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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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.
Epichloë, Endophytic fungi, Genetics