Figure 6.

Localisation of GlaA at the hyphal septa increases following arfA overexpression. Strains expressing a GlaA-dtomato reporter protein, and enabling doxycycline mediated wildtype, reduced, and overexpression of arfA (MF31.2, MF45.5, and FH1.1, respectively), were grown at 22°C for 2 days on MM plates. After 1 h of incubation with liquid MM supplemented with respective concentrations of doxycycline, confocal microscopy of GlaA-dtomato was performed and Z-stack series were taken (dark red = low GlaA-dtomato signal, bright yellow = high GlaA intensity signal. (A) Exemplar fluorescent image of growing colonies demonstrated GlaA localizes to hyphal septa in the control and overexpression strain FH1.1 using 10 μg/ml doxycycline. (B) Septum localized GlaA-dtomato fluorescence of 12 hyphal septa from projected Z-stacks was quantified for MF31.2 and FH1.1 grown in the presence of 10 μg/ml doxycycline, which confirmed increased expression of GlaA, especially at the hyphal septum following arfA overexpression. Standard error bars are shown from 12 replicates. A schematic overview of the region analyzed in this analysis is depicted in (C), whereby we took the septum as the central point and measured up to 2.4 μm from this point either side, giving a total length of 4.8 μm.