Functional genomic characterization of germination and early infection of wheat by the fungus Zymoseptoria tritici.

Providing enough food for all people on the planet is a major issue facing humanity in the 21st Century. As the Earth's population tops six billion the need to increase production of staple crops e.g. rice, wheat, banana is real. As the population grows to an anticipated nine billion by 2030 that demand will be even higher. In the UK wheat is a major crop, with 12.1 million tonnes, valued at more than £1.5 billion, produced in 2013. However, fungal disease, in particular septoria tritici blotch (STB), caused by Zymoseptoria tritici, is a major constraint on production. In untreated trials an average 20% of yield is lost, and in some cases field losses of 50% have been reported. Control of this disease could contribute an extra 2.4 - 6 million tonnes to the UK wheat harvest, and if applied to the global wheat harvest in 2013 would have delivered an extra 140 million tonnes into the food supply. There is no doubt that STB is a serious and immediate threat to global food security, and new control measures are urgently required.

To initiate STB infection spores of Z. tritici alight on a leaf surface and germinate, beginning the disease process. This is followed by polarized growth development and passage into the interior of the leaf, usually within 12 hours. In susceptible wheat cultivars two days post-infection (dpi) the fungus has produced lateral branching and by 6-8 dpi this has developed to circumscribe a large area beneath the point of entry. At this stage the fungus has not yet invaded wheat cells and little evident symptomology is seen on the plant. However, 8-10 dpi symptoms begin to appear, concomitant with development of an incipient asexual reproductive structure, death of plant cells and a switch to parasitic growth. Around 14-21 dpi mature asexual spores are released, and the process starts again. This results in epidemic infections and is one reason why yield loss is so high. The hypothesis underpinning our planned research is that disruption of the very earliest events in infection e.g. Z. tritici spore germination, leaf penetration or initial hyphal development will prevent establishment of infection and initiation of disease. Thus targeting fungicide development to gene products essential for these developmental programs should deliver compounds that prevent disease and hence reduce yield loss.

We have established a collaboration with Syngenta, a large Swiss based agribusiness to address our principle aim, which is to characterize the early events (0-4 dpi) in establishment of infection and initiation of disease by Z. tritici on wheat. Specifically we will: (1) Define the genes that are switched on by Z. tritici spores as they germinate on and initiate infection in the compatible wheat cultivar Avalon. This will provide a catalogue of genes that play a role in the initiation of disease, and that will be characterized in this project; (2) Characterize the cellular biology of both Z. tritici and wheat during early infection, and thus link specific genes to defined infection stages; (3) Undertake a combined bioinformatics and modelling assessment to prioritize Z. tritici genes involved in early infection for further analysis. This will allow us to select one hundred genes for analysis and (4) we will determine where the proteins encoded by the 100 genes are located within the fungus during infection. We will then inactivate each in turn and determine if this blocks infection, and if so at what stage? If lack of a protein results in the inability to cause infection then this protein has potential as a target for fungicide development.

Taken together these experiments will not only reveal substantial new biology they will also identify proteins that can be utilized for rational target selection within the context of fungicide discovery.

Disruption of early events in Z. tritici infection e.g. germination, stomatal penetration or sub-stomatal hyphal development will prevent disease. Thus targeting fungicide development to proteins essential for these developmental programs should deliver compounds that prevent disease and hence reduce yield loss. The aim of this IPA with Syngenta is to characterize early infection.

To identify genes involved in spore germination and establishment of disease we will perform an in planta RNA-Seq time series experiment (WP1). To characterize the cell biology of disease establishment we will quantitatively define temporal and developmental events in initiation of infection including Z. tritici germination, stomatal penetration and sub-stomatal hyphal development. Simultaneously we will measure alterations in wheat subcellular dynamics and Z. tritici-induced defence responses (WP2). For wild type Z. tritici these host responses will be subtle but their quantification will provide a baseline for functional analysis of mutants in WP4. Using a combination of bioinformatics and modelling (WP3) we will link the expression data to the cell biology to create a coherent framework and select 100 genes identified in WP1, that represent all developmental stages from WP2, for null mutant construction. We will construct the null mutants and determine the impact of gene loss on infection (WP4). We will then select ten genes whose inactivation causes infection to stall at different stages and characterize their role in detail, addressing the questions. Is correct temporal expression of the gene required for proper function? Does altered expression induce aberrant host responses? What expression level is required for proper function?

These experiments will provide the first in-depth analysis of the early events of Z. tritici infection on wheat, revealing novel biology and proteins that that can be exploited in the context of fungicide discovery.

We will characterise the very early stages of infection on a compatible wheat host by the fungal pathogen Zymoseptoria tritici and determine the role that selected components play in in initiation and establishment of disease. This work will be of immediate use to other scientists within academia, research institutes and the commercial sector interested in plant pathology, spore germination and fungal developmental biology. In the medium to longer term it will be of substantial interest to UKplc, including Syngenta, as we will identify novel targets that can be exploited for the discovery of new fungicides. We will mentor the staff employed on the grant to "train the next generation of scientists". Finally we will pursue outreach and educational activities emphasizing the risk of increasing fungal disease of food crops and the rise of fungicide resistance. We will focus our Impact activities on the areas described below.

1. Engagement with the scientific community. To ensure that the academic and industrial scientific communities are aware of, and have full access to the data generated, methodology developed and strains constructed we will make our data public as promptly as possible using standard communication and engagement activities (publishing in appropriate open access journals, conference attendance, and presenting scientific seminars).

2. Intellectual Property (IP) and commercialization. If awarded the University of Exeter and Syngenta will develop a formal collaboration agreement. Substantial contacts already exist between the two organisations and KH & Dr Mike Csukai (MC) from Syngenta co-supervise a BBSRC CASE student. The two PI's will meet with MC regularly to discuss potential IP, this will be an agenda item on all meetings. KH & MJD will meet with the University's Research & Knowledge Transfer (RKT) IP specialist, Rachel Baird, on a six monthly basis to appraise these discussions and to identify potential IP arising from the project. One likely source of IP, and potential commercialization, is the identification of novel fungicide targets in Z. tritici. Novel fungicide targets that may be discovered will be developed for use through collaboration, licensing or the creation of a spinout company.

3. Training and Capability. Dr Cairns will receive excellent multidisciplinary training in microbiology, molecular biology, biochemistry, cell biology, large data acquisition & analysis, fungicide discovery and functional genomics. These skills will enhance his career opportunities and fulfil RCUK's goal of training the next generation of scientists. Dr Cairns will contribute substantially to our Pathways to Impact activities and will be given appropriate training. He will be involved in all aspects of IP protection and commercialization. Bioscience's embeds impact activities into the professional development of PDRAs, providing them with valuable insights into potential career opportunities in the bio-economy. He will spend two months working at Syngenta to further strengthen this understanding. In addition to receiving training to enhance his technical skills Mr Yogesh Chaudhari will be given increasing responsibility within the project, taking on aspects of health & safety provision, record and data management and methods development.

4. Public understanding of Science. The PI is fully committed to developing the public understanding of science. We will undertake school educational and outreach activities under the auspices of Dr Nicky King, who oversees school liaison for Biosciences. In collaboration with the Bisociences "Press Gang" we will develop bespoke activities, including professional quality hand-outs and "factsheets" around "food security and fungi".

5. Resources for the Activity. For travel to local schools, plus visits by students to the Haynes lab we request £1,000. To allow the production of professional quality hand-outs and factsheets to promote the work we request £2,000.