Structural and functional genomics of perennial ryegrass-derived fungal endophytes

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Applied Systems Biology, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

Perennial ryegrass is often associated with clavicipitaceous fungal endophytes that include Epichloë and Neotyphodium isolates. The research described in this thesis was focused on assessment of genetic diversity of ryegrass-derived endophytes, generation and characterisation of reporter gene-containing endophytes, evaluation of the capacity to introduce multiple endophytes into a single host and up-regulation of the biosynthetic pathway associated with peramine production. Genetic diversity of 20 selected ryegrassderived endophyte strains was evaluated based on 7 full-length gene sequences derived from whole genome sequence data. In addition to the well-characterised taxa (N. lolii and LpTG-2), two other novel taxa were identified in this study, which were designated as LpTG-3 and LpTG-4. Three endophyte strains were selected on the basis of taxonomic category, bioactivity profile, anti-fungal activity and mating type variation to generate derivatives containing the reporter genes gfp and DsRed. Binary vectors constructed for the constitutive expression of GFP (green fluorescent protein) and DsRed were successfully transformed using Agrobacterium tumefaciens-mediated technology. Expression of GFP and DsRed was obtained in all three endophyte strains, as detected by fluorescence microscopy and further comprehensive characterisation of transformants was achieved through use of multiple molecular and bioinformatic tools. Reporter genecontaining endophyte strains were used to exploit a novel in-house artificial seed-based inoculation system in order to evaluate the capacity to introduce multiple endophytes into a single host based on complementary reporter gene signals. Subsequently, a detailed characterisation of the perA gene from 20 ryegrass-derived endophyte strains was performed. Presence of the entire perA gene was identified in peramine-producing strains, and a partially deleted perA gene associated with miniature inverted-repeat transposable elements (MITEs) and premature stop codons was observed in peramine non-producing genotypes. A functional copy of the perA gene was introduced by A. tumefaciens-mediated transformation into endophyte strains with different genetic backgrounds in order to introduce and enhance insect feeding deterrence. The results obtained in this study will provide the basis for detailed understanding of both endophyteendophyte and endophyte-host interactions, and for enhancing the bio-protective properties of the symbiotum.