The endophytic fungus Epichloë festucae systemically colonizes the intercellular spaces of temperate grasses to establish mutualistic symbiotic associations. We have previously shown that reactive oxygen species produced by a specific NADPH oxidase isoform, NoxA, and associated regulators, NoxR and RacA, have a critical role in regulating hyphal growth in the host plant to maintain a mutualistic symbiotic interaction. We also identified BemA and Cdc24, homologues of polarity establishment proteins of yeast, as interactors of NoxR. In this study, we investigated culture developmental phenotypes of 'knockout' mutants of noxA and noxB and their associated regulators, noxR, racA and bemA. On nutrient-rich medium, all of the mutants except racA, which had undulating hyphae, hyphal swellings and increased branching, had a colony growth phenotype similar to the wild type strain. In contrast, on water agar, noxA, noxR and bemA mutants had disorganized hyphal growth and distorted instead of straight hyphae. These changes in hyphal growth characteristics indicate that NoxA and associated regulators have a crucial role in polarized growth under conditions of nutrient starvation. Conidiation in the noxA mutant was greater than wild type, and further enhanced in the noxA/noxB double mutant suggesting ROS negatively regulates asexual development. In contrast, deletion of noxR had no effect on conidiation. Hyphae of the wild type and noxB mutant of E. festucae had frequent vegetative hyphal fusions, whereas noxA, noxR and racA mutants totally lost this ability and fusions in the bemA mutant were significantly reduced. These results indicate that NoxA, NoxB and their associated regulators have distinct or overlapping functions for the regulation of different hyphal morphogenesis processes.
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