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A metabolomic analysis of G-protein signalling mutants of Stagonospora nodorum

Gummer, Joel (2012) A metabolomic analysis of G-protein signalling mutants of Stagonospora nodorum. PhD thesis, Murdoch University.

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Stagonospora nodorum is the causal agent of Stagonospora nodorum blotch (SNB) of wheat. This fungus has cost the Australian grains industry upwards of 100 million dollars (AUD) p.a. in recent growing seasons, making it one of the most agriculturally damaging pathogens in Australia.

Disease severity is governed by the polycyclic lifecycle of S. nodorum, requiring a succession of spore inoculum arising from the asexual fruiting body of the fungus, known as the pycnidium. The resultant fungal density will determine the level of damage and ultimately influence the grain yield of the plant. G-protein signalling through the heterotrimeric G-protein is a biochemical mechanism used by S. nodorum in the host-pathogen interaction and has been linked to important biological processes including asexual sporulation. In this work, the unique phenotypes of three mutant strains of S. nodorum; each lacking either the Gα (Gna1), Gβ (Gba1), or Gγ (GgaA) subunit of the heterotrimeric G-protein were explored, and the biochemistry underpinning the phenotypes assessed by metabolomics.

The mutant strain S. nodorum ggaA was created by homologous recombination of the GgaA gene for comparison with the previously created gna1 and gba1 strains. All strains possessed developmental defects and reduced pathogenicity on the wheat plant. Growth assays uncovered differences in carbon source utilisation between the strains. Asexual sporulation was monitored by light microscopy; with the differentiation of mutant mycelia into pycnidia found to occur only after a comparatively longer culture time than in wild type, and at a reduced temperature. Until this time, asexual sporulation is completely abolished in the mutant strains. The matured pycnidia also possessed an irregular morphology. These results identified an association of all three G-protein subunits in asexual sporulation in S. nodorum.

Metabolites were isolated from S. nodorum mycelia for gas chromatography-mass spectrometer (GC-MS) analysis. An assessment of existing metabolomic methods identified some key steps in the sample preparation employed prior to injection into the GC-MS. Quenching the fungal metabolism upon harvesting, drying the fungal mycelia prior to metabolite extraction and isolation, and lyophilisation of the fungal metabolites in preparation for chemical derivatisation; each improved the metabolite recovery and overall reliability of the metabolomic analyses. These methods were applied to the metabolomic characterisations that followed.

Metabolite extracts from the in vitro cultured fungal strains were analysed using a single-quadrupole GC-MS and the recorded analytes cross-refereces to purchased metabolite standards for identification. Changes in the accumulation of various carbohydrates were apparent in the mutant metabolomes. Of those, the altered abundances of the metabolites glucose and trehalose are believed to in part explain or be consequential to the sporulation phenomena of these strains. Metabolomic analysis of the mutant strains in differentiating from a non-sporulating to a sporulating phenotype revealed the specific association of a number of metabolites with each of the two phenotypic classifications. Many of which have been targeted for identification in future studies. Among those identified was again trehalose, providing further evidence for it having a role in the asexual sporulation of this fungus.

These results have demonstrated the requirement for Gna1, Gba1 and GgaA in regulating developmental processes and the pathogenesis of S. nodorum, and added significantly to the biochemical dissection of asexual sporulation in this fungus.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Veterinary and Biomedical Sciences
Supervisor(s): Solomon, Peter, Trengove, Robert and Oliver, Richard
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