PROJECT SUMMARY The pathogenic fungus Candida albicans can form biofilms on both abiotic and mucosal surfaces. The presence of biofilms is a significant cause of morbidity and mortality for individuals with C. albicans infections. Biofilm formation in C. albicans is mediated through a core network of 6 transcription factors, one of which is the zinc cluster transcription factor, Rob1. We discovered that a deletion of one ROB1 allele results in heterozygous rob1?/ROB1 isolates with two distinct and disparate phenotypes. One set of rob1?/ROB1 isolates had decreased biofilm formation and decreased hyphae production while the second set of rob1?/ROB1 isolates had increased biofilm formation and hyphae production exceeding that of wild type. We analyzed the ROB1 ORF in SC5314 and identified a single nucleotide polymorphism (SNP) that distinguishes the two alleles of ROB1 and codes for either a serine or proline at amino acid position 946. The rob1?/ROB1 mutants containing Ser946 have increased hyphae and biofilm formation, while mutants with Pro946 have decreased hyphae and biofilm formation. Based on this data we developed the hypothesis that the SNP in ROB1 impacts the activity of Rob1. To address this hypothesis we propose the following specific aim: Aim 1: Determine the functional significance of ROB1 allelic heterozygosity in C. albicans. We next analyzed whole genome sequencing data from 20 clinical isolates of C. albicans to identify additional mutations in ROB1 and found multiple variants of ROB1 relative to the SC5314 reference sequence. These clinical isolates also have varying ability to form biofilms. Based on these observations, we hypothesize that mutations in ROB1 influence the ability of clinical isolates to form biofilms. To test this hypothesis we propose the following specific aim: Aim 2: Characterize the role of ROB1 indels and nonsynonymous SNPs on the ability of C. albicans clinical isolates to form biofilms. We performed an in silico preliminary screen of 82 Zn(2)Cys6 transcription factors in C. albicans and identified 52 genes with allelic heterozygosity. We hypothesize that these allelic variations influence transcription factor function. We will test this hypothesis in the proposed specific aim: Aim 3: Identify and characterize Zn(2)Cys6 transcription factors with functionally significant allelic heterozygosity. This study will be the first to screen and characterize allelic heterozygosity of zinc cluster transcription factors in C. albicans and to characterize the isoforms of Rob1 in different C. albicans clinical isolates.