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

Lead Research Organisation: Rothamsted Research
Department Name: Biointeractions and Crop Protection

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Related Projects

Project Reference Relationship Related To Start End Award Value
BB/N011686/1 31/03/2016 31/12/2017 £297,804
BB/N011686/2 Transfer BB/N011686/1 02/01/2018 01/12/2019 £157,031
 
Title Fusarium Postcards 
Description A collection of postcard highlighting the importance of Fusarium and the research on-going at Rothamsted were prepared for the Fusarium one-day event held at Rothamsted in July 2016. These postcards were disseminated to the wider public and key stakeholders through this event and follow-on activities. 
Type Of Art Artwork 
Year Produced 2016 
Impact No known measurable impacts. 
 
Title How to control Fusarium cartoon 
Description A Fusarium cartoon was produced that portrayed the risk of Fusarium-borne diseases to cereals and the problems associated with current control strategies. The cartoon progresses to explain new GM and non-GM mediated approaches to stop Fusarium. In addition, the cartoon depicts the mechanism behind how host-induced gene silencing can be used to combat fungal disease, in a simplified manner assessable to the general public. 
Type Of Art Artwork 
Year Produced 2016 
Impact This cartoon has now been translated into Portuguese and is to be used by Embrapa-Trigo at a national agricultural show. 
URL http://www.rothamsted.ac.uk/system/files/How%20to%20sustainably%20control%20Fusarium.pdf
 
Description 1) A transcriptomic investigation of Fusarium Head Blight on wheat showed how the fungal pathogen, Fusarium graminearum, regulated the expression of its genes to firstly establish symptomless colonisation, and then to promote destructive symptomatic infection. This revealed that the genes involved in the production of the fungal toxin (deoxynivalenol), and other metabolites, where turned on during symptomless infection, to inhibit plant defences. Then later, hundreds of fungal enzymes were secreted during symptomatic infection to promote plant cells death and the acquisition of nutrients from the host plant. This showed that the fungal pathogen coordinates virulence strategies during wheat infection, pointing to the importance of fungal environment sensing within the host plant. This work was published in Molecular Plant Pathology 2017 (https://bsppjournals.onlinelibrary.wiley.com/doi/full/10.1111/mpp.12564).

2) A comparative bioinformatic analysis of G-protein coupled receptors (GPCRs) in fungal pathogens of humans or plants defined ten distinct classes of GPCRs in fungi, while also revealing the expansion of class X receptors in plant pathogens, pointing to their importance in promoting plant diseases including Fusarium Head Blight. This study was published in Nature Microbiology 2018 (https://www.nature.com/articles/s41564-018-0127-5). This work identified the F. graminearum genome to encode 123 GPCRs. Numerous class X receptors were found to be more highly expressed in F. graminearum during wheat infection, again implicating their involvement in promoting disease.

3) A fungal genetics study of F. graminearum generated a collection of fungal mutants lacking individual GPCRs. Class X GPCRs were identified to contribute to Fusarium Head Blight disease on wheat, promoting symptomless infection through their regulation of fungal membranes, mycotoxins and secreted proteins. Disruption of GPCR host sensing activated an enhanced wheat defensive response to infection. This amounts to increased plant cell wall biosynthesis, resulting in apoplastic and vascular occlusions that impede the progression of symptomless infection. These non-classical class X GPCRs were confirmed to be bona fide G-protein interactors, and specific receptors domains were required for virulence. This work was published in Plos Pathogens 2019 (https://journals.plos.org/plospathogens/article?id=10.1371%2Fjournal.ppat.1007666).

4) A collaboration with the University of Sao Paulo, Brazil, was forged during this fellowship on fungal GPCRs from a comparable system, namely the food spoilage mould and toxin producer Aspergillus nidulans. This study showed how fungal class V GPCRs, incorporated the sensing of sugars and light in the regulation of sexual development and toxin production. This work was published in Plos Genetics 2019 (https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1008419).

