Summary Epichloë festucae is an endophytic fungus that forms a symbiotic association with Lolium perenne. Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates of E. festucae. A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from uninfected and infected L. perenne. Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem MS was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using MarVis in combination with the BioCyc and KEGG databases, and an in‐house Epichloë metabolite database. We were able to identify the known endophyte‐specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novel Epichloë‐derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressed in planta. Comparative MS analysis of apoplastic wash fluid from wild‐type‐ vs mutant‐infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

Although lipid signaling has been shown to serve crucial roles in mammals and plants, little is known about this process in filamentous fungi. Here we analyse the contribution of phospholipase D (PLD) and its product phosphatidic acid (PA) in hyphal morphogenesis and growth of Epichloë festucae and Neurospora crassa, and in the establishment of a symbiotic interaction between E. festucae and Lolium perenne.Growth of E. festucae and N. crassa PLD deletion strains in axenic culture, and for E. festucae in association with L. perenne, were analysed by light-, confocal-and electron microscopy. Changes in PA distribution were analysed in E. festucae using a PA biosensor and the impact of these changes on endocytic recycling and superoxide production investigated. We found that E. festucae PldB and the N. crassa ortholog, PLA-7, are required for polarized growth, cell fusion and ascospore development, whereas PldA/PLA-8 are dispensable for these functions. Exogenous addition of PA rescues the cell-fusion phenotype in E. festucae. PldB is also crucial for E. festucae to establish a symbiotic association with L. perenne. This study identifies a new component of the cell-cell communication and cell fusion signaling network that controls hyphal morphogenesis and growth in filamentous fungi.