Investigation of the molecular basis of symbiosis between Epichloë festucae and perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand

Abstract

The symbiosis between the endophytic filamentous fungus Epichloë festucae and its plant host, perennial ryegrass (Lolium perenne), is a highly regulated mutualistic interaction which represents a good model system for the investigation of plant-fungal mutualism. Fungal signalling pathways play a crucial role in regulation of this interaction. While genes involved in the production of reactive oxygen species (ROS), as well as a member of the MAP kinase signalling pathway, have been shown to regulate maintenance of the mutualistic interaction, the signalling pathways responsible for regulation of this symbiosis are still relatively poorly understood. In pathogenic fungi, members of calcium signalling pathways, such as Ca2+/calmodulinregulated kinases (CaMKs) and phosphatase (calcineurin), are required for normal hostpathogen interactions. Three genes encoding multifunctional CaMKs, cmkA, cmkB and cmkC, were identified in E. festucae, as well as one gene encoding the catalytic subunit of calcineurin, cnaA. Targeted replacements of these genes have identified a novel role for the fungal cmkB in the regulation of ion homeostasis and an important role for calcineurin for both culture growth and symbiosis maintenance. However, unlike the pathogenic fungi, E. festucae CaMKs do not appear to have a role in the regulation of the mutualistic interaction. In order to identify new genes regulating the symbiosis, T-DNA mutagenesis was used to generate symbiotically defective E. festucae mutants. Two mutants, Ag51 and Ag212, with both in culture and in planta phenotypes, were identified. A detailed molecular analysis showed that Ag51 had a complex T-DNA insertion while Ag212 had a deletion of ten genes. Ag212 failed to establish plant infection and complementation experiments using cosmids identified candidate genes for both the in culture and in planta phenotype. Analysis of the colonization process showed that this mutant is defective in establishing a specific interaction between hyphal and plant cell walls, essential for the plant colonization. This work provides new insights into calcium signalling in fungi and increases our understanding of plant-fungal mutualism.