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.
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.
Content removed from thesis due to copyright restrictions: Schwelm, A., Barron, N. J., Zhang, S. & Bradshaw, R. E. (in press). Early expression of aflatoxin-like dothistromin genes in the forest pathogen Dothistroma septosporum.