PROJECT SUMMARY Candida albicans is both a component of the human mycobiome as well as one of the most important human fungal pathogens, capable of causing disease in both immunocompetent and immunocompromised people. The pathogenic cycle that leads to invasive C. albicans disease can be divided into three steps: 1) transition from mucosal colonization to sub-epithelial invasion; 2) filamentous morphogenesis within the sub-epithelium leading to tissue damage; and 3) intravascular dissemination to target organs. Infections that stop after the first two steps lead to mucosal diseases such as oropharyngeal candidiasis (OPC) and vulvovaginal candidiasis while those that progress to the third step cause life-threating candidemia and deep organ disease. Although all three steps are required for the development of invasive disease, the third has been the most extensively studied through the widely used tail-vein inoculation mouse model of disseminated infection. In this application, we propose to use our barcoded collections of transcription factor (TF) and protein kinase (PK) mutants in competitive fitness assays to genetically define the global regulatory networks and key downstream effectors required for OPC mucosal infection (Aim 1). This aim will be facilitated by our recent development of an in vivo RNA-seq approach to characterize the transcriptional profile of C. albicans during OPC. In Aim 2, we will characterize the functions of the TF networks and identify the upstream PK signaling pathways that regulate in vivo filamentation. We will identify PK substrates critical for in vivo filament initiation and elongation. The successful execution of these aims will provide an unprecedentedly detailed in vivo profile of the regulatory mechanisms for the first two steps in C. albicans pathogenesis. Since PKs have emerged as important therapeutic targets across multiple areas of medicine, identification of PK networks with critical roles in C. albicans pathogenesis also holds promise to provide new insights with relevance to antifungal drug development.