A Systems Approach to Disease Resistance Against Necrotrophic Fungal Pathogens

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

The fungal pathogens Botrytis cinerea and Sclerotinia sclerotiorum have broad host ranges and cause serious disease on many horticultural crops. Both fungi can cause substantial losses on field-grown and protected lettuce crops, an industry worth almost £200 M annually in the UK. B. cinerea is a particular problem post-harvest, whereas S. sclerotiorum can result in up to 50% crop loss pre-harvest. Chemical control is problematic as few effective compounds are available, the number of sprays is restricted and timing is difficult. Moreover, the fungicides are medium to high risk for development of resistance. Development of durable resistance in the crop is a more sustainable solution, but has been an intransigent problem for lettuce breeders. The objective of this proposal is to demonstrate that a novel approach to breeding for pathogen resistance is possible.

We will apply genomic and systems biology (computational) approaches in lettuce, and combine this with quantitative genetics studies to identify novel genes for increasing the resistance of lettuce to both B. cinerea and S. sclerotiorum. This will provide a foundation to develop similar resistance to these pathogens in other horticultural crops.

We have two hypotheses we want to test. Firstly, that we can identify genes which confer resistance to both B. cinerea and S. sclerotiorum, two necrotrophic fungal pathogens. Genome sequencing of these fungi has indicated they share a range of genes associated with infection and colonization of plants, hence host resistance mechanisms against one pathogen might also confer resistance to the other.

Secondly, we want to test the feasibility of applying systems biology research into horticultural crop species. We have used systems biology approaches to generate network models of how genes interact during the defence response of Arabidopsis to infection by B. cinerea. We combined large-scale gene expression data with mathematical modelling to predict the key resistance genes. In this work, we will carry out network analysis of the lettuce defence response and test whether the same genes are involved in disease resistance, and/or whether the hub genes in the network are important. This is a completely new approach to crop improvement, relying on gene-gene interactions during defence against pathogen infection. We will also look for conservation of disease resistance genes in tomato and Brassica, key crops affected by these pathogens.

At the same time we will employ a more traditional quantitative genetic analysis to identify regions of the lettuce genome that influence resistance against both of these pathogens. We will screen nearly 100 lettuce accessions and cross accessions with the greatest resistance to a standard cultivar to generate mapping populations. A pre-existing mapping population (known to be segregating for disease resistance) will be screened for disease resistance to both B. cinerea and S. scerotiorum to identify important genomic regions for these traits.

Finally we will integrate our quantitative genetic analysis and results from network analysis to generate lettuce lines and markers for use in breeding programmes. This project is possible because of the lettuce genome sequence that is available, as well as the extensive lettuce germplasm and genetic and genomic resources that Warwick has generated. The work will be exploited primarily through A.L.Tozer to develop lettuce varieties with increased resistance to B. cinerea and S. sclerotiorum fungal pathogens.

Technical Summary

We will use systems biology approaches in lettuce combined with quantitative genetics studies to identify novel genes for increasing the resistance of lettuce to both Botrytis cinerea and Sclerotinia sclerotiorum, two important fungal pathogens. In a previous project, we generated network models predicting regulatory interactions between Arabidopsis genes during infection by B. cinerea. This network analysis significantly improved our detection of major defence genes and demonstrated the power of systems biology to predict the targets governing a particular trait. We will now test whether this approach can be used in crop plants using lettuce as our exemplar, where breeding for resistance against B. cinerea and S. sclerotiorum has not been very successful.

Firstly we will profile lettuce gene expression over time following B. cinerea infection and use this data in network inference to identify the key lettuce hub genes. We will screen a lettuce diversity collection for resistance against both pathogens and use the accessions with extreme phenotypes to test whether expression of the key genes is correlated with disease resistance. We will use RNAi and overexpression to directly test the function of a small number of key genes in lettuce, and ask whether it is possible to identify sources of resistance against both pathogens using network analysis. We will also test whether hub genes are conserved in lettuce, Arabidopsis, tomato and Brassica and hence broadly applicable.

Secondly, we will screen a lettuce mapping population (generated from parents differing in disease resistance) to identify quantitative trait loci (QTL) for resistance against B. cinerea and S. sclerotiorum, as well as eQTL for expression of the lettuce network hub genes. Integrating QTL with eQTL, hub gene location and polymorphisms will enable us to identify markers for beneficial lettuce alleles, and produce pre-breeding material for the development of disease resistant lettuce cultivars.

