Project Summary Opportunistic fungal infections can be life-threatening and difficult to treat. Identifying the genetic and molecular mechanisms that enable fungi to persist in humans could have major health benefits for society, potentially even enabling the development of more effective antifungal therapies. The model organism Saccharomyces cerevisiae is itself an opportunistic human pathogen, with many strains isolated from clinical infections. The ability to infect and persist within humans is not universal among S. cerevisiae strains. Clinical S. cerevisiae isolates tend to be highly heterozygous diploids that can grow at higher temperatures and invade into surfaces. However, rigorous genetic dissection of S. cerevisiae’s persistence and pathogenicity within mammalian hosts is needed. To begin such work, we used chromosomally- encoded barcodes and lineage tracking to phenotype a panel of genotyped haploid progeny from a budding yeast cross in mice. The specific cross employed was between a haploid derivative of a clinical isolate and the reference strain. Linkage mapping identified dozens of loci influencing fungal persistence within a mammalian host, many of which lack previously identified candidate genes and show host organ-dependent effects. Following our work, major questions remain unanswered, including the genetic, molecular, and physiological mechanisms underlying yeast persistence and yeast-host interactions; how alleles at causal loci shape the phenotypes of highly heterozygous diploids resembling clinical isolates; the role of surface attachment and invasion in persistence and pathogenicity; and whether the effects of causal loci contributing to fungal pathogenicity have effects that depend on host genotype. Here, we will extend our work by (1) studying mechanisms causing yeast persistence in particular organs by cloning causal genes in yeast, as well as by using cutting-edge microscopy and RNA-seq to analyze yeast-host interactions; (2) testing how combinations of pathogenicity alleles combine in highly heterozygous diploid yeast strains; (3) analyzing how the ability to attach to and invade into surfaces influences the pathogenicity of cross progeny; and (4) examining the genetics of fungal pathogenicity across genetically distinct mouse hosts. Our proposal will utilize the untapped potential of the budding yeast model system to provide concrete insights into the genetics and molecular mechanisms underlying opportunistic fungal pathogenicity.