Metabolomic analysis of the plant pathogenic microbes Fusarium graminearum and F. culmorum

Many micro-organisms specialise in attacking plants and agricultural crops to complete their lifecycle. Some microbial infections lower crop yields, whilst others lower the quality of the harvested product, for example, causing spots on apples. Both these situations can seriously affect farm profit margins and may in certain countries lead to food shortages. Another group of micro-organisms which attack plants are of even greater concern. This is because during the infection of plant tissue they produce specific toxins, called mycotoxins, which are harmful to human and animal health even when eaten in only modest quantities. The accurate testing for the presence of these mycotoxins in bulk grain as well as raw fruit and vegetable products is very difficult and is also expensive. A micro-organism called the Fusarium scab fungus is of growing international concern (http://www.scabusa.org) because it infects the flowers of all cereal plant species (wheat, barley, maize and rice) and during these infections the cereal grain becomes contaminated with a highly toxic, water soluble, mycotoxin called DON, commonly known as vomitoxin because of the effects it causes on humans when ingested. Interestingly the Fusarium fungus only produces mycotoxins when it grows inside plant tissue or under conditions in the laboratory which mimic the carbon and nitrogen rich interior of the plant. In this project we wish to understand how the Fusarium fungus switches on mycotoxin production. This has already been explored, by determining whether the expression of each of the 11,640 Fusarium genes is specifically switched on, off or do not alter under mycotoxin inducing conditions. To complement this study, we plan to investigate under precise laboratory conditions what happens to the biochemistry inside the Fusarium fungus as DON production is switched on. We plan to explore in detail the global changes in the small water soluble chemical molecules made inside the Fusarium fungus. To increase our understanding of this process, we also plan to test under the identical conditions up to 100 different mutant strains of the Fusarium fungus which each have a single gene removed. The Fusarium fungus is predicted to have in total 11,640 genes. Therefore we intend only to focus on gene deletion strains which remove different key internal regulators, and other which are known to compromise DON production or plant infection processes. To help interpretation the complex Fusarium results we will modelling the changes in small molecules observed and thereby generate working hypotheses to test by subsequent experimentation. In addition to assist us in interpreting the Fusarium results we will include some comparable experiments with two well/studied species of fungi, namely baker's yeast which grows by budding and a free living filamentous fungus called Neurospora. The research should help us to devise various ways either to stop the Fusarium fungus from infecting cereal crops or from producing mycotoxins in the developing grain.

Most species of fungi are free living. However, a few species specialise in the invasion of other organisms, causing detriment (parasitism), benefit (mutualism) or no effect of their chosen hosts (commensalism). In the last eight years, full genomic sequence information has become available for over fifteen fungi. Comparative genomics has revealed that fungi which grown by budding have fewer genes than filamentous fungi (~6,000 vis ~10,000). Many of these additional genes, some located in high density gene clusters, are probably involved in the generation of a wide range of secondary metabolites. In the post-genomics era, the inter-linking of the transcriptome, proteome and metabolome of different fungi with the model yeast species S. cerevisiae and the model filamentous fungus Neurospora crassa will yield unprecedented insights into the common and unique mechanisms regulating the biochemistry of eukaryotic cells as well as the potential for cross talk between different regulatory networks controlling growth, differentiation, development and responses to external stimuli. This proposal will explore Fusarium species which are now causing serious disease epidemics in cereal crops throughout Europe, North America, Asia and Australia. Floral infections lead to the harvested cereal grain becoming contaminated with trichothecene mycotoxins which when eaten are harmful to human and animal health. The F. graminearum genome has been fully sequenced and aligned to the four chromosomes. Affymetrix oligoarrays are in use to define the Fusarium transcriptome under five different biological situations, include the switch to DON mycotoxin production in vitro. The Fusarium metabolome will be investigated and directly compared to the metabolome of S. cerevisiae and N. crassa growing under the identical conditions. Various wild-type and single gene deletion strains in each organism will be included to attempt to link the synthesis of specific metabolites to specific regulatory networks. We will explore in vitro, three different 'switch' conditions which mimic particularly important stages during the Fusarium host infection process, namely, (1) deoxynivalenol (DON) mycotoxin inducing production, (2) nutrient starvation, and (3) decreasing water availability. These metabolome studies will involve 1H-NMR profiling and follow up GC-MS and LC-SPE-NMR-MS analysis. To explore the details of the underlying regulatory networks and potential for cross talk, up to fifty additional single gene deletion mutants will be provided by the global Fusarium community for metabolic profiling under one or more biologically relevant conditions. In addition, up to thirty unknown mutants recovered from forward genetic experiments either compromised or restored for disease causing ability and / or mycotoxin production, will be investigated in an attempt to define additional regulatory networks, feedback loops and cross talk mechanisms. To further define the common and unique mechanisms regulating the biochemistry of Fusarium cells, the metabolome information will be directly related to the available Fusarium in vitro and in planta generated transcriptome data on DON mycotoxin production. Overall this proposal should greatly expand our understanding of the regulatory networks and metabolites in Fusarium cells controlling the production of trichothecene mycotoxins and required for pathogenicity. The comparative studies involving yeast and Neurospora, should define which regulatory controls are Fusarium specific and which are in common and could therefore be analysed in a model species.