During plant infection, Magnaporthe grisea elaborates a specialised infection cell known as an appressorium (Talbot, 2003). This cell functions by accumulating large amounts of compatible solute, glycerol, which is responsible for generation of enormous turgor (de Jong et al., 1997) that is, then, translated into mechanical force to bring about plant infection. In order to understand the process of appressorium development, we have extracted soluble proteins from infection structures at various times during their development and separated these by isoelectric focussing and polyacrylmide gel electrophoresis. The resulting 2D separations have then been subjected to spot cutting in gel trypsin digestion and maldi-ToF mass spectrometry. In this way it has been possible to identify and characterise a large number of proteins that are specifically required by the fungus in order to elaborate an appressorium. Comparative 2-D proteomic analysis has been carried out using a wild-type strain of the fungus, Guy11, and mutants which are affected specifically in appressorium morphogenesis.

A particular interest has been comparative analysis with a pmk1 MAP kinase mutant (Xu and Hamer, 1996). PMK1 is required by the fungus in order to regulate the morphogenetic pathway responsible for infection cell formation and, therefore, proteins which are altered in expression can tell us significant amounts about the nature of Pmk1 MAP kinase signalling during appressorium formation. So far, we have been able to resolve and characterise a 2D proteome map of Magnaporthe grisea of approximately 400 proteins and have analysed the abundance levels of a large sub-set of this group of proteins. This work has been carried out in collaboration with the group of Professor Al Brown at the University of Aberdeen COGEME Proteomics Unit and protein extractions and electrophoresis has been carried out by Laura Selway, bioinformatic analysis of protein abundance by Dr Zhi Kang Yin and maldi-tof MS by Dr David Stead. High throughput gene functional analysis of the corresponding genes is being carried out at present.

Appressoria of Magnaporthe grisea generate very high internal turgor pressure which is used mechanically to breach the rice cuticle. In order to generate this pressure, the cells draw water into the appressorium by producing an osmotic gradient with the external environment. M. grisea appressoria accumulate very high internal concentrations of glycerol in order to achieve this. Current research is aimed at determining the importance of glycerol accumulation and the formation of other compatible solutes within the appressorium and identifying elements of the glycerol biosynthetic pathway and the manner in which it is genetically controlled (Thines et al., 2003). Magnaporthe appressoria form on the rice leaf surface in the absence of external nutrients. The major storage products within conidia are glycogen, lipid and trehalose. We are, therefore, studying the manner in which these storage compounds are transported to the appressoria during morphogenesis and how turgor generation is accomplished (Wang et al., 2005).

Lipid bodies are found in abundance in the appressorium and have been shown previously to be transported to the apex of the extending germ tube and, then, to coalesce before take-up by the vacuole during appressorium turgor generation (Thines et al., 2000; Weber et al., 2001). This is accompanied by a large increase in triacyl glycerol lipase activity within the appressorium. TAG lipase activity is regulated by the cyclic AMP response pathway, and occurs at a much lower level in cpka mutants, which lack protein kinase A activity. Furthermore, lipid transport to the appressorium appears to require the pmk1 MAP kinase pathway. A large number of lipase-encoding genes are present in the Magnaporthe genome (Dean et al., 2005) and we have been systematically characterising the role of these lipases in conditioning the ability of the appressorium to degrade lipid rapidly during the onset of turgor generation. One of the consequences of rapid lipolysis in the appressorium is the activation of the fatty acid beta-oxidation pathway and the glyoxylate pathway (Wang et al., 2003; 2005). A number of components of the fatty acid beta-oxidation pathway are currently being characterised in the laboratory and evidence of the involvement of a carnitine acetyl transferase encoded by the pth2 gene has recently been characterised, indicating that fatty acid metabolism and the resulting transport of acetyl CoA from the peroxisome is important in the physiology of infection cells. Current research is aimed at identifying further components of the beta oxidation pathway, investigating how this pathway differs from that deployed by other pathogenic and free-living fungi, and determining the precise roles of the distinct acetyl CoA pools which exist within appressoria.

Glycogen reserves are extremely abundant in appressoria in spores and are degraded very rapidly during spore germination. The resulting glucose is then available for metabolism and, also, for production of appressorial glycerol through the action of NADH-dependent glycerol 3-phosphate dehydrogenase, or NADPH-dependent glycerol dehydrogenases, both of which have been shown, previously to be present within appressoria (Thines et al., 2000). We are currently investigating the role of amyloglucosidase and glycogen phosphorylase enzymes, which are activated during spore germination. Mutants lacking either of these enzymes are severely attenuated in virulence, indicating a role for glycogen mobilisation in appressorium turgor control.

