Functional analysis of genes encoding hydrolytic enzymes in the interaction of Epichloë festucae with perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand

Abstract

Hydrolytic enzymes degrade macromolecules into smaller components. These enzymes are important in fungal nutrition and have been implicated in the pathogenicity and virulence of pathogenic fungi towards their hosts. However, it is unknown if hydrolytic enzymes play important roles in mutualistic symbioses. In this study, the function of two different classes of hydrolytic enzymes was examined in the mutualistic symbiosis between the fungal endophyte Epichloë festucae and perennial ryegrass (Lolium perenne cv. Nui). Nine members of a gene family encoding subtilisin-like proteases were identified in E. festucae. The prt2, prt3 and prt5 genes encode putative extracellular proteins belonging to the proteinase K subfamily 1, and prt1 and prt6 encode putative extracellular proteins belonging proteinase K subfamily 2. The prt7 and prt8 genes encoded pyrolysin-like enzymes from subfamilies 1 and 2. The prt4 gene encodes a putative vacuolar protease, while the kex2 gene encodes a putative proprotein convertase. Expression analysis showed that the prt1, prt3, prt5, prt4 and kex2 genes, but not the prt2 gene, were expressed in culture. The prt1 and prt3 genes appeared to be up-regulated in planta compared to culture. The function of prt1 and prt2 in the symbiotum between E. festucae and perennial ryegrass was characterised by expressing these genes under the control of the Aspergillus nidulans gpdA or the E. festucae F11 ltmM promoters. No major differences in hyphal or plant morphology were observed between symbioses containing wild type E. festucae or endophyte strains containing the prt1 or prt2 transgenes. The gcnl gene, which encodes a β-1,6-glucanase, was identified immediately downstream of the prt2 gene. The function of the gcnl gene was characterised by gene replacement and testing the phenotype during growth in culture and in planta. E. festucae ∆gcnl strains grew normally on glucose-containing media. On media containing the β-1,6-glucan pustulan, ∆gcnl strains did not form aerial hyphae or hydrolyse pustulan, which the wild type strain did. This phenotype was partially complemented by growth of the ∆gcnl mutant in close proximity to wild type strains, and fully complemented by insertion of the gcnl gene. This suggests that the gcnl gene encodes the major β-1,6-glucanase activity of E. festucae.