Candida albicans is among the most significant human fungal pathogens. It can undergo two well-studied cell type transitions, yeast-hyphal and white-opaque. The yeast-hyphal transition is required for both virulence and biofilm formation; the white-opaque transition is required for mating. Each transition occurs in distinct growth conditions, and the regulatory network that governs the transitions seems structured to ensure that the transitions are mutually exclusive. Our recent studies indicate that two white-opaque regulators, Wor1 and Wor3, have unexpected positive roles in biofilm and hypha formation and in host cell damage and virulence. Importantly, we find that Wor3-regulated genes are not the typical hypha-associated genes that are already known to promote biofilm, hyphae, and virulence. Our objective is to use this newly discovered role of Wor3 to define new mediators of biofilm and hypha formation and virulence, and to use the analytical strategies that were successful for Wor3 to test the roles of other white-opaque regulators in these central pathogenicity traits. The analysis of Wor3 has exploited two genetic interaction strategies. One is an overexpression- rescue assay, in which we ask if increased expression of one gene can overcome the phenotypic defect of a deletion mutant in another gene. The second is a synthetic defect assay, in which as ask if a deletion mutation in one gene causes a unique or prominent phenotype when combined with a deletion mutation in another gene. Our proposed studies apply these assays systematically to the other Wor regulators in combination with well-known regulators of hypha and biofilm formation to define new gene functions and targets. Our specific aims are to define functional roles of Wor regulators in biofilm and hypha formation, and to define functional roles of Wor regulators in host interaction and virulence. Our longer-term goal is to use novel targets of Wor regulators to define new determinants of pathogenicity and biofilm persistence.