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

Abstract

Summary Epichloë 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.

An Epichloë festucae homologue of MOB3, a component of the STRIPAK complex, is required for the establishment of a mutualistic symbiotic interaction with Lolium perenne

“…Endophytic E. festucae hyphae exit the host cuticle layer via an expressorium to form an epiphyllous hyphal network on the surface of the host plant. The formation of these structures requires functional NADPH oxidase NoxA and NoxB complexes (Becker et al ., ). The inability of nox mutants to develop expressoria results in the formation of extensive subcuticular growth of hyphae, which eventually breach the surface of the leaves.…”

“…i). However, once on the cell surface, the cell walls of mutant hyphae, like WT hyphae, were remodelled with both cell wall and septa binding WGA‐AF488 (captured in blue pseudocolour), whereas only the septa of endophytic hyphae bind WGA‐AF488 (Becker et al ., ).…”

“…However, a recent study has shown that E. festucae does form appressoria‐like structures, named expressoria, that allow endophytic hyphae to breach the cuticle from the inside of the leaf to form a net of epiphyllous hyphae on the surface of the leaf. Deletion of noxA and noxR abolishes expressoria formation in E. festucae and, instead, the mutant hyphae form convoluted branched structures under the surface of the leaf cuticle (Becker et al ., ). Although some WT‐like expressoria structures were observed on the adaxial surface of L. perenne leaves infected with the Δ mobC mutant, more commonly, subcuticular, highly convoluted structures were observed, suggesting that MobC is important, but not absolutely required, for expressoria development.…”

“…Blue pseudocolour (PMT1, 498–551 nm) was assigned to emission fluorescence from WGA‐AF488 excited with the 488‐nm argon ion laser. Two pseudocolours were assigned to emission fluorescence from aniline blue and plant autofluorescence (PMT2, 571–633 nm, green; PMT3, 661–800 nm, red) as the result of excitation with the 561‐nm DPSS laser (Becker et al ., ). For TEM, pseudostem sections were fixed in 3% glutaraldehyde and 2% formaldehyde in 0.1 m phosphate buffer, pH 7.2, for 1 h, as described previously (Spiers and Hopcroft, ).…”

Apoplastic fungal effectors in historic perspective; a personal view

“…Ectomycorrhizal, endophytic fungi and extracellular fungal plant pathogens colonize the apoplast – the free diffusional space outside the plasma membrane of plant cells – of their hosts (Lo Presti et al ., ; Pellegrin et al ., ; Becker et al ., ). In this review, I focus mainly on the search for Avr factors secreted by fungal plant pathogens that colonize the apoplastic space.…”