Molecular and bioinformatic analysis of the perA locus in Epichloë : this thesis is presented as a partial fulfilment of the requirements for the degree of Master of Science (M.Sc.) in Genetics at Massey University, Palmerston North, New Zealand
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Date
2011
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Massey University
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Abstract
Fungal endophytes of the Epichloë genus form largely mutualistic symbioses with coolseason
grasses, systemically colonising the intercellular spaces of the host in a strictly
regulated fashion. The endophyte receives protection and sustenance from the host, and
in return provides benefits such as increased growth, drought resistance and protection
against herbivores. Protection against herbivory is mediated through the production of
bio-protective fungal secondary metabolites (SM). Examples of these SMs include
lolitrem B, the causative agent of ‘ryegrass staggers’ in stock, and the insect feeding
deterrent peramine.
The genes responsible for the production of each of these SMs are usually found
clustered together in the genome, and are often closely associated with a range of
transposon relics. SM gene expression occurs only when the endophyte is growing in
planta, indicating the presence of plant-fungal signalling. This study investigated the
locus structure and organisation of the gene perA that encodes the non-ribosomal
peptide synthetase PerA, which is both essential and sufficient for production of
peramine. It was found that perA and its flanking intergenic sequences exhibit
considerable transposon-mediated variability across Epichloë, and that this transposon
activity is likely responsible for the taxonomically discontinuous production of
peramine both within and across Epichloë spp.
The major facilitator superfamily transporter gene EF102 is divergently transcribed
from and co-regulated with perA (EF103). Transcriptome data were used to identify
transcription start sites for both genes. Comparative analysis of the intergenic sequence
separating EF102/perA from 10 Epichloë isolates covering six different species refined
the perA translation start site, and identified conserved regions in the promoters of both
genes proposed to be important for regulation. A motif search identified a conserved
DNA motif present multiple times in the promoters of both genes.
Deletion analysis of EF102 revealed the gene probably does not encode a peramine
transporter, as was hypothesised; however the four independent ΔEF102 mutants
exhibited a reduction in peramine production relative to wild type, resulting in an
alternative hypothesis that EF102 encodes a transporter for a PerA substrate precursor
molecule such as glutamate.
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Keywords
Epichloë, Endophytic fungi, Genetics