yEvo: Experimental evolution in high school classrooms selects for novel mutations and epistatic interactions that impact clotrimazole resistance in S. cerevisiae.

Antifungal resistance in pathogenic fungi is a growing global health concern. Non-pathogenic laboratory strains of Saccharomyces cerevisiae are a useful model for studying mechanisms of antifungal resistance that are relevant to understanding the same processes in pathogenic fungi. We developed a series of lab modules in which high school students used experimental evolution to study antifungal resistance by isolating azole-resistant S. cerevisiae and examining the genetic basis of resistance. All 99 sequenced clones from these experiments possessed mutations previously shown to impact azole resistance, demonstrating the efficacy of our protocols. We additionally found recurrent mutations in an mRNA degradation pathway and an uncharacterized mitochondrial protein (Csf1) that have possible mechanistic connections to azole resistance. The scale of replication in this high school-led initiative allowed us to identify epistatic interactions, as evidenced by pairs of mutations that occur in the same clone more frequently than expected by chance (positive epistasis) or less frequently (negative epistasis). We validated one of these pairs, a negative epistatic interaction between gain-of-function mutations in the multidrug resistance transcription factors Pdr1 and Pdr3. This high school-university collaboration can serve as a model for involving members of the broader public in the scientific process to make meaningful discoveries in biomedical research.