Defining the signalling network linking pathogen infection and asparagine accumulation in wheat grain

Lead Research Organisation: Rothamsted Research
Department Name: Sustainable Soils and Crops

Abstract

This project arises from discoveries that an amino acid called asparagine accumulates in wheat grain in response to disease and that the plant's response to floral infection by a disease-causing fungus called Fusarium graminearum (Fg) involves a protein called SnRK1. SnRK1 is a master regulator of plant metabolism and it controls the activity of genes encoding an enzyme called asparagine synthetase that is responsible for making asparagine. The project will involve a multidisciplinary team from Rothamsted Research, with collaboration from a team from University College Dublin (not eligible for BBSRC funding but fully involved in the project through the sharing of resources, expertise and data analyses). It will define what we are calling a signalling hub (a control point within a network) involving SnRK1 and partner proteins that links pathogen infection with asparagine synthesis and accumulation in wheat grain. We believe that the increase in asparagine concentration induced through the activation of this hub upon Fg infection is an important part of how plants defend themselves when under attack from disease-causing organisms.

Fg causes Fusarium head blight disease, which reduces yield and grain quality, and contaminates grain with toxic compounds called mycotoxins, of which the most common is called deoxynivalenol (DON). SnRK1 is involved in the regulation of defence mechanisms when wheat is infected by Fg and a protein that partners with SnRK1, called TaFROG, has also been shown to contribute to Fg and DON resistance. Recent work has shown that Fg infection and DON treatment both affect SnRK1 but in different ways, with Fg infection causing the SnRK1 protein to be divided into smaller proteins in a way not seen with DON on its own. Subsequently, a protein that is secreted by the fungus, called OSP24, has been shown to partner with SnRK1 and to cause SnRK1 to be broken down. TaFROG, on the other hand, competes with OSP24 for partnering with SnRK1 and protects SnRK1 from degradation. These fascinating discoveries mean that this project can focus directly on the signalling hub and its relationships to other proteins in SnRK1's wider network. That network likely includes several proteins called bZIP transcription factors. These proteins control the activity of some target genes, possibly including asparagine synthetase genes involved in asparagine synthesis, and have characteristics suggesting that they could be controlled by SnRK1.

We aim to identify all the components of this signalling hub linking Fg infection with asparagine accumulation in wheat grain. We will dissect the hub in different types of wheat using different strains of Fg, such as strains that do not make DON and/or the OSP24 protein. We will use a technique called RNA-seq that will enable us to identify all of the genes affected by infection by Fg or treatment with DON, looking in particular for those that could be involved in making or breaking down asparagine. We will use protein-based studies to identify additional hub components, and find out if the bZIP transcription factors we are interested in do control the activity of asparagine synthetase genes. Finally, we will see if the ability of different Fg strains to cause disease is linked to their effect on the signalling hub and asparagine. These experiments will enable us to model the signalling hub and perform further experiments to target the genes involved in the hub so that we can test whether our model is correct.

SnRK1 has been implicated in other plant defence mechanisms, including those against herbivores, viruses and bacteria, as well as other fungi. In addition, the amount of asparagine in wheat grain has implications for food safety because asparagine can be converted into a cancer-causing contaminant called acrylamide during baking. This means that the project, while focussed on basic science, will have potential impact for a range of stakeholders in the agrifood sector.

Technical Summary

This project arises from discoveries that free asparagine (Asn) accumulates in wheat grain in response to pathogenic fungi, and that responses invoked by Fusarium graminearum (Fg) and its mycotoxin, DON, involve the protein kinase, SnRK1, a regulator of plant metabolism that controls Asn synthetase gene expression. Another protein, TaFROG, interacts with SnRK1, contributing to Fg/DON resistance. SnRK1 becomes more active in response to DON treatment, and wheat becomes more sensitive to DON when SnRK1 is silenced. Fg infection (but not DON on its own) causes a loss of SnRK1 protein integrity. A fungal secreted protein, OSP24, promotes degradation of SnRK1, with TaFROG protecting SnRK1 by competing with OSP24 for binding. Furthermore, several bZIP transcription factors (TFs) are potential targets for SnRK1, and Asn synthetase gene promoters contain bZIP binding sites.

This project will define this signalling hub linking Fg infection with Asn synthesis. It will identify hub components, separate the effects of Fg and DON on the hub, and characterise the hub's targets and interactions with wider signalling systems. It will identify SnRK1 breakdown proteins induced by Fg, use RNA-seq, phosphoproteomics and TurboID to identify additional hub components and investigate the interaction of different TFs with Asn synthetase genes. The ability of Fg to cause disease will be linked to its interactions with the hub, and a model of how the hub works will be produced and validated by knocking components out using VIGS.

The project will involve teams from Rothamsted and University College Dublin. It will test the hypothesis that the increase in Asn concentration in wheat grain upon Fg infection is part of the plant's defence against the disease. It will also enable us to assess the potential for targeting the hub for Fg control and the prevention of Asn accumulation. This has food safety implications because Asn can be converted to the carcinogen, acrylamide, during baking.

