A genetic strategy to allow detection of F-actin by phalloidin staining in diverse fungi

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 fungi <i>Aureobasidium pullulans</i> and <i>Aspergillus nidulans</i> by introducing the point mutation <i>act1^V75I</i> at the native <i>ACT1</i> locus confers phalloidin binding while retaining actin function. This strategy should enable characterization of F-actin in a wider range of fungi.IMPORTANCEHigh-resolution tools to visualize filamentous actin networks are critical to the investigation of organisms' cell biology. The gold standard tool is fluorescent phalloidin, a mushroom toxin. However, several fungi have actin that fails to stain with phalloidin. Here, we describe a way to reverse that failure, rendering the invisible actin visible.