5) New technologies were required to permit the study of fungal GPCRs and the environmental signals they detect. Two such approaches were successfully developed. Fungal GPCRs were produced in Xenopus oocytes to permit electrophysiology experiments aimed at identifying signals that interact with fungal GPCRs. Additionally, engineered fungal GPCRs were produced to permit their purification in lipid nanoparticles. This preserves the native membrane surrounding the fungal GPCR and therefore permits the assessment of its true membrane-bound structure. This permits the development of studies to interrogate the dynamics of fungal GPCRs structures and how they change when they interact with their signals they detect.
Exploitation Route 1) The transcriptomic study provided novel insights into the combined spatial temporal coordination of functionally characterized and hypothesized virulence strategies, which is a valuable resource for the Fusarium community, facilitating the selection of fungal gene targets for functional characterisation. It also represents a comparative resource to investigate the regulation of fungal genes, from other non-Fusarium species, during pathogenic interactions.

2) The comparative analysis of fungal G-protein coupled receptors (GPCRs) set out a framework for the characterisation of distinct classes of GPCRs in fungi from different ecological environments. This will facilitate the development of improved understandings of fungal GPCR evolution and function.

3) The Fusarium graminearum GPCR study showed that fungal GPCR signalling is important for the establishment of Fusarium Head Blight disease on wheat. The discovery of fungal GPCRs and specific extracellular domains that influence sterol membrane and mycotoxin biosynthesis, while contributing to virulence, opens new avenues for biotechnology to minimise diseases in crop species.

4) The Aspergillus nidulans collaboration revealed how fungal GPCRs regulate sexual development and toxin production. Inhibition of fungal GPCRs can therefore impact on the fungal sex, genetic recombination and spore dispersal. Impeding these traits would impede the evolution and spread of problematic antifungal-resistant pathogens. Therefore, this study provides new avenues to protect the efficacy of our existing antifungal compounds. Similarly, the discover of GPCRs which regulate toxin production provides new avenues to develop approaches to combat fungal toxin contamination in human food supply chain.

5) The developed approaches will facilitate the discover of the environmental signals fungal GPCRs detect, and their visualisation in lipid nanoparticles. This will reveal how dynamic GPCR structures change to modulate their function and to regulate fungal biology. This knowledge is paramount for the development of any new GPCR-targeting compounds that could help combat the spread of fungal diseases and toxin contamination related issues.
Sectors Agriculture, Food and Drink,Environment,Pharmaceuticals and Medical Biotechnology

URL https://journals.plos.org/plospathogens/article?id=10.1371%2Fjournal.ppat.1007666
 
Description Dynamic fungal membranes: regulators of receptor function, sex & disease
Amount £20,000 (GBP)
Funding ID RGS\R2\202128 
Organisation University of Bath 
Sector Academic/University
Country United Kingdom
Start 09/2020 
End 09/2021
 
Description FAPESP - Postdoctoral Research Internships Abroad (BEPE) program
Amount $21,776 (USD)
Organisation São Paulo Research Foundation (FAPESP) 
Sector Public
Country Brazil
Start 06/2017 
End 12/2017
 
Description Global challenges research fund - scientist exchange
Amount £3,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2017 
End 03/2017
 
Description One year university scholarship
Amount £18,000 (GBP)
Organisation Beijing Academy of Agriculture and Forestry Sciences 
Sector Academic/University
Country China
Start 02/2017 
End 02/2018
 
Description Research England GCRF (University of Bath): New tools to uncover novel regulators of fungal sex, disease & toxin contamination
Amount £31,985 (GBP)
Organisation University of Bath 
Sector Academic/University
Country United Kingdom
Start 12/2018 
End 07/2019
 
Description University of Bath - FAPESP SPRINT award: Fungal receptor-mediated glucose sensing regulates light-dependent development and toxin production
Amount £14,320 (GBP)
Organisation University of Bath 
Sector Academic/University
Country United Kingdom
Start 02/2019 
End 02/2021
 
Description University of Bath URSA PhD studentship: Nutritional immunity: The importance of micronutrient acquisition to fungal disease and toxin contamination in plants
Amount £55,220 (GBP)
Organisation University of Bath 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 09/2022
 
Title Fusarium G-protein coupled receptor mutant collection 
Description A collection of Fusarium graminearum strains lacking individual G-protein coupled receptors (GPCRs) has been generated, including multiple independent single gene deletion strains for 6 classical and 19 non-classical GPCRs. 
Type Of Material Biological samples 
Provided To Others? No  
Impact The biological resource has permitted the identification of Fusarium graminearum receptors involved in the establishment of disease on wheat plants. 
 
