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    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
    (Massey University, 2018) Candy, Alyesha Valerie Joy
    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.
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    The role of the G protein and cAMP/PKA signalling pathway in establishment and maintenance of the mutualistic Epichloë festucae - ryegrass association : 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
    (Massey University, 2017) Bisson, Alexander
    Growth of the fungal endophyte Epichloë festucae in mutualistic symbiotic association with Lolium perenne (perennial ryegrass) is highly regulated and synchronised with the growth of the host plant leaf. To maintain this pattern of fungal growth in planta, specific signalling between symbiont and its host grass is required. To sense the extracellular environment and respond to changes, filamentous fungi rely on G protein-coupled receptors (GPCRs), which transmit signals predominantly via heterotrimeric G proteins to downstream pathways such as the cAMP/Protein Kinase A (PKA) and MAP kinase signalling pathways. In phytopathogenic fungi, G protein signalling and the associated cAMP/PKA pathways are often essential for a normal host interaction. Signal transduction using the second messenger cAMP to activate the PKA activity is finely balanced through a regulatory feedback loop for signal attenuation regulated by 3’-5’-cyclic nucleotide phosphodiesterases (PDE). Using a blast-based bioinformatic approach, a total of 40 genes encoding putative GPCRs were identified in the genome of E. festucae, grouping into 13 of 14 classes of the recent classification system for fungal GPCRs. Among genes encoding components of the G protein signalling cascade, nine of these GPCRs including the two cAMP receptor-like GPCRs, Gpr1b and Gpr2, show significant transcriptional up-regulation in association with the host compared to the respective expression level when grown in axenic culture. A reverse genetics approach was used to functionally characterise the identified cAMP receptor-like GPCRs (Gpr1a, Gpr1b and Gpr2). While deletion of gpr1a was unsuccessful, plants infected with an E. festucae Δgpr1b mutant showed a severe breakdown of the E. festucae–ryegrass association, whilst no effects were observed for Δgpr2-inoculated plants. Among numerous other genes putatively involved in G-protein and cAMP/PKA signalling, two putative PDEs involved in regulation of the cAMP-mediated signal were also identified. Deletion of pdeH (ΔpdeH), a gene encoding a PDE with high affinity towards cAMP, had a dramatic effect on the endophyte-plant association. In contrast, deletion of pdeL (ΔpdeL), a gene encoding a PDE with low affinity towards cAMP, had no effect on the host interaction phenotype, while primarily modulating the intracellular cAMP level during nutrient-induced activation of the cAMP/PKA ii signalling pathway in axenic culture. Finely balanced cAMP levels are crucial for various cellular processes including hyphal growth, cellular differentiation of asexual development and conidiogenesis. This research identified Gpr1b as an important receptor involved in maintenance of the highly restricted endophytic growth pattern of E. festucae in association with the host, potentially regulated by plant-derived molecules. These signals are mediated by the cAMP/PKA signalling pathway for an appropriate alteration in gene expression and subsequent cellular adaptation. Intracellular cAMP levels triggering these concentration-dependent processes are regulated by PdeH, which specifically acts in planta. This study shows that G-protein and cAMP/PKA signalling play an important role in regulating environmental signalling for establishment and maintenance of the mutualistic association between E. festucae and L. perenne.