Trehalose is a non-reducing disaccharide found in abundance inside the conidia of M. grisea. During spore germination this disaccharide is rapidly degraded (Foster et al., 2003). Genetic evidence has shown that trehalose synthesis is required for pathogenesis, although this appears to be associated with a role in sugar signalling. We are currently investigating the role of trehalose-6-phosphate synthase as a control point for the regulation of both sugar signalling and nitrogen utilisation pathways within the rice blast fungus and determining in detail how trehalose metabolism is involved in the control of plant infection by M. grisea. These projects involve genetic analysis, biochemical analysis and, most recently, the utilisation of high throughput metabolomic techniques, which are being used to trace the profile of metabolites within germinating spores and infection cells produced by the fungus.

The cell biology of appressorium development by M. grisea is being investigated using both forward and reverse genetic approaches. Mutants specifically affected in the ability to make appressoria have been identified using Restriction Enzyme-Mediated Insertion (REMI) and Agrobacterium-mediated transformation. A number of developmental mutants has so far been identified in this manner, including PDE1 P-type ATPase gene, which belongs to the DRS2 class of aminophospholipid translocases. PDE1 is involved in infection hypha production by M. grisea which appears to be due to a role in regulating membrane phospholipid balance during a time of severe membrane stress during plant infection (Balhadère and Talbot, 2001). The identification of PDE1 has led to further investigation of the roles of aminophospholipid translocases in M. grisea which are in progress. Genes which show elevated expression during plant infection, or those regulated by the PMK1 MAP kinase– a central regulator of fungal pathogenesis –have also been selected and characterised. A number of interesting genes have been characterised to date including the CYP1, cyclophilin-encoding gene which may play a role in control of appressorium turgor during plant infection (Viaud et al., 2002), and the metallothionein-encoding gene MMT1 which is necessary for pathogenesis (Tucker et al., 2004).

The MMT1-encoded metallothionein appears to be involved in cell wall modifications associated with appressorium development and may act as an anti-oxidant, or as a means of spatially regulating oxidative cross-linking of proteins in thee cell wall. This has led to a series of investigations into the role of reactive oxygen species in M. grisea and the function of NADPH oxidases in plant infection by the rice blast fungus.

Another focus of interest in the laboratory is the role of the cell cycle in regulating appressorium morphogenesis and re-establishment of polarity during plant infection. Current research is aimed at identifying the checkpoints that regulate appressorium development in M. grisea.

Finally, we have a longstanding interest in the role of morphogenetic proteins called fungal hydrophobins which are involved in aerial morphogenesis by fungi (Elliott and Talbot, 2004) and plays significant roles in fungal attachment and cellular differentiation. We have investigated the MPG1 hydrophobin, which is involved in appressorium development and have recently investigated the role of the four disulfide linkages that are present in all hydrophobins. These appear to be required for efficient secretion of fungal hydrophobins and there are pronounced developmental phenotypes associated with preventing disulfide linkage formation in Mpg1 (Kershaw et al., 2005). Industrially-funded research has focused on the commercial application of hydrophobins.

Genome data is currently being analysed from twenty-four sequenced fungal species and comparative studies are underway that focus on identifying the genetic determinants associated with fungal pathogenesis. The laboratory undergoes annotation work and incorporation of data into publicly available databases (Dean et al., 2005; Giles et al., 2003). As a bioinformatics resource centre of the BBSRC Consortium for Genomics of Microbial Eukaryotes (http://www.cogeme.man.ac.uk) project, we are collating publicly held data from a large number of pathogenic fungi into a relational database that can be queried by the molecular plant pathology community. EST and genomic sequnece information has been incorporated into a MySQL relational database for effective comparison of gene sets (Soanes et al., 2002; Soanes and Talbot, 2005; 2006). The database is available on this site (http://cogeme.ex.ac.uk).

Our current research is part of the BBSRC e-fungi project which is aimed at incorporating diverse forms of functional genomic information (transcriptomic, proteomic, gene functional analysis, metabolomic studies) into an accessible data warehouse using GRID technology that will also provide intuitive linkage to key metabolic and regulatory pathways with in-built links to enzyme and structural protein information and associated gene information. The e-fungi fuGIMS (Fungal Genome Information Management System) data warehouse will be a dynamic e-science resource for the fungal genomics research community.

Other bioinformatics research includes phylogenetic analysis which is aimed at investigating the evolutionary histories of gene families in pathogenic fungi and the ancestral genome-level relationships between fungi and other taxonomic groups. We are also assessing the incidence of lateral gene transfers between fungi and other groups both experimentally and at the informatic level.