Analysis of Epichloë festucae membrane lipid composition and its role in Nox complex assembly : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand

Abstract

Epichloë festucae is a filamentous fungus that forms a highly regulated mutualistic symbiosis with perennial ryegrass. The spatially and temporally controlled production of reactive oxygen species (ROS) by the fungal NADPH oxidase (Nox) complex regulates this interaction by restricting fungal growth in planta. Whilst much is known about the importance of ROS in plant-fungal interactions, comparatively little is known about how its production is regulated. In plant and mammalian systems, production of ROS by the Nox complex is regulated via lipid signalling. Cytosolic Nox components containing lipid-binding domains are targeted to specific phosphoinositide enriched at certain locations within in the plasma membrane. Once assembled, specific lipids then directly activate the Nox complex. It is hypothesised that similar regulation also occurs in fungi. This study investigated whether lipid signalling could play a role in regulating the fungal Nox complex. The lipid-binding PH domain of Nox protein Cdc24 was found to be necessary for membrane localisation, supporting a role for lipid signalling in fungal Nox complex regulation. To identify potential lipid targets for the cytosolic Nox proteins, a comprehensive analysis of E. festucae membrane lipid composition was carried out using a suite of biosensors. These biosensors consisted of mammalian lipid binding domains of known specificity fused to a fluorophore, enabling live cell imaging of phosphoinositide localisation both in culture and in planta via fluorescence microscopy. Phosphatidylinositol 4,5-bisphosphate was detected in the plasma membrane and septa in culture and in planta. A striking asymmetric gradient was observed at the hyphal tip, with enriched fluorescence in the sub-apical region. Similar to yeast, phosphatidylinositol 4-phosphate was localised to golgi vesicles in culture. In contrast, phosphatidylinositol 3-phosphate was found in vacuolar and endosomal membranes. Biosensors for phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate, localised to the cytoplasm in culture and in planta, suggesting that these phospholipids were absent under the growth conditions examined. These results confirm a role for lipid signalling in fungal Nox complex assembly and provide insight into membrane lipid composition, identifying candidate phosphoinositide targets for assembly.