The filamentous fungus Aspergillus fumigatus is quickly becoming a major threat to immunocompromised individuals such as transplant and chemotherapy patients, at a time when there are astonishingly few potent antifungal compounds available to control or cure these life-threatening infections. The goal of this Phase II project is to enable and improve screening for antifungal hit compounds that will yield novel classes of potent antifungal drugs. Under Phase I funding for this grant, we established proof-of-principle for a novel method of identifying essential genes in A. fumigatus based on conditional expression of random antisense (AS) RNA. Under Phase II funding we will apply this technology systematically, with the goal of defining and cataloging a significant number of essential genes in this organism. We will also exploit the regulated expression of target-specific AS-RNA to develop hypersensitive whole-cell drug screens capable of detecting target-specific inhibitors of fungal growth. The molecular techniques developed for A. fumigatus under Phase I funding have allowed Elitra to extend our previous Candida albicans-based target identification and validation efforts to a second important fungal pathogen. The availability of promoter replacements of particular genes of interest in both organisms will greatly expand our options for primary and secondary screening under Phase II funding and will thus greatly enhance our ongoing efforts to develop novel, broad-spectrum antifungal drugs. [unreadable] [unreadable]