A functional genomics toolkit for the wheat pathogen Mycosphaerella graminicola

Lead Research Organisation: University of Exeter
Department Name: Biosciences

Abstract

Wheat is the principle food crop in Europe, including Britain where the annual value of wheat production is approximately £1.4 billion. With the expectation that planet's population will increase to over 9 billion by 2050, we urgently need to find ways to provide more food to support this growth. One of the major threats to wheat production is fungal disease. The pathogen Mycosphaerella graminicola is the major disease-causing agent of wheat in the UK and is one of the principle reasons why over £500 million of fungicides are applied to protect cereal crops in the UK alone. Despite this there are still major yield losses. Hence there is an urgent need for new durable disease control strategies, including novel fungicides. The main difficulty in developing novel fungicides is the inability to determine how new molecules work to kill the fungus. In the model fungus brewer's/baker's yeast, Saccharomyces cerevisiae, powerful tools have been generated, such as sets of yeast strains; lacking each fungal gene; that have each protein tagged with a fluorescent protein, or where each individual protein is expressed at a high level. These resources have allowed large-scale determination of how a wide range of chemicals function to alter the biology of yeast. We intend to construct a similar complete set of tools that will allow us to perform these assays, and hence determine how novel fungicides work, in the wheat pathogen M. graminicola. Specifically, we will place each of its ~10,000 genes into a mobile genetic element (plasmid) that will allow easy transfer into a large range of other plasmids (that we will also create) that can be used to inactivate, fluorescently tag and over-express each gene, both individually and as a pool. We will also construct a series of M. graminicola strains that will allow us to determine how the natural fungus invades wheat, and we will develop a laboratory model of wheat infection to achieve this. This will significantly add to the vue of the current investment in sequencing the wheat genome. We will then use these resources to determine how fungicides impact on the infection process, and hence circumvent the principle impediment to the development of new control measures. To ensure that these resources are made widely available we will develop a repository for these plasmids and strains, place them in international depositories, write standard operating procedures to allow maximal exploitation and create a website to advertise and allow access to the resources. Finally, we will interact with stakeholders, both in the South-West (eg the new integrated farm platform at BBSRC North Wyke), and nationally (eg Rothamsted Research, Sainsbury Laboratory, and Syngenta) to increase the impact and dissemination of the tools generated.

Technical Summary

The project aims to establish and disseminate functional genomics resources that will facilitate rapid characterization of the wheat pathogenic fungus Mycosphaerella graminicola. The fungus causes Septoria leaf blotch disease, which is a severe constraint on wheat production. There is an urgent need for new durable disease control strategies. The project will generate the complete set of M. graminicola predicted gene sequences as a Gateway compatible ORFeome library allowing rapid generation of tagged alleles for protein localization, protein interaction studies and to facilitate rapid targeted gene deletion. In parallel, the project will generate a suite of destination vectors; a set of validated inducible promoters to modulate gene expression in a controllable manner; a comprehensive set of M. graminicola strains in which each major cytoskeletal component and sub-cellular compartment is fluorescently tagged to allow live cell imaging studies that can address infection-associated development and the functional analysis of virulence genes. These resources will be made freely available via an Exeter repository. All strains will also be deposited at the Fungal Genetics Stock Center, Kansas, USA. A full set of experimental protocols will also be developed and bioinformatic resources publicly disseminated through the European Bioinformatics Institute Ensembl repository, and via a web resource created in this work. When considered together, this work will provide the tools to circumvent the major obstacles to characterizing the biology of this economically significant pathogen, allowing rapid progress to be made in understanding one of the world's most important cereal pathogens and developing new methods for disease control. In this way it will significantly add to the investment in sequencing the wheat genome. Furthermore, combating the disease will constitute a fundamental advance in addressing global food security, a BBSRC strategic priority.

Planned Impact

Wheat is one of the three major cereals grown worldwide and the staple food source for the majority of the world's population. Global wheat production in 2010 was 651 million tonnes and in 2008, UK wheat production was valued at $1.67 billion. However, this vital commodity is prone to disease, and fungal pathogens typically cause 10% yield losses in UK-grown crops. We aim to study the fungal pathogen Mycosphaerella graminicola, which causes the disease septoria leaf blotch and is the principal cause of wheat loss in the UK. The purpose of the work is to support the development of next-generation fungicides by providing a 'toolkit' of genomic resources. The market in the UK is currently worth about $700 million and the development of new approaches to manage this serious pest is crucial to maintain production levels and ensure food security. To ensure that the resources generated are fully exploited we intend to focus part of our Pathways to Impact activities on ensuring that the user community is aware of, and has full access to, the 'toolkit' that we will provide. Provision of this set of tools and resources will, in the long-term, yield new, durable crop protection strategies and as such will indirectly benefit the UK population through increased food security. This specifically addresses the BBSRC priority area of food security. More directly it will benefit UK plc, as it will enable mode of action studies to be carried out on novel fungicide chemistries identified in high throughput screens, a current stumbling block to developing new crop protection strategies. In addition it will encourage the scientific community to adopt M. graminicola as a model for plant pathogenic and cell biological analyses, and facilitate these on a global sclae, which is currently not possible. As such, the work will be of immediate use to other scientists within academia, research institutes and the commercial sector. Furthermore we will train two PDRAs and a technician in functional genomics, plant pathology and host interaction biology. This, in addition to the transferable skills training at Exeter, will ensure that they complete the project with the expertise that will enable them to contribute significantly in many areas of Biosciences, or elsewhere within the economy.

Publications

10 25 50

publication icon
Kilaru S (2015) A gene locus for targeted ectopic gene integration in Zymoseptoria tritici. in Fungal genetics and biology : FG & B

publication icon
Steinberg G (2015) Cell biology of Zymoseptoria tritici: Pathogen cell organization and wheat infection. in Fungal genetics and biology : FG & B

publication icon
Kilaru S (2015) Conditional promoters for analysis of essential genes in Zymoseptoria tritici. in Fungal genetics and biology : FG & B

publication icon
Devran Z (2015) Development of molecular markers tightly linked to gene in pepper using next-generation sequencing. in Molecular breeding : new strategies in plant improvement