Planned Impact

Food security is currently a major research challenge and the yield and economic losses associated with plant diseases continue to have a great impact on our ability to ensure the production of good quality vegetable crops. Reducing the inputs required for production is a high priority for increasing the sustainability of food production. This project aims to address this by identifying and mapping novel alleles associated with increased resistance to Botrytis cinerea and Sclerotinia sclerotiorum in lettuce. New genetic resources, markers and the knowledge generated in this research will accelerate the ability of the industry partner A.L. Tozer and other breeders to develop commercially acceptable lettuce cultivars incorporating this valuable resistance trait. This will result in significant economic gains for both growers and breeders as well as environmental benefits. For UK growers, a 50% reduction in disease due to B. cinerea/S. sclerotiorum would save at least £10M p.a. given an average crop loss of 10%. As more than 90% of UK lettuce crops (22,000 ha) are treated with fungicides targeted at these pathogens (2-3 sprays per crop), a 50% reduction in these applications due to the deployment of more resistant lettuce cultivars would result in total savings of >£7.1M p.a. Moreover, there would also be a concomitant reduction in pathogen inoculum (particularly a reduction in sclerotia returned to the soil by S. sclerotiorum) which would benefit disease management in many of the other susceptible crops in rotations. The associated environmental benefits would therefore include a reduction in crop waste, and more efficient use of resources and inputs such as land, water, pesticides and fuel. Consumers would also then have access to good quality lettuce grown in a more sustainable way.

Crucially, demonstration of network analysis as a successful method for gene discovery in a horticultural crop would provide a framework for similar approaches in other crops, and even, if key disease resistance genes are conserved, candidate genes to immediately test. Collaboration with East Malling Research (transformation of diploid strawberry) and Syngenta (the SAMUTAGENE tomato TILLING population) will be sought to build on our results in lettuce and initiate direct testing of hub genes in strawberry and tomato. We will also exploit the Brassica resources available at Warwick in further funding applications. B. cinerea and S. sclerotiorum are pathogens with wide host ranges, hence the potential applicability of our generated data and approach is applicable to a broad range of crops. Integration of our systems knowledge with existing QTL phenotyping can accelerate the identification of other beneficial alleles.

An important aspect of our proposed research is the training that the PDRA on the project would receive. Warwick is recognised for its expertise in interdisciplinary training and the Systems Biology MSc and Doctoral Training Centre have successfully trained biologists, mathematicians, and computer scientists to be systems biologists working in an interdisciplinary manner. The PDRA would be exposed to this environment, have the opportunity to take modules of the various MSc courses at Warwick (including Systems Biology, Food Security, and Sustainable Crop Production) and also receive training in various transferable skills.
The next generation of young scientists will benefit from knowledge gained from this project and learning about the combined experimental and theoretical approaches used to add value to crop research. Furthermore, the integration of up to date network analysis with applied crop science is likely to catch the imagination of students from high school to undergraduate level and help spark interest in plant science. It is essential we build interest in plant science amongst young people if we are to build a generation of capable of meeting the global food security challenge.

Publications

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Related Projects

Project Reference Relationship Related To Start End Award Value
BB/M017877/1 18/05/2015 12/06/2016 £471,161
BB/M017877/2 Transfer BB/M017877/1 13/06/2016 30/09/2018 £337,163
 
Description • Identified genetic variation for susceptibility to Sclerotinia sclerotiorum and Botrytis cinerea across diversity set of cultivated and wild lettuce.
• Generated F2 seed from crosses between wild lettuce and/or cultivated lettuce with varying resistance levels
• QTL conferring resistance (assessed by lab assay) have been identified in a PI251246 x Armenian L. serriola population.
• Established robust field trial protocol
• A mapping population has been generated where the two parents differ for field resistance against S. sclerotiorum.
• Transcriptome-based network analysis has predicted candidate genes for enhancing resistance in lettuce and shown conservation of host response to both pathogens
• Gene editing protocol has been established for lettuce
• Two lettuce genes have been shown to confer enhanced disease resistance in Arabidopsis
• Candidate genes for future testing of disease resistance potential
• Mapping population and protocol to map field resistance against S. sclerotiorum
• Identification of genetic sources of resistance
• Validation of transcriptome network strategy in a crop
Exploitation Route Our demonstration of succesful use of time series transcriptome data and modelling for gene discovery could be used for other traits and in other species. The work is being taken forward in a separate proposal with Rijk Zwaan.
Sectors Agriculture, Food and Drink

