A nuclear protein NsiA from Epichloë festucae interacts with a MAP kinase MpkB and regulates the expression of genes required for symbiotic infection and hyphal cell fusion.

The endophytic fungus Epichloë festucae systemically colonizes the intercellular spaces of cool-season grasses to establish a mutualistic symbiosis. Hyphal growth of the endophyte within the host plant is tightly regulated and synchronized with the growth of the host plant. A genetic screen to identify symbiotic genes identified mutant FR405 that had an antagonistic interaction with the host plant. Perennial ryegrass infected with the FR405 mutant were stunted and underwent premature senescence and death. The disrupted gene in FR405 encodes a nuclear-localized protein, designated as NsiA for nuclear protein for symbiotic infection. Like previously isolated symbiotic mutants the nsiA mutant is defective in hyphal cell fusion. NsiA interacts with Ste12, a C2H2 zinc-finger transcription factor, and a MAP kinase MpkB. Both are known as essential components for cell fusion in other fungal species. In E. festucae, MpkB, but not Ste12, is essential for cell fusion. Expression of several genes required for cell fusion and symbiosis, including proA/adv-1, pro41/ham-6, ham7, ham8, and ham9 were downregulated in the nsiA mutant. However, the NsiA ortholog in Neurospora crassa was not essential for hyphal cell fusion. These results demonstrate that the roles of NsiA and Ste12 orthologs in hyphal cell fusion are distinctive between fungal species.

Differential roles of NADPH oxidases and associated regulators in polarized growth, conidiation and hyphal fusion in the symbiotic fungus Epichloë festucae.

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.