3 results for Berry, Daniel, Thesis

  • 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

    Berry, Daniel

    Thesis
    Massey University

    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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  • 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

    Berry, Daniel

    Thesis
    Massey University

    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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  • Characterisation of novel secondary metabolism genes in plant-endophytic Epichloë fungi : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Manawatu, New Zealand

    Berry, Daniel

    Thesis
    Massey University

    Plant-­‐endophytic fungi of the genus Epichloë are symbionts of cool season grasses. Epichloë infections are generally asymptomatic, with endophyte and host growth synchronised to form an interconnected hyphal network in the intercellular spaces separating host cells. In return for the sustenance received from the host apoplast, Epichloë spp. produce a range of bioprotective secondary metabolites (SM) that deter both vertebrate and invertebrate herbivores. Peramine is an Epichloë-­‐ derived insect-­‐deterring SM produced by the two-­‐module non-­‐ribosomal peptide synthetase (NRPS) PerA, encoded by the gene perA. The perA gene is widespread across Epichloë, but peramine production is much more discontinuous. Through an Epichloë-­‐wide survey it is shown that the peramine chemotype of Epichloë isolates can be accurately predicted through a combination of PCR and sequencing methods. Furthermore, the protein encoded by the widespread peramine-­‐negative perA-­‐ΔR allele is analysed in detail. The perA-­‐ΔR allele has a transposable element inserted into the 3’ end of the gene, and was previously assumed to be non-­‐functional. Evidence for ongoing perA-­‐ΔR functionality is presented, with the PerA-­‐ΔR protein shown to exhibit altered substrate specificity relative to PerA. A PerA/PerA-­‐ΔR domain swap is shown to restore peramine production to PerA-­‐ΔR, providing insight into the functional changes that separate this protein from PerA. Another major project investigated the role SM genes may play in Epichloë sexual development. Many Epichloë spp. are transmitted vertically through infection of the host seed. However, sexual Epichloë spp. may also undergo a reproductive cycle that involves formation of pre-­‐sexual stromata structures over developing host inflorescences. This sexual cycle culminates in horizontal transfer of the endophyte to endophyte-­‐free host plants. Deletion of the symbiotically regulated endophyte gene irlA induced a symbiosis-­‐defective phenotype in planta, and this observation led to the identification of a novel Epichloë SM cluster. Synteny analysis and comparison to previously characterised fungal SM gene clusters defines a five-­‐gene cluster centred on irlA that is shown to be upregulated in stromata. A model is proposed whereby this SM cluster controls initiation of the proliferative hyphal growth associated with stromata formation.

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