is controlled by these receptors will be identified by comparing the expression of fungal genes during wheat infection. Fluorescent proteins and cutting-edge microscopy will be used to visualise how these receptors are activated upon detection of the plant host and how they interact with other proteins within the fungus to control disease.

These studies will provide an understanding of how fungal pathogens regulate disease and toxin production in response the "touch and taste" of their environment. This new knowledge, and the state-of-the-art techniques developed, will be transferable to other fungal pathogens of plants and humans.

Approximately 40% of modern pharmaceuticals target similar receptors. Therefore, the identification of fungal receptors needed for disease would represent novel drugable targets to combat a fungal pathogen, improving safe food production and protecting human health.

DO G-PROTEIN COUPLED RECEPTORS REGULATE PATHOGENESIS AND MYCOTOXIN BIOSYNTHESIS IN FILAMENTOUS PHYTOPATHOGENIC FUNGI?
G-protein signalling performs a central role in the coordination of fungal development and virulence. G-protein coupled receptors (GPCRs) sense changes in the environment and initiate signalling events that direct the appropriate cellular/molecular response, yet the role of GPCRs and their activating ligands in promoting fungal virulence is largely unknown.

The globally important cereal pathogen, and mycotoxin producer, Fusarium graminearum possesses more GPCRs than other model fungi. F. graminearum deploys a complex array of virulence mechanisms to cause disease on wheat, which are spatio-temporally regulated during infection. To define the involvement of GPCRs and their ligands in the regulation of F. graminearum virulence mechanisms, I will:

1) Create 20 GPCR-deficient strains by split marker transformation. Assess the impact on virulence using microscopy, DON mycotoxin ELISAs, Megazyme assays and western blots with Fgl1/SSP6 antibodies. Select two GPCRs for further study via RNAseq to reveal their transcriptional influence.
2) Identify associated Galpha protein by BiFC and by complementing GPCR-deficiency with Galpha possessing an activating or interfering mutation. Monitor nuclear shuttling of downstream GFP-tagged kinases.
3) Develop a novel FIAsH-TC technique to monitor GPCR conformational activation by FRET, facilitating ligand identification. Confirm GPCR-ligand activation by assessing Galpha(CFP)-Gbeta(YFP) interaction via FRET and by monitoring cAMP levels, PKA activity, MAPK phosphorylation, and kinase nuclear shuttling.
4) Use site-directed mutagenesis to define the binding site of the FIAsH-TC tagged GPCR. Assess ligand induced GPCR conformational change by FRET, and the impact on the virulence phenotype.

These studies will provide fundamental knowledge on how fungi regulate virulence in response to plant or fungal signals.

DO G-PROTEIN COUPLED RECEPTORS REGULATE PATHOGENESIS AND MYCOTOXIN BIOSYNTHESIS IN FILAMENTOUS PHYTOPATHOGENIC FUNGI?
Cereals are the most important source of human calories, and wheat provides one fifth of the world's total calorific intake. Fungal diseases have a major, negative impact on cereal grain yields, thereby detrimentally affecting food security and human health. Fusarium ear blight (FEB) is a devastating fungal disease of cereals, including wheat. Direct crop losses are compounded by pre- and post-harvest contamination of the grain with a fungal mycotoxin, making the harvest unsuitable for human or animal consumption. Costly disease control strategies are ineffective at combating FEB and mycotoxin contamination. G-protein coupled receptors (GPCRs) are the target of 40% of modern pharmaceuticals. By identifying the fungal GPCRs required for disease in the cereal pathogen, and toxin producer, Fusarium graminearum, this proposal will uncover novel, drugable targets to combat fungal pathogens. Thus, the outcomes of this project will have immediate and long-term impacts on improving safe food production and the welfare of the wider public.

This project has a numerous and diverse range of beneficiaries. The knowledge generated in this proposal may be exploited as a resource to ignite collaborations with Agri-tech and pharmaceutical industries, leading to the development of a non-GM approach to dually curtail Fusarium infection and mycotoxin contamination. The genus Fusaria contains many pathogens of importance to agriculture, horticulture, forestry and human health. If effective, a GPCR-interfering approach could be effective against other problematic cereal infecting and post-harvest spoilage Fusaria, such as F. culmorum, F. langsethiae and F. verticillioides. The identified GPCR-interfering compound may also be effective against Fusaria, which cause a spectrum of superficial, locally invasive, or disseminated human infections, such as F. solani and F. oxysporum. Thus, the private sector could directly derive economic benefits from the results of the proposal in the longer term through the development of non-GM approaches to reduce Fusarium infections and mycotoxin contamination.

The study of how GPCRs regulate aspects of fungal biology in other species will be facilitated by the techniques developed in this proposal. Academics in the fungal pathology and industrial mycology will benefit from the newly developed cell biology techniques for studying GPCR and the novel insights into how GPCRs regulate virulence in response to environmental cues. Potential areas for the expansion of GPCR investigations are: 1) Plant pathogens possessing GPCR homologues to those required for full virulence in F. graminearum. 2) Mycotoxin regulation of plant and animal infecting pathogens. 3) Mycotoxin-producing post-harvest and food spoilage fungi. 4) Fungal quorum sensing and biofilm formation in pathogenic fungi. 5) Investigations that aim to identify the receptors/signals that regulate other virulence mechanisms deployed by fungi, including morphological adaptations, reproduction and the secretion of proteinaceous effectors. The improved understanding of how GPCRs mediate cell-to-cell, or host-pathogen, communication and in turn regulate virulence traits (i.e. biofilm formation, host penetration, virulence factor secretion) may lead to additional GPCR-interfering fungal disease control strategies.

In turn, this project will have an impact on a range of beneficiaries including academics and industries related to the agricultural and pharmaceutical sectors, enhancing the bioeconomy. These beneficiaries and governmental policy makers will assist in the transfer of this new fundamental knowledge into practical solutions for social issues, such as food security, agricultural and forestry sustainability, and fungal infections of humans and farmed animals, contributing to the creation of a sustainable urban and rural society.