Title Yeast split ubiquitin membrane protein interaction assay 
Description The establishment of the use of the heterologous yeast split ubiquitin assay to identify membrane protein interactions for the study of Fusarium graminearum G-protein coupled receptors and their interacting G-proteins. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact The establishment of this tool successfully showed a non-canonical G-protein coupled receptor in Fusarium graminearum interacts at the cell membrane with intracellular G-proteins. This success was incopporated within a manuscript accepted for publication in Plos Pathogens 2019. 
 
Title FgMutantDB 
Description FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Through the use of FgMutantDB missing annotations were feedback into larger multispecies fungal genomic databases including, FungiDB, Ensembl and PHI-base. 
URL https://www.sciencedirect.com/science/article/pii/S1087184518300021
 
Title Fusarium graminearum genome-wide transcriptional study of symptomless and symptomatic wheat infection 
Description A Fusarium graminearum genome-wide transcriptional study of symptomless and symptomatic wheat infection revealed the spatial temporal coordination of characterised and putative virulence strategies during the establishment of wheat infection. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact The Fusarium graminearum symptomless wheat infection transcriptomic dataset permitted the refined selection of fungal G-protein coupled receptors (GPCRs) for biological characterisation via the generation of fungal strains lacking individual GPCRs. This facilitated the discover of fungal receptors involved in the establishment of wheat infection. 
 
Description Comparative study of fungal G-protein coupled receptors 
Organisation Flanders Institute for Biotechnology
Country Belgium 
Sector Charity/Non Profit 
PI Contribution This is a collaboration between three research team in the UK, Brazil and Belgium, studying the function and mechanism of fungal G-protein coupled receptors (GPCRs) in different fungal pathogens, namely Candida, Aspergillus and Fusarium species. Neil Brown proposed, designed and undertook the preparation of an analytical review of GPCRs in fungal pathogens. This comparative review has been invited for submission to Nature Microbiology. Neil Brown is first and corresponding author on this research.
Collaborator Contribution Prof. P. van Dijck (VIB-KU Leuven Center for Microbiology) has focused on the review of Candida species. Prof. G. H. Goldman (University of Sao Paulo-Riberiao Preto) has focused on the review of Aspergillus species.
Impact This collaboration has resulted in a successful application for a Global Challenges Research fund grant for a Scientist Exchange, to bring Prof. Goldman (University of Sao Paulo) to Rothamsted in March 2017.
Start Year 2016
 
Description Comparative study of fungal G-protein coupled receptors 
Organisation Universidade de São Paulo
Country Brazil 
Sector Academic/University 
PI Contribution This is a collaboration between three research team in the UK, Brazil and Belgium, studying the function and mechanism of fungal G-protein coupled receptors (GPCRs) in different fungal pathogens, namely Candida, Aspergillus and Fusarium species. Neil Brown proposed, designed and undertook the preparation of an analytical review of GPCRs in fungal pathogens. This comparative review has been invited for submission to Nature Microbiology. Neil Brown is first and corresponding author on this research.
Collaborator Contribution Prof. P. van Dijck (VIB-KU Leuven Center for Microbiology) has focused on the review of Candida species. Prof. G. H. Goldman (University of Sao Paulo-Riberiao Preto) has focused on the review of Aspergillus species.
Impact This collaboration has resulted in a successful application for a Global Challenges Research fund grant for a Scientist Exchange, to bring Prof. Goldman (University of Sao Paulo) to Rothamsted in March 2017.
Start Year 2016
 
Description Developing a multidisciplinary approach to assess the role of extracellular receptors in mycotoxigenic fungi 
Organisation Rothamsted Research
Department Rothamsted Insect Survey
Country United Kingdom 
Sector Private 
PI Contribution Neil Brown developed the concept behind this multidisciplinary collaboration to evaluate G-protein coupled receptor (GPCR)-ligand interactions in multiple mycotoxigenic fungi, namely Aspergillus and Fusarium species. Neil Brown identified putative Aspergillus and Fusarium GPCRs important to each species fungal biology and/or pathogenicity. Now Neil Brown is developing molecular tools to further study GPCR-mediated signalling.
Collaborator Contribution Prof. Goldman (University of Sao Paulo-Ribeirao Preto) characterised GPCRs in Aspergillus species. Also Prof. Goldman developed the split-ubiquitin assay for studying Aspergillus GPCR-G-protein interactions, which will now be used by Neil Brown to study Fusarium receptors. Dr Jing-Jiang Zhou (Rothamsted Research, BCCP) developed the Xenopus oocyte voltage-clamp method for the assessment of insect GPCR-ligand interactions.
Impact This collaborations has resulted in the application for two grants for visiting workers to come to Rothamsted to perform the GPCR-ligand interactions studies using the Xenopus voltage clamp method. This included the successful application for a one-year scholarship from the Beijing Forestry University (awarded £18000) focusing on Fusarium receptors, and a FAPESP - Research Postdoctoral Internships Abroad (BEPE) program (applied for $21776) focusing on Aspergillus receptors.
Start Year 2016
 
