Characteristics of gel-phase conidiophores in <i>A. fumigatus.</i>

<p>Pictures of gel-phase conidiophores were taken from slide culture (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074805#pone.0074805.s002" target="_blank">Figure S2</a>) of strain AF293 embedded in GMM agar media from 48 to 120 hour after inoculation. Bars  =  50 µm. (A) Time lapse tracking of a conidiophore developing secondary conidiophores. Elongated phialides (black arrowheads) and secondary conidiophores (white arrowheads) were observed at 78 hour and 96 hour after inoculation, respectively. (B) Time lapse tracking of embedded phialides associated with a vesicle (v). An extensively elongated phialide than the others (at 120 hour) is marked with black arrowheads. (C-E) Various morphologies of conidiophores in the gel-phase environment. (F-G) Conidiophores in transition stage between gel and air (basal stalks are embedded; but vesicles, phialides and conidia are exposed to air). Arrows indicates boundary interface between aerial and agar layer.</p

Oxygen dependent development of <i>A. fumigatus</i> conidiophore.

<p><i>A. fumigatus</i> (AF293) colonies grown on thin agar blocks of GMM using the sandwiched culture method (see Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074805#pone.0074805.s002" target="_blank">Figure S2</a>). Series of pictures were concatenated by using photomerge function of Adobe Photoshop Elements 9. Pictures were taken 72 hours after inoculation. The locations of embedded vesicles are indicated by black arrowheads. Bars  = 100 µm. (A) Colonies in ambient air. (B) Colonies in oxygen-saturated agar block.</p

Conidiophore development of the mutants lacking regulatory genes under gel-phase and aerial environments.

<p><i>A.fumigatus</i> mutants and WT (AF293) strains grown in gel-phase with or without oxygen (left and middle panel, respectively, bars  =  30 µm) and under aeriform environment (right panel, bars  =  200 µm). Pictures were taken 96 hours after inoculation. The locations of embedded vesicles are indicated by black arrowheads.</p

Phialide development and ROS production in the <i>ΔracA</i> mutant were partially restored at temperature shifting condition.

<p><b>(A)</b> Conidiophore development of the <i>racA</i> mutant grown at 37°C only (top) or initially at 37°C, then switched to 25°C (bottom). Bars = 10 μm. <b>(B)</b> ROS distributions in the <i>ΔracA</i> mutant conidiophores after temperature switching from 37°C to 25°C. Bars = 20 μm.</p

Treatment of NADPH-oxidase inhibitor partially mimics the <i>ΔracA</i> mutant phenotype.

<p><b>(A)</b> Four representative developmental stages of AF293 phialides are shown in the top panel (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149548#sec007" target="_blank">Results</a> 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>(B)</b> Four representative developmental stages of the <b><i>Δ</i></b><i>racA</i> 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.</p

Expression of central regulatory genes of <i>A. fumigatus</i> in response to oxygen and physical environments.

<p>Total RNA extracted from <i>A. fumigatus</i> colonies grown in gel-phase or aeriform environment with or without oxygen (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074805#pone-0074805-g005" target="_blank">figure 5</a>). Colonies were cultured on GMM agar media from upside (aeriform colony) or downside (gel-phase colony) of a cellophane membrane for 96 hours at 30°C. Bars are mean values and error bars are standard deviation of relative expression levels normalized with Ct values of β-tubulin. n  =  3. Environmental conditions for the expression analysis are as follows; (A) aeriform and low-oxygen condition, (B) aeriform and high-oxygen condition, (C) gel-phase and low-oxygen condition, and (D) gel-phase and high-oxygen condition.</p

Expression of central regulatory genes are reduced in the <i>ΔracA</i> mutant.

<p>Total RNA extracted from AF293 and the <i>ΔracA</i> 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.</p

Suggested Model for conidiogenesis regulation in <i>A</i>. <i>fumigatus</i>.

<p>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.</p

ROS accumulates in conidiophore vesicles of <i>A</i>. <i>fumigatus</i>.

<p>Agar blocks including the embedded conidiophores were prepared from an <i>A</i>. <i>fumigatus</i> 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; <b>(A)</b> vesicle stage, <b>(B)</b> after phialide budding, <b>(C)</b> and phialide elongation stage. Bars = 100 μm.</p

The <i>A</i>. <i>fumigatus ΔracA</i> is defective in phialide development.

<p>Agar blocks including aerial or embedded conidiophores were prepared from an <i>A</i>. <i>fumigatus</i> colony grown on GMM agar plate (30°C, 4 days after inoculation). Vesicles of the <i>ΔracA</i> are indicated with black arrowheads. <b>(A and B)</b> Aerial conidiophores of the wild type (A) and the <i>ΔracA</i> mutant (B). Bars = 50 μm. <b>(C and D)</b> Conidiophores of the wild type (C) and the <i>ΔracA</i> mutant (D) stained with 25 μM Calcofluor White. Bars = 15 μm. <b>(E and F)</b> Agar-embedded conidiophores of the wild type (E) and the <i>ΔracA</i> mutant (F). Bars = 50 μm. <b>(G—I)</b> Embedded conidiopohres of the wild type (G) and the <i>ΔracA</i> mutant (H and I) stained with 0.5 mM NBT.</p