The endophytic symbiont Epichloe festucae establishes an epiphyllous net on the surface of Lolium perenne leaves by development of an expressorium, an appressorium-like leaf exit structure

Epichloe festucae forms a mutualistic symbiotic association with Lolium perenne. This biotrophic fungus systemically colonizes the intercellular spaces of aerial tissues to form an endophytic hyphal network. E. festucae also grows as an epiphyte, but the mechanism for leaf surface colonization is not known. Here we identify an appressorium-like structure, which we call an expressorium that allows endophytic hyphae to penetrate the cuticle from the inside of the leaf to establish an epiphytic hyphal net on the surface of the leaf. We used a combination of scanning electron, transmission electron and confocal laser scanning microscopy to characterize this novel fungal structure and determine the composition of the hyphal cell wall using aniline blue and wheat germ agglutinin labelled with Alexafluor-488. Expressoria differentiate immediately below the cuticle in the leaf blade and leaf sheath intercalary cell division zones where the hyphae grow by tip growth. Differentiation of this structure requires components of both the NoxA and NoxB NADPH oxidase complexes. Major remodelling of the hyphal cell wall occurs following exit from the leaf. These results establish that the symbiotic association of E. festucae with L. perenne involves an interconnected hyphal network of both endophytic and epiphytic hyphae.