Epimutations driven by RNAi or heterochromatin evoke antifungal drug resistance in human fungal pathogens

Antimicrobial resistance is a global health threat emerging through microbe adaptation, driven by genetic variation, genome plasticity or epigenetic regulation. This study investigates how the Mucor circinelloides species complex adapts to the antifungal natural product FK506. In Mucor bainieri , most resistant strains exhibit unstable phenotypes without genetic changes. Approximately ∼50% of FK506-resistant isolates acquire resistance via RNAi-dependent epimutation, where small interfering RNAs (siRNAs) silence fkbA transcription. The remaining isolates undergo heterochromatin-mediated silencing via H3K9 methylation and siRNAs spreading, repressing fkbA and neighboring genes. One isolate retained only heterochromatin marks without detectable siRNAs. A similar mechanism operates in Mucor atramentarius , where FK506 resistance is mediated by ectopic heterochromatin associated with siRNA. Strikingly, heterochromatin-based epimutation inheritance remains stable following in vivo infection. These findings reveal that antifungal resistance can arise through distinct, heritable epigenetic pathways involving RNAi, heterochromatin, or both highlighting adaptive strategies employed by ubiquitous eukaryotic microbial pathogens infecting humans.