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

Summary

Scientists discovered that many fungi cannot be stained with phalloidin, a widely-used fluorescent dye that helps visualize actin filaments in cells. They traced this problem to a single amino acid difference in fungal actin proteins. By changing this one amino acid back to its original form using genetic engineering, they successfully enabled phalloidin staining in previously incompatible fungi, providing researchers with better tools to study fungal cell biology.

Background

Phalloidin is the gold standard fluorescent reagent for detecting polymerized actin in fixed cells, but many ascomycete fungi fail to bind phalloidin despite actin being highly conserved across eukaryotes. This limitation has hindered progress in studying actin cytoskeleton dynamics in diverse fungal systems.

Objective

To identify the molecular basis for phalloidin binding failure in ascomycete fungi and develop a genetic strategy to restore phalloidin binding capability in fungal species.

Results

A single amino acid change at position 75 (isoleucine to valine) in actin correlates with phalloidin binding failure. Reverting this change via the V75I point mutation restored phalloidin binding in both A. pullulans and A. nidulans while maintaining normal actin function and cellular morphology.

Conclusion

This genetic strategy enables phalloidin-based F-actin visualization in fungal species previously inaccessible to this gold-standard technique, significantly expanding tools for investigating actin cytoskeleton organization across diverse fungi.
  • Published in:mSphere,
  • Study Type:Research Study,
  • Source: PMID: 41020595, doi: 10.1128/msphere.00517-25
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