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

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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.
Epichloe festucae, Endophytic fungi, Perennial ryegrass, Lolium perenne, Epichloe festucae genetics