Description Developing a multidisciplinary approach to assess the role of extracellular receptors in mycotoxigenic fungi 
Organisation Universidade de São Paulo
Country Brazil 
Sector Academic/University 
PI Contribution Neil Brown developed the concept behind this multidisciplinary collaboration to evaluate G-protein coupled receptor (GPCR)-ligand interactions in multiple mycotoxigenic fungi, namely Aspergillus and Fusarium species. Neil Brown identified putative Aspergillus and Fusarium GPCRs important to each species fungal biology and/or pathogenicity. Now Neil Brown is developing molecular tools to further study GPCR-mediated signalling.
Collaborator Contribution Prof. Goldman (University of Sao Paulo-Ribeirao Preto) characterised GPCRs in Aspergillus species. Also Prof. Goldman developed the split-ubiquitin assay for studying Aspergillus GPCR-G-protein interactions, which will now be used by Neil Brown to study Fusarium receptors. Dr Jing-Jiang Zhou (Rothamsted Research, BCCP) developed the Xenopus oocyte voltage-clamp method for the assessment of insect GPCR-ligand interactions.
Impact This collaborations has resulted in the application for two grants for visiting workers to come to Rothamsted to perform the GPCR-ligand interactions studies using the Xenopus voltage clamp method. This included the successful application for a one-year scholarship from the Beijing Forestry University (awarded £18000) focusing on Fusarium receptors, and a FAPESP - Research Postdoctoral Internships Abroad (BEPE) program (applied for $21776) focusing on Aspergillus receptors.
Start Year 2016
 
Description Dynamic fungal membranes: regulators of receptor function, sex & disease 
Organisation University of Bath
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We are adapting newly developed biochemical approaches from other systems to identify novel protein and lipid interactions with fungal sex receptors in their native fungal membrane environment. Our contribution is to engineer fungal receptors for purification in fungal membrane lipid nanoparticles, and performing downstream biophysical analyses.
Collaborator Contribution Our collaborator in the Department of Chemistry (Prof. K. Edler) is providing expertise and training in the use of lipid nanoparticle to isolate fungal receptors in native fungal membrane nanodiscs, to facilitate biophysical studies.
Impact This collaboration resulted in the successful application for a Royal Society Research grant (£20K) awarded to Dr N. Brown (Sept. 2020-21).
Start Year 2020
 
Description Fungal receptors regulate development and toxin production 
Organisation Universidade de São Paulo
Department Ribeirão Preto School of Medicine
Country Brazil 
Sector Academic/University 
PI Contribution This is a FAPESP-Bath Sprint award. This enabled scientist exchanges between the two institutes, Dr Brown (Bath) and Miss Johns (Bath PhD student) visited the USP-Ribeirao Preto in 2019. There they performed fungal transformation experiments, which yield newly engineered fungal strains that will now be used in follow-on collaborative studies looking at how fungal receptors interact with other proteins in the cell membrane. Miss Johns learned new skills in fungal transformation, which Dr Brown and Miss Johns used during a taught MSc Research practical at the University of Bath in 2019-20 on molecular fungal biology. Dr Brown collaborated in the finalisation of a research manuscript on fungal receptors published in 2019.
Collaborator Contribution Prof. Goldman (USP) visited Bath in 2019 where he participated in the teaching of the MSc Research practical, giving guest lectures on the impact of fungal disease on human and ecosystem health. Prof. Goldman also contributed plasmids and fungal strains used in the MSc practical. Prof. Godman and his research team collaborated in the finalisation of a research manuscript on fungal receptors published in 2019.
Impact dos Reis T et al. Brown NA*. 2019. GPCR-mediated glucose sensing system regulates light-dependent fungal development and mycotoxin production. Plos Genetics. 15(10): e1008419.
Start Year 2019
 
