Abstract:
Epichloëfestucae is an agronomically-important endophytic fungus that grows symbiotically within the intercellular spaces of temperate grass species such as Loliumperenne and L. arundinaceum. Colonization of host seedlings by E. festucae occurs when hyphae in the shoot apex invade developing host leaves and extend via intercalary hyphal growth, a highly unusual mechanism of division and extension in non-apical compartments. We hypothesise that intercalary hyphal growth is stimulated by mechanical stretch imposed by attachment of hyphae to elongating host cells, and that this stress is sensed by mechano-sensors located on the hyphal membranes. Genome analysis revealed that homologues of known mechano-sensors in Saccharomyces cerevisiae such as Mid1 (a stretch activated calcium ion channel), Wsc1 and Mid2 (cell wall integrity sensors) are present in the E. festucae genome. Gene replacement studies of mid1 and wsc1 in E. festucae reduced radial growth rate in axenic culture confirming the role of both genes in hyphal growth. In axenic culture both ∆wsc1 and ∆mid1 mutants were sensitive to fungal cell wall modifiers such as Calcofluor White, supporting their role in cell wall integrity. Preliminary plant infection studies with ∆wsc1 and ∆mid1 mutants revealed a hyper-branched unsynchronized growth pattern within the host (Loliumperenne), and ∆wsc1 also caused severe stunting in most plants suggesting a disruption in the symbiosis. A technique to stimulate intercalary growth under in-vitro conditions through mechanical stretch is being optimised to test the ability of Mid1, Wsc1 and Mid2 to sense mechanical stress and initiate intercalary growth.
