A Systems Approach to Disease Resistance Against Necrotrophic Fungal Pathogens
Lead Research Organisation:
University of York
Department Name: Biology
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.
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.
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.
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
Hickman R
(2017)
Architecture and Dynamics of the Jasmonic Acid Gene Regulatory Network.
in The Plant cell
Buchanan-Wollaston V
(2017)
Harnessing diversity from ecosystems to crops to genes
in Food and Energy Security
Beacham A
(2018)
Addressing the threat of climate change to agriculture requires improving crop resilience to short-term abiotic stress
in Outlook on Agriculture
Oates CN
(2021)
Insect egg-induced physiological changes and transcriptional reprogramming leading to gall formation.
in Plant, cell & environment
Alvarez-Fernandez R
(2021)
Time-series transcriptomics reveals a BBX32-directed control of acclimation to high light in mature Arabidopsis leaves.
in The Plant journal : for cell and molecular biology
Zhang R
(2022)
A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis.
in Genome biology
Pink H
(2022)
Identification of genetic loci in lettuce mediating quantitative resistance to fungal pathogens.
in TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik
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 successful use of time series transcriptome data and modelling for gene discovery could be used for other traits and in other species. We are taking this forward with an iCASE PhD studentship with Tozer Seeds. The work is also being progressed in a collaborative work with North Carolina State University. The establishment of gene editing protocols for lettuce led to the award of an Innovate UK KTP award with Tozer Seeds to embed this new technology within their company. |
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 started in Oct 2019 working on lettuce disease resistance. A second iCASE studentship looking at conservation of defence regulators across different vegetable crops will begin in Oct 2023. We have also submitted a NSF-BBSRC proposal focusing on the changing interaction between lettuce and B. cinerea under climate change. The project provided fantastic genetics and breeding training for the postdoc fellow and two PhD students 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 IAA Apr 2018- Mar 2022 BB/S506795/1 |
Amount | £21,050 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2020 |
End | 07/2020 |
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 | 04/2019 |
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 | 07/2017 |
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 | 07/2017 |
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 | 07/2018 |
End | 07/2019 |
Description | Transformations to Regenerative Food Systems |
Amount | £6,292,511 (GBP) |
Funding ID | BB/V004581/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2021 |
End | 01/2026 |
Description | Varietal Innovation for Vertical Farming |
Amount | £34,988 (GBP) |
Organisation | Innovate UK |
Sector | Public |
Country | United Kingdom |
Start | 12/2022 |
End | 03/2023 |
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 | Invited speaker - Ist International Plant Systems Biology Conference, Jacques Monod Conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Invited speaker |
Year(s) Of Engagement Activity | 2018 |
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 | 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 | 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 | Times Radio "Explainer" interview about genome editing |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Times Radio "Explainer" about genome editing in light of Defra consultation on regulation. https://www.thetimes.co.uk/radio/show/20210118-4163/2021-01-18 (2:52). |
Year(s) Of Engagement Activity | 2021 |
URL | https://www.thetimes.co.uk/radio/show/20210118-4163/2021-01-18 |
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/ |