Erg251 has complex and pleiotropic effects on azole susceptibility, filamentation, and stress response phenotypes.

Ergosterol is essential for fungal cell membrane integrity and growth, and numerous antifungal drugs target ergosterol. Inactivation or modification of ergosterol biosynthetic genes can lead to changes in antifungal drug susceptibility, filamentation and stress response. Here, we found that the ergosterol biosynthesis gene is a hotspot for point mutations during adaptation to antifungal drug stress within two distinct genetic backgrounds of . Heterozygous point mutations led to single allele dysfunction of and resulted in azole tolerance in both genetic backgrounds. This is the first known example of point mutations causing azole tolerance in Importantly, single allele dysfunction of in combination with recurrent chromosome aneuploidies resulted in azole resistance. Homozygous deletions of caused increased fitness in low concentrations of fluconazole and decreased fitness in rich medium, especially at low initial cell density. Dysfunction of resulted in transcriptional upregulation of the alternate sterol biosynthesis pathway and , a Zinc transporter. Notably, we determined that overexpression of is sufficient to increase azole tolerance in . Our combined transcriptional and phenotypic analyses revealed the pleiotropic effects of on stress responses including cell wall, osmotic and oxidative stress. Interestingly, while loss of either allele of resulted in similar antifungal drug responses, we observed functional divergence in filamentation regulation between the two alleles of ( and ) with exhibiting a dominant role in the SC5314 genetic background. Finally, in a murine model of systemic infection, homozygous deletion of resulted in decreased virulence while the heterozygous deletion mutants maintain their pathogenicity. Overall, this study provides extensive genetic, transcriptional and phenotypic analysis for the effects of on drug susceptibility, fitness, filamentation and stress responses.