N.J. Talbot, D.M. Soanes, J.M. Jenkinson, Z.Y. Wang, L.J. Holcombe, M.J. Gilbert, and G. Bhambra,
School of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter, EX4 4QG, UK

The rice blast fungus, Magnaporthe grisea causes one of the most serious diseases of cultivated rice, and understanding the early events of the infection is of paramount importance if durable control measures are to be developed. Magnaporthe grisea develops a specialised infection structure called an appressorium which is used to penetrate the tough outer cuticle of rice leaves allowing the fungus entry to the underlying tissue. Appressoria are melanin pigmented, dome shaped cells which accumulate massive intracellular turgor. Turgor is generated by accumulation of a compatible solute within the appressoria to very high concentrations. M. grisea appressoria accumulate glycerol as a major compatible solute during appressorial turgor generation, and understanding the mechanisms by which glycerol is synthesised within appressoria and how this process is genetically regulated is one of the primary aims of our research. Appressoria of Magnaporthe grisea form in dew drops on the surface of rice leaves in the absence of exogenous nutrients. Therefore, glycerol is synthesised from precursors that are present within un-germinated spores of the fungus. We have been examining the role of trehalose, glycogen and lipids as sources for glycerol biosynthesis. Trehalose is synthesised by the trehalose-6-phosphate synthase complex, and TPS1 encoding the enzyme T6P synthase is required for pathogenicity of M. grisea and turgor generation. However, trehalose breakdown, which would be required for glycerol synthesis, is dispensable for appressorium turgor generation, and, therefore, it seems more likely that trehalose accumulation contributes to appressorium function, either in a protective capacity or by directly contributing to turgor. TPS1 also exhibits a regulatory effect on glycolysis, and tps1 mutants are unable to grow on glucose. We have been examining the mechanism by which this occurs by using a genetic screen and metabolite profiling, and then determining how this may impact upon appressorium function. Glycogen breakdown is catalysed by glycogen phosphorylase and amyloglucosidase. Functional analysis of the GPH1 and AGL1 genes encoding these enzymes shows a small effect on virulence indicating that glycogen degradation via this route is, probably, not a significant contributor to appressorial turgor generation.
Triacylglycerol lipase activity is highly induced during appressorium development, and lipids are mobilised specifically to the appressorium during their maturation, consistent with the role for this polymer as a source for appressorial glycerol. The Magnaporthe grisea genome contains several putative intracellular lipases and we have initiated a programme of functionally characterising intracellular lipases and will present information concerning the likely functions of these genes in lipid metabolism in the fungus and appressorium turgor generation. The availability of the Magnaporthe genome and better methods for gene functional analysis are allowing a more systematic multi-disciplinary approach to be initiated to investigate the biology of appressorium mediated plant infection in the rice blast fungus.
Foster, A.J., Jenkinson, J.M., Talbot, N.J. (2003 Trehalose synthesis and metabolism are required at different stages of plant infection by Magnaporthe grisea. EMBO J. 22: 225-235.
Wang, Z.Y., Thornton, C.R., Kershaw, M.J., Debao, L. and Talbot, N.J. (2003) The glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic fungus Magnaporthe grisea. Molecular Microbiology 47: 1601-12
Talbot, N.J. (2003) On the trail of a cereal killer: investigating the biology of Magnaporthe grisea. Annual Review of Microbiology 57: 177-202