Investigating the role of the Sln1 turgor sensor kinase in the rice blast fungus Magnaporthe oryzae

Rice blast is a devastating disease which affects rice cultivation all over the world. Blast is caused by a fungus called Magnaporthe oryzae and this project aims to investigate mechanisms required by the fungus to infect rice plants. The rice blast fungus produces a specialised infection structure called an appressorium, which is a dome-shaped cell that can generate enormous pressure of up to 8MPa (or 40 times the pressure of a car tyre). This pressure is applied as physical force at the leaf surface to puncture the plant cuticle. This remarkable infection process enables the fungus to cause disease.

We aim to investigate how the enormous pressure generated inside the appressorium is translated into physical force at the base of the infection cell. We have identified a sensor protein called Sln1, which is necessary for the infection cell to regulate turgor pressure. Without this protein, the appressorium continues to expand under pressure, but cannot generate physical force and break its way into a rice leaf.

In this project we will investigate how Sln1 functions in the rice blast fungus. We will investigate how Sln1 is activated during appressorium development. We will identify proteins that physically interact with Sln1 and may therefore contribute to its function, and we will study the proteins that are likely to be activated, or repressed in function by the activity of Sln1.

In this way, we aim to define the turgor-sensing network that is regulated by Sln1 and understand how the appressorium functions. This research may lead to the development of new and novel disease control strategies that are focused on preventing fungal pathogens, like the blast fungus, from being able to gain entry to plant tissue.

The project will investigate the mechanism of appressorium-mediated plant infection by the causal agent of rice blast, the filamentous fungus Magnaporthe oryzae. Appressoria of the rice blast fungus generate enormous turgor which is applied at the rice leaf surface as physical force to breach the rice leaf cuticle and gain entry to plant tissue.

The project aims to determine how a pressure-dependent sensor kinase called Sln1 is able to control the generation of turgor in the appressorium, in order to re-establish polarised growth. Sln1 is a histidine-aspartate kinase and the temporal dynamics of Sln1 phosphorylation will be determined during appressorium development along with its putative transfer and response regulator proteins, Ypd1 and Ssk1. To identify Sln1 protein-protein interactions, proximity-dependent biotinylation coupled to mass spectrometry will be used and interacting partners functional analysed by targeted gene replacement.

Sln1-dependent changes in protein phosphorylation will then be analysed during appressorium development, using an optogenetic approach to generate a conditional Sln1 mutant that can be light-inactivated, so that rapid changes in Sln1-dependent phosphorylation can be determined to order the sensing pathway. Initial data-dependent acquisition of phospho-peptide enriched samples will be used to build a spectral library of phospho-peptides, which will enable us to quantify phosphorylation events by Parallel Reaction Monitoring. This information will be used to define the Sln1 turgor-sensing pathway and individual components then functionally characterised and studied using live cell imaging of appressorium turgor generation.

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.