PROJECT SUMMARY Aspergillus fumigatus is a major human fungal pathogen and an important threat to a variety of patient groups. A few other closely related species in Aspergillus section Fumigati, are also pathogenic. In contrast, most other closely related species in the section are either not known to be pathogenic or very rarely cause disease. Pathogenic species appear to have originated repeatedly from non-pathogens, suggesting that pathogenicity has evolved multiple times independently in this lineage. Furthermore, species on the pathogenic end of the spectrum tend to be found in the environment in comparable numbers with species on the non-pathogenic end, suggesting that the observed differences in pathogenicity are genetic. Several key traits are known to be associated with Aspergillus pathogenicity, such as the ability for fast growth at high temperatures or in nutrient-limiting conditions and the ability to produce small, bioactive molecules known as secondary metabolites. Similarly, several genes have been associated with Aspergillus fumigatus virulence, although more await discovery. Surprisingly, little is known about how pathogenicity-related traits and genes vary across pathogenic and non-pathogenic species belonging to the Aspergillus section Fumigati. Hypothesizing that detailed functional and genomic comparisons between and within species on the pathogenic end of the spectrum and species on the non-pathogenic end will identify new genetic factors and key adaptations associated with the ability to cause human disease, this project aims to compare and contrast Aspergillus species and strains spanning the pathogenicity spectrum at multiple levels of biological and chemical complexity. We will achieve the goals of this project by carrying out three specific aims. In the first aim, we will examine variation in the response to a range of key infection-relevant environments between and within species spanning the pathogenicity spectrum. In the second aim, we will use the same set of strains to discover their genomic and gene expression differences that control whether they are able to cause human disease or not. In the third and final aim, we will describe the roles distinct genes play in fungal pathogenicity and analyze how specific, or general, those contributions to fungal pathogenicity are to organisms across the section Fumigati. The proposed experiments will shed light on the biological, chemical, and genomic similarities and differences between a set of very closely related species and strains spanning the pathogenicity spectrum, thus advancing our understanding of the molecular foundations of the evolution of virulence in the genus Aspergillus, one of the major genera associated with human fungal disease. More generally, our comparative and diverse methods will aid in the establishment of a broader approach for understanding fungal disease and how major pathogens can evolve from historically innocuous organisms.