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
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Date
2014
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Massey University
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Abstract
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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Keywords
Fungal gene expression, Dothistroma septosporum, Dothistromin, Biosynthesis, Red band needle blight, Research Subject Categories::NATURAL SCIENCES::Biology::Cell and molecular biology::Genetics