Wirshing, A. C.; Colarusso, A. V.; Lew, D. J.

Actin is highly conserved across eukaryotes. This versatile protein builds cytoskeletal networks central to diverse cellular processes, including cell division and cell motility. The most potent and broadly used reagents to detect polymerized actin distribution in fixed cells are fluorescently conjugated derivatives of the basidiomycete-derived toxin, phalloidin. However, despite its conservation, actin in many ascomycete fungi fails to bind phalloidin. Here we trace the failure to bind phalloidin to a single amino acid change in a phalloidin-binding residue in actin. Reverting this change in the fungus Aureobasidium pullulans by introducing the point mutation act1V75I at the native ACT1 locus confers phalloidin binding without disrupting actin function. We describe a simple genetic technique to introduce this point mutation that may be effective in other fungal systems. This strategy should enable characterization of F-actin in a wider range of fungi.