Analysis of the role of velvet and laeA in the molecular interactions between grass and Epichloë fungal symbionts

Abstract

Epichloë species form agriculturally important symbioses with many cool season grasses. However, little is known about the molecular details of the interaction and the key regulatory systems involved. VelA and LaeA are two wellknown regulators of different fungal development and metabolic processes. Some of these include fungal cell wall characteristics and hyphal morphology that have been previously implicated as important for compatible interaction between Epichloë and grasses. In addition they regulate secondary metabolism, which is an important feature of the symbiosis given the bioprotection conferred by fungal alkaloids. In this thesis, the velA and laeA homologues in the model Epichloë species E. festucae were deleted and functionally characterised in vitro and their roles in the E. festucae interaction with its host Lolium perenne (perennial ryegrass) were determined. Results showed that velA and laeA are required in E. festucae in vitro for normal hyphal morphology, resistance to stress, and conidiation in nutrient limited conditions. In planta studies showed the requirement for both genes in establishing and maintaining a compatible interaction, with high levels of seedling death and a drastically reduced infection rate after inoculation with mutants. In surviving infected plants, different endophytic and epiphytic morphologies were observed between infected plants with ΔvelA and ΔlaeA mutants, suggesting they are involved to some extent in different processes required for benign interaction. Methods to detect plant responses after inoculation were developed and these showed increased plant responses such as H2O2 production in inoculated seedlings with both mutants. A set of comparative transcriptomics analyses identified a range of biological processes influenced by VelA and LaeA, some of which were inferred as required for a mutualistic interaction. Investigation of the ryegrass transcriptome in compatible versus incompatible interactions also revealed host processes that may be influential in the interaction.