 
Description This successful project has generated knowledge and resources to advance the breeding of fungal disease resistance in lettuce, as well as developed a productive ongoing relationship between Prof. Denby's group and A L Tozer. The tools and resources we have generated (for example, gene editing protocol, field testing protocol, identification of two key defence regulators, novel mapping populations) will provide a strategic advantage to Tozer in future breeding and will be jointly exploited in future collaborative work. It has also provided validation of our transcriptome network strategy for gene discovery in crops. Further awards: • BBSRC Follow on Fund "Targeting plant pathogens through LPMO gene silencing" (BB/S018735/1) PI £250k • Innovate UK Knowledge Transfer Partnership with A. L. Tozer Ltd. Innovative technologies to enhance speed and efficiency of vegetable breeding £230k • • BBSRC IAA "Proof of resistance to Sclerotinia disease in a new lettuce mapping population" PI £21,050 York BBSRC AgriFood Technology ISCF Catalyst seed funding award in Jan 2018 for ~£15k to progress a mapping population to a further generation and develop high-throughput markers for analysis of recombinants. • Direct funding from Tozer from Feb 2019 until June 2019 to progress transgenic lettuce analysis and carry out field trial in 2019. • BBSRC-funded iCASE studentship awarded to KD and Tozer for entry in Oct 2019, to progress mapping population with field resistance, progress finer mapping of QTL in PIxArmenian population, test candidate resistance genes emerging from network and transcriptome analysis in Arabidopsis and lettuce. Self funded PhD student on full scholarship will also start in Oct 2019 working on lettuce disease resistance. • Successful Innovate UK KTP award with Tozer to develop gene editing protocols for key crops (lettuce, cucurbits, parsnip and celery) as well as speed breeding protocols to reduce generation time. • BBSRC IPA proposal under development with Rijk Zwaan focusing on S. sclerotiorum resistance and analysis of the pathogen during infection. Intend to submit to April 2019 responsive mode call. The project provided fantastic genetics and breeding training for the postdoc fellow as well as state-of-the-art omics skills such as genotype by sequencing, large scale transcriptome data analysis and network modelling. The postdoc fellow was subsequently employed as a bioinformatician by a clinical company.
First Year Of Impact 2018
Sector Agriculture, Food and Drink
Impact Types Societal,Economic

 
Description A. L. Tozer Ltd
Amount £12,500 (GBP)
Organisation A L Tozer Ltd 
Sector Private
Country United Kingdom
Start 02/2019 
End 10/2019
 
Description BBRC GCRF IAA Soybean improvement in Zambia: breeding for enhanced disease resistance and drought tolerance
Amount £7,465 (GBP)
Organisation University of York 
Sector Academic/University
Country United Kingdom
Start 01/2017 
End 03/2017
 
Description BBSRC DTP - MIBTP
Amount £100,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 09/2019
 
Description BBSRC IAA
Amount £21,050 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2020 
End 07/2020
 
Description Bill and Melinda Gates Foundation. £200k. 2019 - 2020. Global Burden of Crop Pests and Diseases. Led by CABI
Amount £150,000 (GBP)
Organisation Bill and Melinda Gates Foundation 
Sector Charity/Non Profit
Country United States
Start 05/2019 
End 10/2020
 
Description Development of innovative breeding technologies to enhance efficiency of improved vegetable variety production
Amount £230,000 (GBP)
Funding ID KTP 11630 
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 05/2019 
End 10/2021
 
Description ISCF Agri-food Technology Catalyst Seeding Award
Amount £17,000 (GBP)
Funding ID BB/SCA/York/17 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2017 
End 02/2018
 
Description ISCF Agri-food Technology Catalyst Seeding Award
Amount £3,000 (GBP)
Funding ID BB/SCA/York/17 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2017 
End 02/2018
 
Description Testing the feasibility of CrookRNA to silence gene expression in plants and plant pathogens
Amount £5,000 (GBP)
Organisation Argonaute RNA Ltd 
Sector Private
Country United Kingdom
Start 08/2018 
End 07/2019
 
Description A.L. Tozers 
Organisation A L Tozer Ltd
Country United Kingdom 
Sector Private 
PI Contribution Provision of information on disease resistance in lettuce cultivars and genotypic selection.
Collaborator Contribution generating mapping populations of lettuce
Impact Knowledge Transfer Partnership award starting 2019 BBSRC iCASE PhD studentship starting Oct 2019 Self-funded studentship starting Oct 2019
Start Year 2013
 
Description AHDB Horticulture 
Organisation Agricultural and Horticulture Development Board
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Identifying sources of disease resistance in lettuce
Collaborator Contribution Providing industrial connections and steering and financial contribution
Impact in progress
Start Year 2015
 
Description Freshtime 
Organisation Freshtime Ltd
Country United Kingdom 
Sector Private 
PI Contribution identifying sources of disease resistance in lettuce
Collaborator Contribution screening potential resistant lines for commercially relevant traits
Impact in progress
Start Year 2015
 
Description Guido van der Ackerveken 
Organisation Utrecht University
Country Netherlands 
Sector Academic/University 
PI Contribution Collaboration on lettuce transcriptome resources and on Peronospora effusa genomics
Collaborator Contribution Collaboration on lettuce transcriptome resources and on Peronospora effusa genomics
Impact no outcomes as yet
Start Year 2019
 
