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    Regulation of dothistromin toxin biosynthesis by the pine needle pathogen Dothistroma septosporum : a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2014) Chettri, Pranav
    Dothistromin is a virulence factor produced by the fungal pine needle pathogen Dothistroma septosporum. It is similar in structure to a precursor of aflatoxin and sterigmatocystin. Unlike most secondary metabolite genes in fungi, the genes for dothistromin biosynthesis are not clustered but spread over six loci on one chromosome. Another characteristic feature of dothistromin synthesis is that dothistromin is produced mainly during the early exponential growth phase in culture. These unusual features have been proposed to be adaptations for the biological role of dothistromin in the disease process. It was therefore of interest to determine whether the regulation of dothistromin production in D. septosporum differs from the regulation of aflatoxin and sterigmatocystin in Aspergillus spp. and to address the question of whether genes in a fragmented cluster can be co-regulated. The availability of the D. septosporum genome facilitated identification of orthologs of the aflatoxin pathway regulatory genes aflR, aflJ and the global regulatory genes veA and laeA. These genes were functionally characterised by knockout and complementation assays and the effects of these mutations on the expression of dothistromin genes and the production of dothistromin were assessed. Inactivation of the DsAflR gene (?DsAflR) resulted in a 104 fold reduction in dothistromin production, but some dothistromin was still made. This contrasted with ?AflR mutants in Aspergillus species that produced no aflatoxin. Expression patterns in ?DsAflR mutants helped to predict the complete set of genes involved in dothistromin biosynthesis. AflJ was proposed to act as a transcriptional co-activator of AflR in Aspergillus spp. Disruption of DsAflJ resulted in a significant decrease in dothistromin production and dothistromin gene expression. Interestingly the expression of DsAflR was not affected by deleting DsAflJ, while conversely DsAflJ transcript levels increased significantly in a DsAflR mutant compared to the wild type. Heterologous complementation with A. parasiticus, A. nidulans and C. fulvum AflJ failed to revert the dothistromin level to wild type suggesting species-specific function of AflJ. VeA is an important regulator of secondary metabolism and development in fungi. Inactivation of the D. septosporum ortholog (DsVeA) resulted in reduced dothistromin production and showed the influence of DsVeA on the expression of other secondary metabolite backbone genes. Asexual sporulation was reduced but mutants were not compromised in pathogenicity. Overall, D. septosporum DsVeA showed functional conservation of the usual role in fungi. LaeA is a global regulator of secondary metabolism and morphogenetic development, first identified in Aspergillus nidulans. Unexpectedly, DsLaeA exhibited an unusual repressive function on the dothistromin pathway and DsLaeA mutants exhibited an extended period of dothistromin production compare to WT in vitro. The mutation of DsLaeA showed varied responses in expression of other secondary metabolite genes and had differences in sporulation and hydrophobicity compared to the wild type.
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    Investigations of dothistromin gene expression in Dothistroma septosporum and the putative role of dothistromin toxin : a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Molecular Biology at Massey University, Palmerston North, New Zealand.
    (Massey University, 2007) Schwelm, Arne
    Dothistroma septosporum causes pine needle blight, a foliar disease currently causing epidemics in the Northern hemisphere. D. septosporum synthesizes dothistromin, a mycotoxin similar in structure to the aflatoxin (AF) precursor versicolorin B. Orthologs of AF genes, required for the biosynthesis of dothistromin, have been identified along with others that are speculated to be involved in the same pathway. The dothistromin genes are located on a mini-chromosome in Dothistroma septosporum but, in contrast to AF genes, not in a continuous cluster. The aim of this study was to increase knowledge of the biological role of dothistromin, which was previously a suspected pathogenicity factor. To identify putative roles of dothistromin, the dothistromin gene expression was investigated and green fluorescence protein (GFP) reporter gene strains of D. septosporum were developed. Expression analyses of dothistromin genes revealed co-regulation. More surprisingly, dothistromin is produced at an early stage of growth and gene expression is highest during exponential growth. This is fundamentally different to the late exponential/stationary phase expression usually seen with secondary metabolites such as AF. Strains with a dothistromin gene (dotA) promoter-regulated GFP confirmed early expression of the toxin genes, even in spores and germtubes. Parallel studies with transformants containing a GFP-DotA fusion protein suggest spatial organization of dothistromin biosynthesis in intracellular vesicles. The early expression of dothistromin genes led to the hypotheses that dothistromin is either required in the early stage of the plant/fungi interaction, or for inhibiting the growth of competing fungi. Constitutive GFP strains helped to determine that dothistromin is not a pathogenicity factor. However, a putative role of dothistromin in competition with other fungi, including pine-colonizing species, was detected, supporting the second hypothesis. It was shown that dothistromin-producing strains appear to have a competitive advantage which is lacking in dothistromin-deficient strains. However, some competitors were not affected and have potential as biocontrol agents. In summary, this work has led to the discovery of an unusual pattern of regulation of a secondary metabolite, has made substantial progress in identifying the biological role of dothistromin, and has indicated potential for biocontrol of Dothistroma needle blight.