Figure 2.

Creation of the alcA(p)∷GFP-mobA strain. (A) Diagram showing the strategy of the creation of the conditional alcA(p)∷GFP-mobA strain. Note that pLB04B-mobA contains only a 5′ truncated sequence from the mobA gene. The integration of pLB04B-mob1 into the mobA locus in the wild-type genome renders a full-length mobA in fusion with GFP under the control of the alcA promoter. (B) Southern blot analysis showed the hybridization patterns of the control strain and four candidate transformants using the probe indicated in A. The control strain rendered a 2.1-kb band. If the plasmid was integrated once into the mobA locus, two bands of 1.1 and 7.2 kb should be detected. Thus, nos. 10, 15, and 26 transformants were such strains. The no. 10 strain was designated as LBA49 and used for further experiments. (C) The control (GR5) and the alcA(p)∷GFP-mobA (LBA49) strains were grown on rich medium containing glucose (bottom), and on minimal medium with glycerol as the sole carbon source (top). Under repressive conditions (with glucose), LBA49 failed to conidiate, while GR5 demonstrated robust conidiating capacity. Under inducing conditions (with glycerol), the two strains grew identically, indicating that the GFP–MOBA fusion protein was as functional as the wild-type MOBA. (D) Under repressive conditions (with glucose), a GR5 (control) hypha produced a septum (arrow). Under identical conditions, the LBA49 hypha failed to septate for an even longer time. Bar, 10 μm.