Investigating the biology of appressorium-mediated plant infection by the rice blast fungus Magnaporthe grisea

The aim of this project is to understand how rice plants become infected with a very serious disease called rice blast. Rice blast disease destroys up to 30% of the rice harvest each year and is a serious and recurrent threat to food security. Rice blast is caused by a fungus and this project is aimed at determining why this fungus is able to cause such a serious disease and defining which of its genes and their associated products are necessary for disease to occur. This project aims to study this disease from the point at which the fungus lands on the leaf surface until it breaches the cuticle and gains entry to the leaf. The rice blast fungus develops a special pressure cell that it uses to puncture the leaf cuticle and gain entry to living plant tissues, in which it grows and feeds. This project will explore the biology that leads to development of the specialised pressure cells produced by the rice blast fungus. Specifially, we will look at the release of reactive oxygen species in these cells, which we have already shown are necessary for disease to occur. We will then go on to study the physiology of infection cells of the fungus and determine how these cells use an enzyme called trehalose-6-phosphate as a sensor of starvation/nutrient availability that is necessary for plant infection. Collectively, this research project, will provide an insight into the biology of plant infection by one of the most important crop diseases in the world today. This 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 this project is also of potential benefit to UK agriculture because similar fungi affect our major cereal crops, barley and wheat, and share a similar underlying biology. The development of broad spectrum, disease control strategies that will benefit agricultural production and the consumer is the long term strategic aim of this research.

The project will explore the biology of appressorium-mediated plant infection by Magnaporthe grisea, the rice blast fungus. We have discovered that two NADPH oxidase-encoding (NOX1 and NOX2) genes are necessary for rice blast disease due to an effect on the biology of appressoria, the specialised cells used by the fungus to bring about plant infection. An oxidative burst occurs during appressorium maturation and reactive oxygen species (ROS) generation requires NADPH oxidase activity. We will characterise the Nox3 NADPH oxidase and examine the regulation of Nox1, Nox2 and Nox3 activity by Rac1, noxR and the Ca2+ signaling pathway. We will use cell wall proteome analysis to identify major changes associated with the onset of the oxidative burst during appressorium maturation. The physical interactions between p67phox, Rac1 and the Nox family, their cellular localisation and the role of Ca2+ in regulation of Nox3 will be investigated. We will then explore the role of trehalose-6-phosphate synthase (Tps1), which is a central regulator of carbon and nitrogen metabolite repression in M. grisea and is essential for pathogenesis. The role of Tps1 in regulating nitrogen source utilization by interaction with the NMR repressor gene family will be explored using gene functional analysis and protein-protein interaction studies. Reporter gene fusions will be used to investigate the temporal regulation of sub-cellular distribution of the Nmr repressors, the Nut1 transcription factor and Tps1. The effect of Tps1 on cytoskeletal organisation during appressorium morphogenesis will also be explored. The effect of Tps1 on cellular NADPH levels and the potential role of Tps1 as a direct sensor of cellular NADPH will be investigated by mutagenesis, direct NADPH-binding assays and metabolite analysis during infection-related development by M. grisea. The effect of Tps1 on the regulation of ROS generation and appressorium function will be determined.