PROJECT SUMMARY Opportunistic fungal infection of immune-compromised individuals is an escalating world health problem, recently highlighted in a report from the World Health Organization. Lethal outbreaks of multi drug-resistant Candida auris in hospitals and the rise of drug resistance in normally benign commensal fungal species like Candida glabrata highlight the severity of the problem. Even severe COVID-19 cases facilitate secondary infection by fungal pathogens like Aspergillus and Candida that can be lethal. Current treatment options for fungal infections are limited to a few antifungal drug classes that are becoming increasingly ineffective. There is a pressing need for new molecular targets for antifungal development to deal with drug-resistant pathogens. Our central objective is to establish auxin-inducible degradation (AID) technology in Candida pathogens to enable functional studies of virulence and drug resistance factors and as a tool to facilitate antifungal target validation in the early stages of antifungal drug discovery. AID provides rapid and specific depletion of target proteins of interest and has key advantages over other common methods for protein functional characterization. In Aim 1 we will engineer molecular biology reagents and strains, and establish protocols, to validate and implement a modified AID system in C. albicans, C. glabrata, and C. auris. Our novel system should be applicable in any strain, including clinical isolates, of Candida pathogen species. Validation experiments will use novel virulence and drug resistance factors identified in our labs. In Aim 2 we will combine AID technology in Candida species with two common animal infection models, Galleria mellonella (waxworm) larvae and immunosuppressed mice, to create systems for early target validation and in vivo simulation of drug effects on pathogenesis. These systems will also be applied to our novel candidate antifungal targets. AID is a powerful functional genomics tool that will enable new research opportunities in fungal pathogens. Reagents and protocols established during the project will be made available to the research community, and the work will establish a blueprint for expanding AID use to other diverse fungal pathogens. Overall, this technological platform will address the pressing need for identification and validation of viable new targets for antifungal therapeutic development.