Publications

10 25 50
 
Description Application of microfluidics chip analyses to Fusarium graminearum research
Amount £5,000 (GBP)
Organisation British Society of Plant Pathoogy 
Sector Learned Society
Country United Kingdom
Start 08/2023 
End 09/2024
 
Description DFW - Designing Future Wheat - Work package 2 (WP2) - Added value and resilience
Amount £7,068,842 (GBP)
Funding ID BBS/E/C/000I0250 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2017 
End 03/2023
 
Description Delivering Sustainable Wheat: Delivering Resilience to Biotic Stress (Rothamsted Research)
Amount £575,550 (GBP)
Funding ID BBS/E/RH/230001B 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2023 
End 03/2028
 
Description X9 High-Throughput Genomics System for new and versatile research capabilities
Amount £158,270 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2023 
End 12/2023
 
Title Identification and functional characterisation of a locus for target site integration in Fusarium graminearum 
Description Background: Fusarium Head Blight (FHB) is a destructive floral disease of different cereal crops. The Ascomycete fungus Fusarium graminearum (Fg) is one of the main causal agents of FHB in wheat and barley. The role(s) in virulence of Fg genes include genetic studies that involve the transformation of the fungus with different expression cassettes. We have observed in several studies where Fg genes functions were characterised that integration of expression cassettes occurred randomly. Random insertion of a cassette may disrupt gene expression and/or protein functions and hence the overall conclusion of the study. Target site integration (TSI) is an approach that consists of identifying a chromosomal region where the cassette can be inserted. The identification of a suitable locus for TSI in Fg would avert the potential risks of ectopic integration. Results: Here, we identified a highly conserved intergenic region on chromosome 1 suitable for TSI. We named this intergenic region TSI locus 1. We developed an efficient cloning vector system based on the Golden Gate method to clone different expression cassettes for use in combination with TSI locus 1. We present evidence that integrations in the TSI locus 1 affects neither fungal virulence nor fungal growth under different stress conditions. Integrations at the TSI locus 1 resulted in the expression of different gene fusions. In addition, the activities of Fg native promoters were not altered by integration into the TSI locus 1. We have developed a bespoke bioinformatic pipeline to analyse the existence of ectopic integrations, cassette truncations and tandem insertions of the cassette that may occurred during the transformation process. Finally, we established a protocol to study protein secretion in wheat coleoptiles using confocal microscopy and the TSI locus 1. Conclusion: The TSI locus 1 can be used in Fg and potentially other cereal infecting Fusarium species for diverse studies including promoter activity analysis, protein secretion, protein localisation studies and gene complementation. The bespoke bioinformatic pipeline developed in this work together with PCR amplification of the insert could be an alternative to Southern blotting, the gold standard technique used to identify ectopic integrations, cassette truncations and tandem insertions in fungal transformation. 
Type Of Material Technology assay or reagent 
Year Produced 2024 
Provided To Others? Yes  
Impact The method has been used in different projects of the lab to complement knock-out fungal strains and to express recombinant proteins in the fungus. 
URL https://fungalbiolbiotech.biomedcentral.com/articles/10.1186/s40694-024-00171-8
 
Title Identification of a locus for target site integration in Fusarium graminearum 
Description We developed a vector system for target site integration (TSI) in an intergenic chromosomic region of the Fg genome. Insertion of the expression cassette in this chromosomic region (TSI locus1) does not alter either fungal growth of fungal virulence. Integrations in the TSI locus1 allow the expression of different genes fusions and activities of virulence specific promoters were not altered by integration into the TSI locus 1. Complementation tests were successfully done on three test genes. Finally, we established a protocol to study protein secretion in wheat coleoptiles using confocal microscopy and the TSI locus1 for stable expression of different gene fusions. 
Type Of Material Technology assay or reagent 
Year Produced 2023 
Provided To Others? Yes  
Impact The TSI locus1 can be used for diverse studies including promoter activity analysis, secretion and gene complementation and localisation studies. Therefore, the vector system has already been used in multiple ongoing BBSRC funded research projects in the laboratory. A PhD student and a postdoctoral researcher have successfully used the vector system for gene complementation studies. The vector system will also be used to study protein secretion in a new collaborative project with US researchers. The project aims to identify fungal secreted proteinases that can be used to engineer fungal resistance in wheat. The new vector system is available from ADDGene. 
URL https://doi.org/10.1186/s40694-024-00171-8
 
Title Additional file 17: of Inter-genome comparison of the Quorn fungus Fusarium venenatum and the closely related plant infecting pathogen Fusarium graminearum 
Description Fusarium venenatum presence of TRI6 Fusarium greaminearum binding sites predicted by Nasmith et al. [39]. Fusarium venenatum BLASTP alignment percentages were added to identify presence or absence. (XLS 61Â kb) 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Placing the chromosome scale, fully annotated F. venenatum genome in the public domain has increased the power of comparative genomics for cereal infecting Fusarium species. 
URL https://springernature.figshare.com/articles/Additional_file_17_of_Inter-genome_comparison_of_the_Qu...
 
