Molecular characterisation of the EAS gene cluster for ergot alkaloid biosynthesis in epichloë endophytes of grasses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand
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Date
2007
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Massey University
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Abstract
Clavicipitaceous fungal endophytes of the genera Epichloë and Neotyphodium form
symbioses with grasses of the family Pooideae in which they can synthesise an
array of bioprotective alkaloids. Some strains produce the ergot alkaloid ergovaline,
which is implicated in livestock toxicoses caused by ingestion of endophyteinfected
grasses.
Cloning and analysis of a plant-induced non-ribosomal peptide synthetase (NRPS)
gene from Neotyphodium lolii and analysis of the E. festucae E2368 genome sequence
revealed a complex gene cluster for ergot alkaloid biosynthesis. The EAS cluster
contained a single-module NRPS gene, lpsB, and other genes orthologous to genes
in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of
Aspergillus fumigatus. Functional analysis of lpsB confirmed its role in ergovaline
synthesis and bioassays with the lpsB mutant unexpectedly suggested that
ergovaline was not required for black beetle (Heteronychus arator) feeding
deterrence from epichloë-infected grasses. Southern analysis showed the cluster
was linked with previously identified ergot alkaloid biosynthetic genes, dmaW and
lpsA, at a subtelomeric location. The ergovaline genes are closely associated with
transposon relics, including retrotransposons, autonomous DNA transposons and
miniature inverted-repeat transposable elements (MITEs), which are very rare in
other fungi.
All genes in the cluster were highly expressed in planta but expression was very low
or undetectable in mycelia from axenic culture, including under nitrogen-, carbonor
phosphate-limited conditions. Comparative analysis of the EAS gene cluster in
four different epichloë strains showed marked differences in gene expression and
ergot alkaloid synthesis. Gene order is conserved in each strain although evidence
for recombination between two MITEs and expansion or reduction of a simple
sequence repeat (SSR) at a single intergenic region was observed. Heterologous
expression of a candidate regulatory gene, laeA, from Aspergillus nidulans, which is
a global regulator of secondary metabolism in aspergilli, did not affect eas gene
expression. This, along with phylogeny and microsynteny analysis, suggests there
is not an orthologue of this gene in epichloë.
This work provides a genetic foundation for elucidating biochemical steps in the
ergovaline pathway, the ecological role of individual ergot alkaloid compounds,
and the regulation of their synthesis in planta.
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Keywords
EAS gene cluster, Molecular characterisation, Epichloë, Endophytic fungi, Ergot alkaloid biosynthesis