VibA, a Homologue of a Transcription Factor for Fungal Heterokaryon Incompatibility, Is Involved in Antifungal Compound Production in the Plant-Symbiotic Fungus Epichloë festucae

Deletion of E. festucae vibA. (A) Physical maps of the E. festucae wild-type vibA genomic region, the integration site of pNPP1 in mutant 830, the linear insert of the replacement construct pNPP151, the E. festucae vibA mutant genomic region, and the insert of the complementation construct pNPP152, showing restriction enzyme sites for BamHI (B), EcoRI (EI), EcoRV (EV), ClaI (C), and XhoI (X). (B) Autoradiograph of a DNA gel blot of EcoRI (EI), ClaI (C), and EcoRV (EV) genomic digests of mutant 830 of E. festucae E437, probed with ³²P-labeled pNPP1. Sizes (in kilobases) of markers are indicated at left. (C) PCR-based verification of vibA mutants. Primer pairs used are indicated to the right. The locations of primers are indicated in panel A. (D) Autoradiograph of a Southern blot of XhoI genomic digests of E. festucae wild-type E437 (WT) and vibA deletion strains (ΔvibA), probed with ³²P-labeled pNPP151. Sizes (in kilobases) of markers are indicated at left.

(A) The deduced protein sequence of E. festucae VibA (EfVibA) was aligned with those of Fusarium oxysporum Vib1 (FoVib1; accession no. FOXB_17342), Magnaporthe oryzae Vib1 (MoVib1; accession no. MGG_00729), Neurospora crassa Vib1 (NcVib1; accession no. NCU03725.5), and Saccharomyces cerevisiae Ndt80p by ClustalW ver. 2.1 (34) with default settings. The NDT80/PhoG DNA binding domain is indicated with a red box. A predicted bipartite nuclear localization signal (NLS) is indicated in a blue box. The C-terminal tail of Ndt80p predicted to make contact with the DNA major groove (54) is underlined, and the arginine residue required for the function of Ndt80p in Candida albicans (55) is indicated by an asterisk. (B) Localization of VibA-GFP in hyphae of E. festucae. VibA-GFP was expressed under the control of a TEF promoter in E. festucae E437 and then monitored by confocal laser scanning microscopy. (Bottom) Hypha of E. festucae expressing VibA-GFP and stained with DAPI. Bars = 10 μm (upper panel) and 2 μm (lower panel).

Effects of vibA gene deletion and overexpression on colony morphology, radial growth, and antimicrobial activity against D. erythrospila. (A) Colony morphologies of E. festucae wild-type strain E437 [WT (E437)], wild-type strain Fl1 [WT (Fl1)], the vibA deletion mutant (ΔvibA-1), a vibA-complemented transformant (ΔvibA/VibA-12), and a vibA-overexpressing strain (Ptef::VibA-1) on PDA before (left) and after (right) pathogen inoculation. The E. festucae strains were allowed to grow on PD agar for 7 days before a mycelial block of the pathogen was placed on the culture plate. (B) Radial growth of E. festucae strains 7 days after inoculation onto PDA. Error bars indicate the standard deviations. Data marked with asterisks are significantly different from the control (WT E437) as assessed by two-tailed Student's t test (**, P < 0.01). (C) Relative expression of vibA in E. festucae strains in axenic culture. Total RNAs were isolated from mycelia of E. festucae strains grown in PD broth for 7 days, and relative expression levels of vibA in endophyte strains were normalized against the actin gene. Different letters indicate significant differences as assessed by two-tailed Student's t test (P < 0.05).

Inhibitory activities of culture filtrates of E. festucae wild-type E437 [WT (E437)], a vibA-overexpressing strain (Ptef::VibA-1), a vibA deletion mutant (ΔvibA-1), and a vibA-complemented transformant (ΔvibA/VibA-12) on conidial germination of D. erythrospila. (A) Percentages of germinated conidia after 12 h of incubation in PD broth or culture filtrate of E. festucae strains. The frequency of germinated conidia was calculated from at least 150 spores. Data are means ± standard errors from 3 biological replicates (>50 spores/experiment). Data marked with asterisks are significantly different from the control (WT E437) as assessed by two-tailed Student's t test (**, P < 0.01). (B) Comparison of inhibitory activities of culture filtrates of E. festucae wild-type E437 and the vibA-overexpressing transformant on conidial germination of D. erythrospila. Morphologies of conidia are shown for D. erythrospila after 12 h of incubation in 100% or diluted culture filtrate of WT E437 or the vibA-overexpressing transformant. Values in the image panels refer to concentrations of endophyte culture filtrate. Percentages of germinated conidia after 12 h of incubation in different concentrations of culture filtrates of E. festucae strains are shown. The frequency of germinated conidia was calculated from at least 150 spores. Data are means ± standard errors from three biological replicates (>50 spores/experiment). Data marked with asterisks are significantly different from the control (WT E437) as assessed by two-tailed Student's t test (**, P < 0.01).

vibA expression is enhanced when E. festucae is challenged by the pathogen. A transformant expressing GFP under the control of the vibA promoter (PvibA::GFP) was subjected to an inhibition assay with D. erythrospila. The micrographs show GFP fluorescence of endophyte hyphae not confronted (left) and challenged (right) by the pathogen.

Inhibitory activity of vibA-overexpressing transformant (Ptef::VibA-1) toward mycelial growth of grass pathogens. Colonies of E. festucae wild-type E437 (left side) and a vibA-overexpressing transformant (right side) were grown for 7 days before a mycelial plug of grass pathogen was inoculated. Each culture was incubated at 23°C for 7 to 14 days, until a clear zone of inhibition was observed.