Figure 2.

ArfA is essential in A. niger, and modifications to arfA expression cause sensitivity to various secretory stressors. An ArfA gain-of-function strain FH1.1 was constructed by placing the arfA gene under control of the doxycycline inducible Tet-on system. For loss-of-function analyses, strain MF45.5 was generated by deletion of the native arfA gene in isolate FH1.1. All growth was compared in biological triplicate to the isogenic progenitor strain (MF31.2). 5 × 10³ spores were inoculated on MM supplemented with various abiotic stressors that cause perturbation of the fungal secretory pathway or cell wall. Plates were incubated at 30°C for 3 days, after which representative images of colony growth were captured. Various concentrations of doxycycline (DOX) enabled titration of arfA expression in conditional expression mutants. Perturbation with congo red (CR) and calcofluor white (CFW) are indicated. Complete lack of growth in MF45.5 lacking DOX indicated ArfA is essential in A. niger.