Fig 6.

Impairment of spore germination and nuclear division by RAC and cell cycle inhibitors in the WT and mutant strains. Due to the lack of conidial production by the Δbcrac and Δbcras1 mutants, the effects of the inhibitors on germinating conidia were determined in conidia of the H1-GFP strain, which is a B05.10 (WT) strain expressing the H1-GFP cassette for live visualization of nuclei by fluorescence microscopy. (A) Conidia of the H1-GFP strain were germinated on a microscope slide in Gamborg's B5 medium with the specified compounds. Samples were analyzed following incubation for 24 h at 22°C. (a to c) Subinhibitory concentrations of NSC23766 (NSC) (RAC inhibitor; 0.5 mM), hydroxyurea (HU) (G₁/S inhibitor; 0.3 M), and benomyl (BEN) (G₂/M inhibitor; 250 μg ml⁻¹). (d to f) Inhibitory concentrations of NSC (0.85 mM), HU (0.5 M), and BEN (500 μg ml⁻¹). (g and h) Combinations of subinhibitory concentrations of NSC (0.5 mM) with HU (0.3 M) (g) and BEN (250 μg ml⁻¹) (h). (i) Control. (B) Mycelia of all strains were cultured for 3 days on PDA medium covered with cellophane, and plugs of fresh mycelium were removed and placed on a glass slide in a droplet of Gamborg's B5 medium containing the desired concentration of the inhibitory compounds: 0.5 mM NSC, 0.3 M HU, 250 μg ml⁻¹ BEN, or the combination of NSC with BEN. The slides with mycelia were incubated at 22°C for 24 h and then inspected microscopically. (j to m) WT; (n to q) CA-BcRAC strain; (r to u) Δbcrac strain; (v to y) Δbcras1 strain. White bars on the bottom left of each image represent the average numbers of nuclei per 10 μm of hypha ± SE. Empty squares represent no detectable growth. Bars = 10 μm.