Description Collaboration with University College Dublin 
Organisation University College Dublin
Country Ireland 
Sector Academic/University 
PI Contribution We are using wheat genotypes and VIGS constructs supplied by UCD in the research.
Collaborator Contribution Professor Fiona Doohan of UCD is a collaborator in the project and has supplied us with wheat genotypes and VIGS constructs. This contribution has been extremely important in enabling us to meet the objectives of the project.
Impact Still ongoing
Start Year 2022
 
Description Fusarium graminearum effector characterisation using global pangenome analyses 
Organisation University of Bath
Department Department of Biology and Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We designed this fungal effector PhD project based on soon to be published Fusarium graminearum pangenome analyses arising from an ongoing now unfunded collaboration with EMBRAPA in Brazil. This included detailed bioinformatics analyses and wet biology verification of the in planta destination location of small secreted candidate effector proteins.
Collaborator Contribution The two partners at the University of Bath will provide specialist transcriptome analyses (Dr Neil Brown) and genome analyses (Dr Hans -Wilhelm Nützmann to this PhD project. In 2023 , Hans -Wilhelm Nützmann moved to The University of Exeter. This collaboration continues with Drs Neil Brown and Hans -Wilhelm Nützmann.
Impact Hiring of the 4 year PhD student Jade Smith
Start Year 2022
 
Description Proximity Labelling methodology - The Sainsbury Lab, Norwich 
Organisation John Innes Centre
Department The Sainsbury Laboratory
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Described and discussed this project goals with this collaborator. Explored the proximity labelling methods developed at TSL Norwich in the group of Professor Jonathan Jones for proximity labelling in Arabidopsis and Nicotiana benthamiana. Methods piloted at Rothamsted and additional positive and negative constructs included in the experimental design Collaboration still on going
Collaborator Contribution Receive two types of detailed non-published proximity labelling protocols for Arabidopsis and Nicotiana benthamiana. .
Impact Still in method development phase at Rothamsted
Start Year 2023
 
Description CRISPR and Plant Genome Editing III, Vienna, Austria, July 8th - 9th 2022, poster and selected speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Dr Navneet Kaur gave oral and poster presentations at the conference
Year(s) Of Engagement Activity 2022
 
Description New Scientist Live Excel London - Oct 2022 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact The Fusarium team in collaboration with the PHI-base, Knetminer, EMBL-EBI, the BSPP and the Wellcome Connecting Science teams devised and presented a large display at New Scientist Live (ExCel London, October 2022), providing posters, hands-on interactive activities, and career advice on the topics of disease and mycotoxin control, DNA extraction, genomics, biocuration, and network analysis. On display table No1 were wheat plants infected with Fusarium head blight, infected and non-infected grains, the chemical structure of the DON mycotoxin as well as wheat plants infected with the Take-all fungus, petri dishes with fungal cultures and a binocular microscope to aid detailed viewing of infected plant material and /or the fungus. On display table 6 was a newly devised interactive game that allowed the visitor to learn about current disease control strategies and future NextGen options based around genomics, functional genomics and/or effector biology for ten globally important arable crop, horticultural and animal husbandry disease problems. FHB disease of wheat was one of the disease problems that could be selected. In total, 21,500 visitors attended the event (one day for school-age children and two days for the general public), and ~1,000 visitors explored our display, with ~20% staying for 1-2 hours. We had a diverse team consisting of post-docs, PhD students and undergraduates across six nationalities. Include in the display team for all 3 days from this project were Martin Urban and Kim Hammond-Kosack.
Year(s) Of Engagement Activity 2022
URL https://live.newscientist.com/
 
Description Presentation at Biennial AAB Presidential Look-Forward: Nature-based and engineered biology solutions to climate mitigation, 1-2nd Nov, 2022, Rothamsted Research, UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Dr. Navneet Kaur presented a poster.
Year(s) Of Engagement Activity 2022
URL https://www.aab.org.uk/event/biennial-aab-presidential-look-forward-nature-based-and-engineered-biol...
 
Description Presentation at International Conference on Food and Nutritional Security (iFANS-2023), January 2023, Mohali, India, invited speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Dr Navneet Kaur delivered an oral presentation at this conference.
Year(s) Of Engagement Activity 2023
 
Description Presentation at UK Flour Millers mycotoxins workshop, December 2022 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Professor Halford gave an oral presentation on the project at this workshop
Year(s) Of Engagement Activity 2022
 
Description participated in AAB-PlantEd Training School: Communicating the Science of Gene-Edited Crops, 28 - 29 April 2022, Rothamsted Research, UK 
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 In this training/workshop, how to communicate science to the public was discussed and practiced.
Year(s) Of Engagement Activity 2022
URL https://www.aab.org.uk/event/aab-planted-training-school-communicating-the-science-of-gene-edited-cr...