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Subject: search.filters.subject.Epichloe festucae

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    Investigation into the role of PacC in Epichloë festucae development and symbiosis with perennial ryegrass : 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, 2014) Lukito, Yonathan
    In order to survive and adapt to the environment, it is imperative for fungi to be able to sense and respond to changes in extracellular pH conditions. In ascomycetes, sensing of extracellular pH is mediated by the Pal pathway which is activated by alkaline pH. The signal is subsequently relayed to changes in gene expression by activation of the transcription factor PacC, which is known to regulate various fungal metabolic pathways; including ion tolerance, cell-wall integrity and secondary metabolism. The role of PacC in regulating fungal virulence and pathogenicity has also been studied in several pathogenic fungi, but to date not in a symbiotic fungus. Epichloë festucae is a biotrophic fungal endophyte that forms a stable symbiosis with the perennial ryegrass Lolium perenne. In this mutualistic interaction, secondary metabolites are produced by the fungus that confer bioprotection for the host, and the host in turn provides nutrients and a means of dissemination for the fungus. In this study, deletion (ΔpacC; acid-mimicking) and constitutively active (pacCCA; alkaline-mimicking) mutants were generated to study the cellular roles of PacC in E. festucae. Deletion of pacC resulted in increased sensitivity to salt-stress and reduction in aerial hyphae formation, but did not affect the ability of the mutant to grow under alkaline pH conditions. The pacCCA mutant on the other hand showed greater sensitivity to cell-wall and temperature stresses, and was able to grow under both acidic and alkaline conditions. Aberrant intrahyphal hyphae formation and abnormal conidiation were additionally observed in the pacCCA mutant in culture, but hyphal fusion was unaffected. The expression of secondary metabolite genes both in culture and in planta was largely unaffected in both ΔpacC and pacCCA mutants, and expression analysis of genes known to be pH- and PacC-dependent in other fungi showed that these genes were generally pH- and PacC-independent in E. festucae. The deletion and constitutive active pacC mutants were still able to sense and modify extracellular pH. Deletion of pacC did not affect the endophyte-host interaction, but ryegrass plants infected with the pacCCA mutant were mildly hypertillered. Further examination of the growth of the pacCCA mutant in the plant revealed formation of aberrant convoluted hyphal structures and an increase in hyphal breakage; possible reasons for the observed host plant phenotype. This study provides novel insights into the role of PacC in the agriculturally important endophyte, E. festucae. In this fungus, PacC regulates salt tolerance and cell-wall integrity, but not secondary metabolism and growth at non-neutral pH conditions. The results also show that PacC is involved in the symbiotic interaction between E. festucae and perennial ryegrass but is not crucial in the establishment and maintenance of the symbiosis.
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    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
    (Massey University, 2011) Mitić, Milena
    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.