Description Richard Michelmore - UC Davis 
Organisation University of California, Davis
Department UC Davis College of Biological Sciences
Country United States 
Sector Academic/University 
PI Contribution We are providing screening of phenotypic traits
Collaborator Contribution UC Davis have provided three mapping populations of lettuce and given us access to the latest lettuce genome sequence
Impact in progress
Start Year 2015
 
Description AHDB KTN Early Career Researcher event, Birmingham 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact AHDB KTN Early Career Researcher event, Birmingham, Adam Talbot gave a talk.
Year(s) Of Engagement Activity 2016
 
Description British Council/Peru Plant Genomics workshop 
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 Adam Talbot and Sarah Harvey (postdocs on two of my BBSRC awards) took part in a British council/Peru workshop on plant genomics in Lima. He presented work and took part in many discussions.
Year(s) Of Engagement Activity 2016
 
Description British Society for Plant Pathology conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presented a talk on our work to breed for disease resistance in lettuce against two major fungal pathogens. Highlighted development of novel approach (combining systems biology with quantitative genetics) and how we are applying this to lettuce.
Year(s) Of Engagement Activity 2015
 
Description Come Dine with the Future 
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 special free event explored food and its sustainability by challenging experts from food-related sectors, in research and industry, to present a menu they think we will be eating in 50 years' time. The panellists will each presented a menu which they think is representative of the future availability and sustainability of our food, and highlighted how current research and innovation in their sectors will shape our future choices. Professor Achim Dobermann, Director of Rothamsted Research, the UK's largest and oldest agricultural research station, reviewed each menu before the chair invited the audience to take part in the discussion.
Katherine Denby was a panel member.
Year(s) Of Engagement Activity 2016
URL http://www.nrn-lcee.ac.uk/events/come-dine-with-the-future-28786
 
Description Compositae Session Talk, Plant and Animal Genomes Jan 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 talk at Compositae session at Plant and Animal Genomics Conference 2017
Year(s) Of Engagement Activity 2017
 
Description Elsom's Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Attended and presented work (via a poster) at Elsom's Open Day for growers and associated businesses.
Year(s) Of Engagement Activity 2015
 
Description Global Plant Council/SEB Stress Resilience Forum 
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 Presented talk and took part in discussions at international stress resilience forum in Iguazu Falls, Brazil. Helped write a White Paper emanating from the workshop.
Year(s) Of Engagement Activity 2015
 
Description Invited speaker. University of Newcastle/N8 AgriFood industry-facing workshop "Translating crop research from lab to field" https://www.n8research.org.uk/translating-crop-research-from-lab-to-field/. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact University of Newcastle/N8 AgriFood industry-facing workshop "Translating crop research from lab to field" https://www.n8research.org.uk/translating-crop-research-from-lab-to-field/.
Year(s) Of Engagement Activity 2017
URL https://www.n8research.org.uk/translating-crop-research-from-lab-to-field/
 
Description Invited talk at International Congress for Molecular Plant Microbe Interactions June 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited research talk at IC-MPMI in Portland June 2016
Year(s) Of Engagement Activity 2016
 
Description Keynote speaker, British Council UK-Kenya-South Africa Legume Improvement Workshop, Stellenbosch, SA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Keynote speaker, British Council UK-Kenya-South Africa Legume Improvement Workshop, Stellenbosch, SA. Workshop to discuss collaboration and develop joint proposals for research projects. Resulted in joint DARA PhD application but this was unsuccessful.
Year(s) Of Engagement Activity 2018
 
Description New Frontiers in Crop Research, SCI event, London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Poster presentation by Adam Talbot at New Frontiers in Crop Research, SCI event, London
Year(s) Of Engagement Activity 2016
 
Description Plant and Animal Genomes Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave a talk at the Plant and Animal Genomes conference in a Systems Genomics workshop.
Year(s) Of Engagement Activity 2016
 
Description Presentation and hosting of British Leafy Salads Association Technical Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation at, and hosting of, British Leafy Salads Association/Babyleaf Salads Association Technical day, University of York, November 2017. Presented our work on lettuce disease resistance, discussed future research ideas and gave a tour of facilities.
Year(s) Of Engagement Activity 2017
 
Description Presentation by Prof. Dave Pink to the Rural Officers of the Church of England and Methodist Church at Stoneleigh, 2017. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation by Prof. Dave Pink to the Rural Officers of the Church of England and Methodist Church at Stoneleigh, 2017.
Year(s) Of Engagement Activity 2017
 
Description YorkTalk 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Katherine presented a talk on "Big Data Driving the development of disease resistant crops" at YorkTalks 2018 https://www.york.ac.uk/research/events/yorktalks/ A day of public engagement talks highlighting world-leading research at University of York. Attended by over 700 people.
Year(s) Of Engagement Activity 2018
URL https://www.york.ac.uk/research/events/yorktalks/