Project Summary Candida albicans is a frequent commensal of the human microbiota and an important opportunistic pathogen. This fungus is chameleon-like in its ability to grow in alternative cellular states and different morphological forms, and this plasticity is critical for infection of diverse niches in the body. Here, we examine the transcriptional regulation of cell fate decisions that drive key developmental programs in C. albicans including filamentation, biofilm formation, and phenotypic switching. Each of these programs contributes to the ability of this fungus to colonize and/or cause disease in the mammalian host. Previous studies have identified multiple transcription factors (TFs) that regulate these developmental programs and showed that they act together in highly coordinated networks to drive gene expression. However, a major knowledge gap exists as to how cell fate-defining TFs act together in a coordinated manner rather than as individual entities. To address this gap, we highlight preliminary data revealing that network TFs can undergo liquid-liquid phase separation (LLPS) and demonstrate that this process enables the formation of complexes containing multiple network TFs. We further show that phase separation is driven by prion-like domains (PrLDs) present in each TF and, critically, that targeted mutation of these domains can abolish both LLPS and TF function. To build on these exciting observations, experiments outlined in Aim 1 will determine how the composition of PrLDs promotes LLPS and the formation of multifactorial complexes in vitro and in cells. We also address how changes in LLPS relate to the function of C. albicans TFs within key networks, including those controlling phenotypic switching and biofilm formation. In Aim 2, we will identify additional regulators of C. albicans cell fate using an overexpression library covering all TFs in the C. albicans genome. Preliminary data indicates that multiple novel regulators can be uncovered by this approach, and newly identified TFs will be integrated into existing transcriptional networks using a variety of approaches including the use of complex haploinsufficiency (CHI) analysis. In Aim 3, we examine how phase separation of C. albicans TFs impacts their function during commensalism and pathogenesis by testing mutant TFs in murine models of infection. These experiments will be facilitated by a barcode sequencing (barcode-SEQ) approach in which multiple strains can be evaluated in parallel for their competitive fitness. Together, these studies will lead to new insights into the fundamental mechanisms by which transcription factors regulate cell fate decisions in C. albicans, with an emphasis on how LLPS enables the formation of functional, multifactorial TF complexes. We will also identify novel TFs in these networks and test these TFs for their role in infectivity. Given the central role of TFs in regulating C. albicans cell fate, these studies will identify new targets...