Fig 1.
rtfA affects A. fumigatus colony growth.
Aspergillus fumigatus wild type (WT), ΔrtfA, complementation (com), and overexpression rtfA (OErtfA) strains were point-inoculated on GMM and incubated for 4 days at 37°C. (A) Photographs of the colonies. (B) Colony growth estimated as colony diameter. Different letters above the bars indicate significantly different values (p ≤ 0.05). Error bars represent standard error.
Fig 2.
Heterologous complementation with S. cerevisiae rtf1 restores wild-type phenotype in the A. fumigatus rtfA mutant.
Aspergillus fumigatus ΔrtfA strain (TRRM3) was transformed with plasmid pRRM4, which contains rtf1 from S. cerevisiae. (A) Effective complementation with rtf1 was confirmed by diagnostic PCR analysis using primers Scer_rtf1Afum_rtfA_f and Scer_rtf1_Afum_rtfA_r. Plasmid pRRM4 was used as a positive control, yielding the expected 1.7 kb PCR product. Aspergillus fumigatus ΔrtfA strain was used as negative control. (B) A. fumigatus strains WT (CEA10), ΔrtfA (TRRM4), and com-rtf1 (TRRM7) were point-inoculated on GMM and incubated at 37°C. (C) Colony diameters were measured after three days of growth. Values represent the average of three replicates. Different letters above the bars indicate significantly different values (p ≤ 0.05). Error bars represent standard error.
Fig 3.
rtfA is a repressor of conidiation in A. fumigatus.
(A) Wild type (WT), ΔrtfA, complementation (com), and overexpression rtfA (OErtfA) strains were grown in liquid stationary cultures and whole mycelial mats were collected at 48 h and 72 h, homogenized, and conidia were quantified with a hemocytometer. Expression analysis of brlA (B) and wetA (C), two key genes in the conidiation regulatory pathway, at 48 h and 72 h. (D) Micrographs showing conidiation in the ΔrtfA strain when grown in liquid shaking cultures. Cultures were grown at 37°C for 72 h at 250 rpm. Scale bars represent 100 μm in the 10x micrographs and 10 μm in the 40x micrographs.
Fig 4.
rtfA regulates protease activity.
Aspergillus fumigatus wild type (WT), ΔrtfA, complementation (com), and overexpression rtfA (OErtfA) strains were point-inoculated on GMM containing 5% skim milk (Difco) and incubated at 37°C. (A) Quantification of proteolytic activity is shown as measured by Azo-casein assay after four days of growth. Different letters above the bars indicate significantly different values (p ≤ 0.05). Error bars represent standard error. (B) Degradation halos (indicated with arrows) at the edge of the colonies growing on skim milk agar plates for four days.
Fig 5.
rtfA is necessary for normal resistance to oxidative stress.
(A) Sensitivity of A. fumigatus wild type (WT), ΔrtfA, complementation (com), and overexpression rtfA (OErtfA) strains to oxidative stress was examined on GMM containing various concentrations of menadione as indicated. Colony formation was observed after 72 h of incubation at 37°C. (B, C) In a separate experiment, the strains were inoculated in liquid GMM with or without 20 μM of menadione (men). Cultures were incubated at 30°C. Mycelia were collected 6 h after the shift for expression analysis of cat1 (B) and cat2 (C). Different letters above the bars indicate significantly different values (p ≤ 0.05). Error bars represent standard error.
Fig 6.
rtfA regulates the production of secondary metabolites.
Plates containing 25 mL liquid GMM were inoculated with 106 spores/mL of A. fumigatus wild type (WT), deletion (ΔrtfA), complementation (com), and overexpression (OErtfA) strains. Cultures were incubated at 37°C. Mycelia were collected at 48 h and 72 h for expression analysis. Supernatant was collected at 120 h for secondary metabolite extraction and analysis. (A) Analysis of ergot alkaloids and fgaPT2 (dmaW), a key gene in this cluster. (B) Analysis of fumiquinazoline C and fmqA, the gene encoding a NRPS involved in fumiquinazoline C biosynthesis. (C) Analysis of pseurotin A and psoA, encoding the PKS-NRPS in this biosynthetic pathway. (D) Analysis of tryptoquivaline F production. Different letters above the bars indicate significantly different values (p ≤ 0.05). Error bars represent standard error.
Fig 7.
Alteration of rtfA expression has a minor effect on cell wall integrity.
Wild type (WT), deletion (ΔrtfA), complementation (com), and overexpression (OErtfA) strains were point-inoculated on GMM supplemented with increasing concentrations of (A) Congo Red or (B) Nikkomycin Z as indicated. Strains were incubated at 37°C for 72 h (A) or 48 h (B).
Fig 8.
rtfA is indispensable for normal pathogenicity.
Larvae of G. mellonella were infected with conidia of A. fumigatus wild type (WT), ΔrtfA, complementation (com), and overexpression rtfA (OErtfA) strains as described in the experimental procedures section. Thirty larvae were infected for each group, including a control group injected with 1X PBS. Survival was first monitored every two hours and eventually extended to every 4 or 8 hours when the larval mortality rate decreased. (A) Larvae infected with 105 spores and (B) 106 spores. A no injection control and a PBS control were included. (C) Six-week old mice were rendered neutropenic by treatment with a cyclophosphamide and kenalog-10. Neutropenic mice were infected with 106 conidia per mouse of wild type (WT), ΔrtfA, or complementation strains and monitored daily for a total of seven days. Two controls were included, one with a group of non-infected mice rendered neutropenic and a second group with mice neither infected with spores nor treated with immunosuppressants. Statistical analysis was carried out by pairwise comparison using a long rank test.