Although much is known about the signals and mechanisms that lead to pathogenic interactions between plants and fungi, comparatively little is known about fungus–plant mutualistic symbioses. We describe a novel role for reactive oxygen species (ROS) in regulating the mutualistic interaction between a clavicipitaceous fungal endophyte, Epichloë festucae, and its grass host, Lolium perenne. In wild-type associations, E. festucae grows systemically in intercellular spaces of leaves as infrequently branched hyphae parallel to the leaf axis. A screen to identify symbiotic genes isolated a fungal mutant that altered the interaction from mutualistic to antagonistic. This mutant has a single-copy plasmid insertion in the coding region of a NADPH oxidase gene, noxA. Plants infected with the noxA mutant lose apical dominance, become severely stunted, show precocious senescence, and eventually die. The fungal biomass in these associations is increased dramatically, with hyphae showing increased vacuolation. Deletion of a second NADPH oxidase gene, noxB, had no effect on the E. festucae–perennial ryegrass symbiosis. ROS accumulation was detected cytochemically in the endophyte extracellular matrix and at the interface between the extracellular matrix and host cell walls of meristematic tissue in wild-type but not in noxA mutant associations. These results demonstrate that fungal ROS production is critical in maintaining a mutualistic fungus–plant interaction.

Summary: While much is known about the biosynthesis of secondary metabolites by filamentous fungi their biological role is often less clear. The assumption is these pathways have adaptive value to the organism but often the evidence to support this role is lacking. We provide the first genetic evidence that the fungal produced secondary metabolite, peramine, protects a host plant from insect herbivory. Peramine is a potent insect feeding deterrent synthesized by Epichloë/Neotyphodium mutualistic endophytes in association with their grass hosts. The structure of peramine, a pyrrolopyrazine, suggests that it is the product of a reaction catalysed by a two‐module non‐ribosomal peptide synthetase (NRPS). Candidate sequences for a peramine synthetase were amplified by reverse transcription polymerase chain reaction. Four unique NRPS products were identified, two of which were preferentially expressed in planta. One of these hybridized to known peramine producing strains. This clone was used to isolate an Epichloë festucae cosmid that contained a two‐module NRPS, designated perA. Nine additional genes, which show striking conservation of microsynteny with Fusarium graminearum and other fungal genomes, were identified on the perA‐containing cosmid. Associations between perennial ryegrass and an E. festucae mutant deleted for perA lack detectable levels of peramine. A wild‐type copy of perA complemented the deletion mutant, confirming that perA is a NRPS required for peramine biosynthesis. In a choice bioassay, plant material containing the perA mutant was as susceptible to Argentine stem weevil (ASW) (Listronotus bonariensis) feeding damage as endophyte‐free plants confirming that peramine is the E. festucae metabolite responsible for ASW feeding deterrent activity.

Key requirements for microbes to initiate and establish mutualistic symbiotic interactions with plants are evasion of potential host defense responses and strict control of microbial growth. Reactive oxygen species (ROS) produced by a specific NADPH oxidase isoform, NoxA, regulate hyphal growth in the mutualistic interaction between the fungal endophyte Epichloë festucae and its grass host Lolium perenne. Unlike mammalian systems, little is known about the fungal NADPH oxidase complex and its response to differentiation signals. We identify an E. festucae p67^phox-like regulator, NoxR, dispensable in culture but essential in planta for the symbiotic interaction. Plants infected with a noxR deletion mutant show severe stunting and premature senescence, whereas hyphae in the meristematic tissues show increased branching leading to increased fungal colonization of pseudostem and leaf blade tissue. Inhibition of ROS production or overexpression of noxR recapitulates the hyperbranching phenotype in culture. NoxR interacts in vitro with the small GTP binding protein RacA and requires a functional RacA binding site to complement the noxR mutant and restore the wild-type plant interaction phenotype. These results show that NoxR is a key regulator of NoxA in symbiosis, where it acts together with RacA to spatially regulate ROS production and control hyphal branching and patterning.