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
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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