Description New tools to uncover novel regulators of fungal sex, disease & toxin contamination 
Organisation Universidade de São Paulo
Department Ribeirão Preto School of Medicine
Country Brazil 
Sector Academic/University 
PI Contribution We will establish a new technology to identify transient membrane protein interactions that regulate GPCR activation state, which is vital to understanding novel GPCR-mediated signalling mechanism. We will generate the construct to use the PUP-IT (pupylation based interaction tagging) system to identify GPCR interactors. We will visit our collaborators at the University of Sao Paulo - Ribeirao Preto (Prof. Goldman) to transform these constructs into Aspergillus nidulans. Our collaborators will use the resulting transformed A. nidulans strains to purify and identify proteins that interact with the GPCR of interest. Collectively, we will analysis the mass spectrometry data to select GPCR-interactors for further study.
Collaborator Contribution Our collaborators at the University of Sao Paulo - Ribeirao Preto (Prof. Goldman) will use A. nidulans strains harbouring the PUP-IT constructs to purify and identify proteins that interact with the GPCR of interest by mass spectrometry.
Impact This new collaboration funded by Research England (GCRF) has already resulted in the successful application for University of Bath - FAPESP SPRINT award entitled: Fungal receptor-mediated glucose sensing regulates light-dependent development and toxin production. This is a two year travel award (£14320) to facilitate the continuation of this successful collaboration, through research and the delivery of higher postgraduate education.
Start Year 2019
 
Description Understanding dynamic fungal receptor structures vital to Fusarium Head Blight on wheat 
Organisation Philipp University of Marburg
Country Germany 
Sector Academic/University 
PI Contribution Our contribution has been to engineer fungal receptors with modified residues to assess the importance of key intramolecule interactions to receptor function and Fusarium Head Blight of wheat.
Collaborator Contribution Our collaborator (Prof. L-O Essen, Marburg, Germany) has used heterologous expression of receptor domains and two-step purification to yield soluble proteins for crystallography studies. These have yielded the first crystal structures for unique extracellular fungal receptor domains.
Impact This collaboration enabled the development of multidisciplinary research that used structural biology to identify leads for functional validation studies using fungal genetics.
Start Year 2020
 
Description Blog - A day in the life of Neil Brown 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact This "A day in the life of Neil Brown" explained what I do and how I ended up being a pmolecular plant pathologist. This was associated with the Reddit ask me anything session "Targeting a crop pathogen's sense of touch and taste". This highlighted to the general public, undergraduates etc what a research career is like and the importance of crop science research.
Year(s) Of Engagement Activity 2016
URL https://www.rothamsted.ac.uk/articles/day-life-dr-neil-brown
 
Description Cereals 2016 (Agricultural show in Royston, Cambridgeshire) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact A Fusarium Head Blight exhibit was situated at the Rothamsted Research stand (Cereals 2016). This exhibit portrayed the impact of Fusarium on wheat production and the associated risk of mycotoxin contamination. It highlighted the need for new approaches to tackle this hazardous fungal disease. During the two day event, knowledge of the approaches taken at Rothamsted, including those within the associated fellowship, were described to farmers, agronomists, the press and industry. This exhibit commonly promoted the discussion of the use of GM and non-GM mediated approaches to control fungal diseases.

As a result of a discussion at this event, Neil Brown was invited to present this research at an International workshop on "Innovative Crop Protection for 21st Century Food Security" at the Weizmann Institute in December 2016. The presentation of this research at the workshop subsequently resulted in the invited submission of a perspective on novel approaches to fight Fusarium head blight and mycotoxin contamination in cereals, which will be published in Pest Management Science.
Year(s) Of Engagement Activity 2016
 
Description Healthy crops, healthy food: How do we stop microscopic fungi affecting crop yields and grain quality? 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This one day Fusarium even, hosted at Rothamsted Research (July 2016), educated the general public and interested stakeholders in the impact of Fusarium-borne diseases and the associated risk of mycotoxin contamination. It highlighted deficiencies is current approaches to prevent Fusarium-borne diseases and the need for new approaches to tackle this hazardous fungal disease. During this event, knowledge of the approaches taken at Rothamsted, including those within the associated fellowship, were described to the general public, farmers and agronomists. This exhibit commonly promoted the discussion of the use of GM and non-GM mediated approaches to control fungal diseases, and also described the background behind the use of host-induced gene silencing as a GM approach to fight fungal disease.
Year(s) Of Engagement Activity 2016
URL http://www.rothamsted.ac.uk/events/healthy-crops-health-food-how-do-we-stop-microscopic-fungi-destro...
 
