Schematic overview of the tetracycline-regulated gene expression system used in this study. The hygromycin resistance gene, hph, is under the control of seven copies of the TetR binding sequence, tetO, linked to a minimal promoter (Pmin). In the tTA-dependent expression system (A), the tTA protein (green circles) promotes hygromycin resistance of A. fumigatus by binding to the tetO promoter and activating transcription of the hph gene. Incorporation of doxycycline into the medium prevents tTA from binding to tetO, resulting in hygromycin sensitivity due to the absence of hph expression. The reverse tTA system (B) takes advantage of a reverse tetracycline transactivator, rtTA (blue circles), that binds to the tetO promoter only in the presence of doxycycline. In this case, rtTA promotes hygromycin resistance of A. fumigatus only when grown in the presence of doxycycline.