Determining the mechanism of septin-mediated plant infection by the rice blast fungus Magnaporthe oryzae

The aim of this project is to understand how rice plants succumb to a very serious disease called rice blast. Each year, rice blast disease destroys up to 30% of the global rice harvest and causes serious epidemics in Sub-Saharan Africa, South-East Asia and South America. It is therefore a continuing threat to global food security.

Rice blast is caused by a fungus called Magnaporthe oryzae and this project aims to determine how the fungus infects rice plants. The rice blast fungus produces a specialised infection structure called an appressorium, which generates enormous pressure (up to 8MPa, or 40 times the pressure of a car tyre) and to apply physical force at the leaf surface to puncture the plant cuticle. In this way the fungus can invade leaf tissue and cause disease. We aim to investigate how pressure inside the appressorium is translated into physical force at the base of the infection cell. We have discovered that a group of protein called septins, are essential for the appressorium to puncture the rice cuticle. Their role is to re-model the cell's internal cytoskeleton so it applies force at the leaf surface and forms a penetration peg that enter the leaf. This project will investigate how septins assemble at the base of the appressorium and how this region of the cell becomes specialised to secrete proteins into plant cells, how the appressorium develops a penetration peg, and how the fungus then rapidly invades the leaf. We will characterise how septin assembly is regulated and, in particular, how the fungus is able to monitor the turgor pressure within the appressorium and determine the optimal point (or trigger) for penetration peg development. We will also determine how this process is regulated in concert with the cell division cycle of the fungus, allowing the plant infection process to be controlled effectively.

When considered together, the objectives of this research project will provide new insight into the biology of plant infection by one of the most important crop diseases in the world today. This information will be used to inform new disease control strategies that are urgently required. In addition to the global significance of rice blast, knowledge gained from the project will also be of value to UK agriculture because many of the most serious diseases that affect our major cereal crops, barley and wheat, share a similar infection mechanism. Disease control strategies emerging from this work are therefore likely to be of broad spectrum for the most important cereal diseases, such as rusts and powdery mildews in addition to rice blast.

The project will investigate the mechanism of septin-mediated plant infection by the rice blast fungus Magnaporthe oryzae. We recently discovered that septin GTPases form a hetero-oligomeric ring complex at the base of the appressorium, which re-models the F-actin cytoskeleton and allows the appressorium to re-establish polarise growth, developing a penetration hyphae that punctures the leaf cuticle and causes infection.

In this project, we will characterise the appressorium pore, where the septin ring forms, and investigate whether the exocyst complex is organised in a septin-dependent manner at the point of plant infection, providing the means by which polarised exocytosis is regulated by the fungus during plant infection. To do this, we will localise fluorescently labelled exocyst gene fusions and generate conditional (temperature-sensitive) mutants by targeted allelic replacement to test their function in pathogenesis. We will then investigate how a pressure-dependent switch operates to trigger septin-dependent appressorium re-polarisation. We will characterise the Sln1 sensor kinase and potential turgor-monitoring proteins with which it appears to physically interact. We will generate kinase-inactive and phosphomimetic alleles of Sln1 to test its function and carry out an extragenic suppressor mutant screen to identify downstream components of the turgor-sensing pathway. Finally, we will define the cell cycle checkpoint which regulates appressorium function. We will test the hypothesis that a morphogenesis checkpoint, mediated by septin assembly, regulates cell cycle progression in concert with turgor control. We will generate conditional mutants and analogue-sensitive kinase mutants of conserved cell cycle regulators to define how the cell cycle checkpoint operates. In parallel, we will use global transcriptional profiling to define the pattern of gene expression associated with appressorium maturation and how these are co-ordinately regulated.

This is a discovery science project, which aims to discover new information about the biology of plant infection by a pathogenic fungus. The results generated will therefore be of immediate interest to scientists within academia, research institutes and the most relevant commercial sector, the agricultural biotechnology industry. Our Pathways to Impact activities will focus on user engagement, intellectual property generation and partnership, communication and public engagement activities. Our partnership activities may include both commercial organisation and charities because of the economic and societal importance of rice blast disease. Pathways to Impact activities will be divided into the following areas.

1. Tools and Resources.
The project will generate a set of new mutant strains, transcriptional profiling data, and cell biological data. All of the information will be made freely available and strains will be archived both locally and at international repositories, as described in out impact plan

2. Engagement with Industry Partners and Intellectual Property Management.
The impact of research from this project will be realised through effective partnership with relevant industry and charity sector organisation. The PI has a strong track record of industrial research, both proprietary contract research and collaborative training programmes and has had continual industrial funding for 20 years carrying out fungicide mode-of-action studies, target identification, target validation work and research for the food biotechnology industry. He also spent a year on sabbatical working for a biotechnology company so has first-hand experience of private sector fungicide discovery challenges. At the start of the project, the PI will meet Research & Knowledge Transfer's IP & Commercialisation unit in order to agree a strategy to protect and manage any intellectual property and potential commercialisation opportunities that may emerge. A plan will be agreed so that potential patent filing can be carried out ahead of publication, as detailed in the plan.

3. Training and Capability
Full training will be provided to the PDRAs in this project in knowledge exchange and intellectual property management. They will be seconded to our Research and Knowledge transfer division for bespoke training activities. We will leo actively encourage outreach activities which will involve both PDRAs.

4. Charities Engagement
Rice blast disease has a very significant societal impact and causes serious economic hardship in the developing world. As such, the PI has significant links with philanthropic trusts to fund rice blast research and training of scientists from these rice-growing regions of the world, including most notably the Halpin Trust which funds researchers from around the world to carry out PhD training in Exeter. We will engage with the charities sector, if developmental opportunities arise from the results of the project, and the project will also have a significant impact on the training of three current Halpin Scholars.

5. Public Understanding of Science
The PI is fully committed to developing the public understanding of science and will undertake communications through the popular press, through social media, and by Schools Engagement Activities which will take place on an annual basis as described. Schools engagement activities will include lectures and hand-on investigative activities for both primary and secondary school students.

6. Milestones
A set of deliverables is specified in the Pathways to Impact plan in order to be able to evaluate the success of impact activities.