Description Invited Speaker at Talent and Collaborations in Agri-Tech, Rothamsted Research, UK, December 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact The objective of this workshop was to present the range of career opportunities to PhD students, primarily through a careers discussion panel for which I represented the academic career path. I provided insights into how I successfully made the transition from PhD student to University lecturer and Principle Investigator. Member of the audience were motivated to subsequently apply for postdoctoral fellowship.
Year(s) Of Engagement Activity 2018
 
Description Invited keynote speaker International Symposium on Fusarium Head Blight, Canada 2020 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This invitation to be the Keynote speaker on future technologies in controlling Fusarium Head Blight will increase the awareness in the research community and industry of how we are developing fungal receptors and other membrane proteins as novel antifungal drug targets.
Year(s) Of Engagement Activity 2020
URL https://www.isfhb.com/
 
Description Invited speaker at British Council Food Security for Vulnerable Populations - The Fungal Threat: Food Security Workshop, Ribeirao-Preto, Brazil, September 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was a international workshop that brought together early career scientists and postdoctoral scientists from the UK and Brazil. It was organised by senior scientists from the area of fungal diseases and food security. The objective of this workshop was to forge new ideas and collaborations. As a result of speaking and participating in this workshop two successful seed research funding (Research England: £32K) and travel grants (University of Bath - FAPESP SPRINT: £14K) were awarded to promote a new collaboration between the Brown lab (University of Bath) and the Goldman lab (University of Sao Paulo - Ribeirao Preto).
Year(s) Of Engagement Activity 2018
 
Description Invited speaker at the Gordon Research Conference: Cellular and molecular fungal biology, 2020 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This invited presentation at the Gordon Research Conference was in the "Sensing, signalling and development" which will increase the awareness of the importance of fungal receptors to sensing the environment and regulating virulence and sexual development. This will highlight the need for more researchers within the community to participate in developing novel fungal proteins as new antifungal drug targets.
Year(s) Of Engagement Activity 2020
URL https://www.grc.org/cellular-and-molecular-fungal-biology-conference/2020/
 
Description Nature Community Blog - Beer, sex and antifungal drugs 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This Nature community Blog entitle "Beer, sex and antifungal drugs" was used to increase the exposure of the related research articles on fungal receptors from our group and others. This highlighted to the research community the potential for fungal receptors to be new targets for the development of antifungal drugs, but noted gaps in our knowledge that need to be addressed.
Year(s) Of Engagement Activity 2018
URL https://naturemicrobiologycommunity.nature.com/users/85459-neil-brown/posts/31692-beer-sex-and-antif...
 
Description Reddit Ask me anything: Targeting a crop pathogen's sense of touch and taste 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact In this Reddit Ask me anything session entitled "Targeting a crop pathogen's sense of touch and taste" I discussed issues related to crop diseases and the control of fungal pathogens with the general public resulting in a greater appreciation of the problem we confront in trying to stop fungal borne disease in crops.
Year(s) Of Engagement Activity 2016
URL https://www.reddit.com/r/science/comments/56rjb2/science_ama_series_neil_brown_here_to_talk_about/
 
Description US Wheat and Barley Scab Initiative - FHB forum 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited to give a seminar to the US Wheat and Barley Scab Initiative - FHB forum 2017, which consisted of around 200 academics and industrial partners, primarily from the USA and Canada, but also Europe. The purpose of the visit was to interact with the International Fusarium research community. This highlighted our published research on Fusarium transcriptomics, while advertising our on-going research on Fusarium G-protein coupled receptors. Interactions with a researcher at the USDA resulted in a joint publication on the development of a new bioinformatics resource for the Fusarium research community.
Year(s) Of Engagement Activity 2017
URL https://scabusa.org/pdfs/nfhbf17_program_web.pdf