Polyketide synthesis in Stagonospora nodorum
Krill, Christian (2012) Polyketide synthesis in Stagonospora nodorum. PhD thesis, Murdoch University.
Stagonospora nodorum is a necrotrophic fungal pathogen of wheat and related grasses. It is the causal agent of Stagonospora nodorum blotch of wheat, a major disease causing upwards of 100 million dollars (AU$) damage in yield loss per annum.
Stagonospora nodorum is a member of the Dothideomycete class of filamentous ascomycetes. Within this class are numerous plant pathogens that rely on secondary metabolite (SM) phytotoxins of the polyketide class as pathogenicity and/or virulence factors. While the production of proteinaceous host specific toxins has been studied extensively in S. nodorum, the role of secondary metabolites in the pathogenic lifecycle of this fungus is completely unexplored.
In this study a combination of bioinformatics, molecular biology and analytical chemistry techniques are used to investigate polyketide synthesis in S. nodorum. In silico analysis of polyketide synthase (PKS) gene and protein sequences was used to catalogue and classify the PKS repertoire of S. nodorum, assign putative functions to PKS genes based on homology, identify PKS gene clusters and elucidate the phylogenetic history of PKSs in S. nodorum. Transcriptomics techniques were used to identify genes active during important stages of the pathogenic lifecycle as candidates for targeted gene deletion experiments. The role these genes play in host colonisation and disease progression was analysed using knockout mutagenesis and in vitro and in planta characterisation of mutant strains. Secondary metabolite extraction and LC-MS analysis techniques were evaluated to identify key compounds produced by the wild type fungus that were differentially abundant in the knockout mutants.
With this approach, a highly conserved alternative melanisation pathway gene cluster involving the putative DHN-melanin synthase MEL1, a putative oxidoreductase and a putative transcription factor has been identified. Further findings highlighted a rapid evolution and plasticity of PKS genes in S. nodorum. Knockout mutants for the SMS1, PKS1 and PKS3 polyketide synthase genes have been generated and were tested for defects in pathogenicity, metabolism and sporulation. No significant differences to the wild type were detected, indicating a menial role for these genes during pathogenesis. An SPE based method for isolating SM from culture filtrate was developed and used to identify compounds produced by S. nodorum in liquid culture, as well as two putative polyketide products absent in cultures of the SMS1 knockout mutant putatively linked to a cryptic mycotoxin pathway.
|Publication Type:||Thesis (PhD)|
|Murdoch Affiliation:||School of Veterinary and Biomedical Sciences|
|Supervisor:||Oliver, Richard, Trengove, Robert and Solomon, Peter|
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