A conserved signalling network regulates Epichloë festucae cell-cell fusion and the mutualistic symbiotic interaction between E. festucae and Lolium perenne : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand

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Epichloё festucae is a filamentous fungus that forms a mutually beneficial symbiotic association with Lolium perenne. The NADPH oxidase complex components noxA, noxR and racA, the transcription factor proA, and the cell wall integrity (CWI) MAP kinases, mkkA and mpkA, are required for mutualistic E. festucae-L. perenne associations and cell-cell fusion. Homologues of these genes in Neurospora crassa, Sordaria macrospora and Podospora anserina are required for cell-cell fusion and sexual fruiting body maturation, thereby establishing a link between self signalling and hyphal network formation in the E. festucae-L. perenne symbiosis. In Podospora anserina, IDC2 and IDC3 are required for cell-cell fusion, crippled growth and fruiting body formation. In S. macrospora and N. crassa, components of the STRIPAK complex regulate cell-cell fusion and fruiting body formation. The aim of this project was to test if E. festucae homologues of IDC2 and IDC3, and the STRIPAK complex protein MOB3, named SymB, SymC and MobC, respectively, are also required for cell-cell fusion and plant symbiosis. Gel shift assays showed the promoters of symB and symC are targets for the transcription factor ProA. In culture, the frequency of cell-cell fusion of ΔmobC was reduced, but in ΔsymB and ΔsymC mutants, totally abolished. All three mutants hyperconidiated and formed intra-hyphal hyphae. Plants infected with these mutants were severely stunted and hyphae exhibited proliferative growth and increased colonisation of the intercellular spaces and vascular bundles. Expressoria formation, structures allowing colonisation of the leaf surface, was reduced in ΔmobC, and abolished in ΔsymB and ΔsymC mutants. Microscopy analyses showed SymB-GFP and SymC-mRFP1 co-localise to the plasma membrane and septa. SymC also localised to highly dynamic punctate structures. Although ΔsymB and ΔsymC phenotypes are identical to ΔmpkA, and the E. festucae pheromone response pathway scaffold ΔidcA mutants, MpkA and MpkB phosphorylation and cellular localisation was unchanged compared to wild-type. Using yeast-two-hybrid assays, an interaction between SymC and the STRIPAK complex associated protein GPI1 was demonstrated. Collectively these results show that MobC, SymB and SymC are required for E. festucae cell-cell fusion and host symbiosis. It is proposed that SymB and SymC interact to form a sensor complex at the cell wall which regulates cell-cell fusion in culture and hyphal network development in planta.
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Epichloe, Lolium perenne, Genetics, Cell hybridization, Plant cellular signal transduction, Plant molecular biology, Research Subject Categories::NATURAL SCIENCES::Biology::Cell and molecular biology