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.

Two closely related Rho GTPases, Cdc42 and RacA, of the en-dophytic fungus Epichloë festucae have contrasting roles for ROS production and symbiotic infection synchronized with the host plant.

Epichloë festucae is an endophytic fungus which systemically colonizes temperate grasses to establish symbiotic associations. Maintaining symptomless infection is a key requirement for endophytes, a feature that distinguishes them from pathogenic fungi. While pathogenic fungi extend their hyphae by tip growth, hyphae of E. festucae systemically colonize the intercellular space of expanding host leaves via a unique mechanism of hyphal intercalary growth. This study reports that two homologous Rho GTPases, Cdc42 and RacA, have distinctive roles in the regulation of E. festucae growth in planta. Here we highlight the vital role of Cdc42 for intercalary hyphal growth, as well as involvement of RacA in regulation of hyphal network formation, and demonstrate the consequences of mutations in these genes on plant tissue infection. Functions of Cdc42 and RacA are mediated via interactions with BemA and NoxR respectively, which are expected components of the ROS producing NOX complex. Symbiotic defects found in the racA mutant were rescued by introduction of a Cdc42 with key amino acids substitutions crucial for RacA function, highlighting the significance of the specific interactions of these GTPases with BemA and NoxR for their functional differentiation in symbiotic infection.

ProA, a transcriptional regulator of fungal fruiting body development, regulates leaf hyphal network development in the Epichloë festucae-Lolium perenne symbiosis.

Transcription factors containing a Zn(II)2 Cys6 binuclear cluster DNA-binding domain are unique to fungi and are key regulators of fungal growth and development. The C6-Zn transcription factor, Pro1, in Sordaria macrospora is crucial for maturation of sexual fruiting bodies. In a forward genetic screen to identify Epichloë festucae symbiosis genes we identified a mutant with an insertion in proA. Plants infected with the proA mutant underwent premature senescence. Hyphae of ΔproA had a proliferative pattern of growth within the leaves of Lolium perenne. Targeted deletion of proA recapitulated this phenotype and introduction of a wild-type gene complemented the mutation. ΔproA was defective in hyphal fusion. qPCR analysis of E. festucae homologues of S. macrospora genes differentially expressed in Δpro1 identified esdC, encoding a glycogen-binding protein, as a target of ProA. Electrophoretic mobility shift assay analysis identified two binding sites for ProA in the intergenic region of esdC and a divergently transcribed gene, EF320. Both esdC and EF320 are highly expressed in a wild-type E. festucae-grass association but downregulated in a proA-mutant association. These results show that ProA is a key regulator of in planta specific growth of E. festucae, and therefore crucial for maintaining a mutualistic symbiotic interaction.

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.