Fig 1.
ROS accumulates in conidiophore vesicles of A. fumigatus.
Agar blocks including the embedded conidiophores were prepared from an A. fumigatus colony grown on GMM agar plate (30°C, 4 days after inoculation) by a described sectioning method (Chi and Craven, 2013), and stained with 0.5 mM nitroblue tetrazolium (NBT). Pictures represent various developmental stages of immature conidiophores; (A) vesicle stage, (B) after phialide budding, (C) and phialide elongation stage. Bars = 100 μm.
Fig 2.
The A. fumigatus ΔracA is defective in phialide development.
Agar blocks including aerial or embedded conidiophores were prepared from an A. fumigatus colony grown on GMM agar plate (30°C, 4 days after inoculation). Vesicles of the ΔracA are indicated with black arrowheads. (A and B) Aerial conidiophores of the wild type (A) and the ΔracA mutant (B). Bars = 50 μm. (C and D) Conidiophores of the wild type (C) and the ΔracA mutant (D) stained with 25 μM Calcofluor White. Bars = 15 μm. (E and F) Agar-embedded conidiophores of the wild type (E) and the ΔracA mutant (F). Bars = 50 μm. (G—I) Embedded conidiopohres of the wild type (G) and the ΔracA mutant (H and I) stained with 0.5 mM NBT.
Fig 3.
Conidiation in the ΔracA mutant is partially restored in some stress conditions.
(A) Aerial conidiophores of the wild type and the ΔracA mutant on minimal media with (top) or without (bottom) 1% glucose. Colonies were grown for 6 days at 37°C. Bars = 100 μm. (B) Colonies of the wild type and the ΔracA mutant on GMM grown at 370°C (top) or initially at 37°C, then switched to 25°C for 24 hours (bottom). White arrow indicates zone of conidiation. (C) The wild type and the ΔracA mutant on GMM with 1 M sorbitol 6 days at 37°C.
Fig 4.
Phialide development and ROS production in the ΔracA mutant were partially restored at temperature shifting condition.
(A) Conidiophore development of the racA mutant grown at 37°C only (top) or initially at 37°C, then switched to 25°C (bottom). Bars = 10 μm. (B) ROS distributions in the ΔracA mutant conidiophores after temperature switching from 37°C to 25°C. Bars = 20 μm.
Fig 5.
Treatment of NADPH-oxidase inhibitor partially mimics the ΔracA mutant phenotype.
(A) Four representative developmental stages of AF293 phialides are shown in the top panel (see Results for detailed description). Bars = 20 μm. After 16 hour treatment in liquid GMM (with or without DPI), the number of conidiophores grouping into each of four defined categories were counted and the ratio of each to the total was calculated (for 0 μM condition, n = 135, 133, and 179; for 1 μM condition, n = 78, 65, and 94, respectively). Means ± s.d. from three biological replicates are displayed under each category. A visualized graph of the ratio is shown in the right panel. Note that ratios determined for 1 μM treatment are significantly different from untreated, as evaluated by t-test (n.s = not significant, **; p<0.05; ***; p<0.01). (B) Four representative developmental stages of the ΔracA phialides are shown in the left panel [note that there is stage 2-M (mature) instead of stage 4 because of aerial induction]. Bars = 20 μm. After 22 hours of air and light exposure, the number of conidiophores falling in each category were counted and calculated in ratio (n = 77, and 127, respectively. Means ± s.d. from two biological replicates were displayed for each category. Visualized graph of the ratio is shown in the right panel.
Fig 6.
Expression of central regulatory genes are reduced in the ΔracA mutant.
Total RNA extracted from AF293 and the ΔracA colonies grown on GMM agar media from on top of a cellophane membrane for 96 hours at 30°C. Bars are mean values from three technical replicates normalized with Ct values of β-tubulin gene.
Fig 7.
Suggested Model for conidiogenesis regulation in A. fumigatus.
Current pictorial model describing the central regulatory pathway in a typical aerial environment is presented. The novel factors identified in this study are highlighted in blue.