The role of TcsC in the stress-induced developmental program leading to a fluffy growth phenotype.

<p>Drop dilution assays were performed on AMM plates (supplemented with ammonium). Panel A: 1% oxygen; B: 2% oxygen; C: 2 mM farnesol; D: 200 µM farnesol; E: 2 mM farnesol; F: 100 mM MgSO₄; G: 100 mM CaCl₂; H: 100 mM MgSO₄; I: 50 mM CaCl₂; J: 500 mM CaCl₂. Side views of colonies from C and D are shown in panels E and F. The depicted colonies were photographed after 48 h at 37°C. AfS35 (top/left); Δ<i>tcsC</i> (middle); complemented strain (bottom/right).</p

Schematic model of the biological activities of <i>A. fumigatus</i> TcsC.

<p>Schematic model of the biological activities of <i>A. fumigatus</i> TcsC.</p

Infection of immuno-compromized mice.

<p>Intranasal infection of cortisone-acetate treated mice infected with 1×10⁶ conidia of the Δ<i>tcsC</i> mutant (n = 20), the parental strain AfS35 (n = 20) and the complemented strain (n = 20). Controls received PBS only. Survival of mice is shown over time.</p

Impact of fludioxonil on <i>A. fumigatus</i> germ tubes.

<p>Conidia of the Δ<i>tcsC</i> mutant (panels A, C, E, G) and its parental strain AfS35 (panels B, D, F, H) were seeded on glass cover slips and incubated overnight in AMM at 30°C. The resulting germ tubes were treated with 1 µg/ml fludioxonil for 2 h (A, B), 4 h (C, D) and 6 h (E–H) at 37°C. A DAPI staining is shown in panels G and H. Arrows indicate lysed cells that lack intracellular nuclei and are associated with amorphous extracellular material. All bars represent 10 µm.</p

The impact of cAMP and light on the fluffy growth phenotype.

<p>Drop dilution assays were performed on AMM plates (supplemented with ammonium). The plates were supplemented or treated as indicated and incubated in incubator. When indicated plates were incubated under white light produced by an LED light source. Pictures were taken after 48 h at 37°C.</p

The Δ<i>tcsC</i> mutant is sensitive to hyperosmotic stress and resistant to fludioxonil.

<p>Drop dilution assays were performed on AMM plates (supplemented with ammonium). Panel A: control; B: 1.2 M sorbitol; C: 1 M KCl; D: 100 µg/ml congo red; E: 1 µg/ml fludioxonil. The depicted colonies were obtained after 48 h at 37°C. Top: AfS35; middle: Δ<i>tcsC</i>; bottom: complemented strain.</p

The role of TcsC in the phosphorylation of SakA.

<p>Protein extracts of resting conidia (RC)(panel A) and germlings (panel B) were analyzed by immunoblot using specific antibodies to phosphorylated SakA and as a loading control mitochondrial MnSOD. Extracts were prepared from germlings treated with 10 µg/ml fludioxonil and 1.2 M sorbitol for 2 and 20 min, respectively. A: parental strain AfS35, B: Δ<i>tcsC</i> mutant, C: complemented mutant.</p

Growth of the Δ<i>tcsC</i> mutant.

<p>Colonies of the AfS35 wild type and the Δ<i>tcsC</i> mutant grown for 72 h on AMM plates are shown in panels A/C and B/D, respectively. Magnifications of the edge of the colonies are depicted in panels C and D. Note the reduced number of extending hyphae in the mutant. Panel E: Quantification of the radial growth of AfS35 (black), Δ<i>tcsC</i> mutant (white) and complemented mutant colonies (gray) on AMM plates after 48 h and 96 h at 37°C or 48°C. Panel F: Quantification of the radial growth after 96 h of AfS35 (black), Δ<i>tcsC</i> mutant (white) and complemented mutant colonies (gray) on AMM plates supplemented with 1.4 M NaNO3 or 0.2 M ammonium tartrate at 37°C. The experiments shown in panels E and F were done in triplicate. Standard deviations are indicated. Student’s <i>t</i>-test: *p<0.005; **p<0.001.</p