Multicellularity in the 1,011 Saccharomyces cerevisiae Genome Panel

The transition from single-celled to multicellular organisms is one of the most important evolutionary transitions in life history. Most microbes are facultatively multicellular– they can live in a single-celled or multicellular form. By studying multicellularity in microbes, we can gain critical insights into how life on earth made the leap from one cell to many. The budding yeast Saccharomyces cerevisiae is capable of forming different kinds of multicellular structures under stressful conditions. Some of these multicellular phenotypes are associated with pathogenicity, and the ability to express the traits varies among strains. This study aims to leverage the power and diversity of the S. cerevisiae 1000 genomes panel to understand the distribution of and the correlation between multicellular phenotypes in a global yeast collection and to identify genetic variation underlying the traits. First, we conducted high-throughput phenotyping assays to screen for five types of multicellular growth: 1) pseudohyphal growth (PSH), 2) invasive growth (IG), 3) plastic adherence (PA), 4) biofilm formation (i.e., complex colony morphology, CCM), and 5) floating mats (flor). Next, we quantified all phenotypes, except for flor, using tailored computational image analysis pipelines. Our results showed that over ⅓ S. cerevisiae strains in the genome panel showed CCM, ⅖ strains displayed pseudohyphae, ⅕ strains grew invasively, ⅕ strain adhered to plastic, and over ⅕ strains formed flor. Employing phylogenetically-corrected correlational analyses, we observed associations between PSH and IG, and flor and PA. Finally, using our phenotyping results, we performed genome wide association studies (GWAS) on normalized values of the traits with corrections for population structure. Analysis of all traits uncovered 4286 significant SNPs, translating to 1960 genes underlying multicellularity expression, supporting a complex basis to, and abundant genetic variation for, multicellularity. A list of 8 genes involved in multicellular traits was narrowed down by focusing on genes that impact multiple phenotypes, contain multiple significant SNPs, and likely represent important targets that contain natural genetic variants. Using our GWAS results to build a protein-protein interaction network, we observed that a couple of genes in this list, MLP2, ELP2, MYO1, and IRA2, interact with over 40 other proteins in the network, further supporting their functional importance in forming multicellularity. Using our GWAS results, Gene Ontology (GO) analysis was conducted to cluster genes enriched in the process of forming multicellularity. Our results show that metabolic, biosynthesis of secondary metabolites, cell cycle, MAPK signaling, and Meiosis pathways are most significantly enriched. Ongoing work includes functionally validating the 8 genes identified through GWAS in the wet lab. Our results show there exists broad phenotypic variation and abundant genetic variation in multicellular traits across the 1000 genomes panel. Understanding the distribution, correlation, and the genetic basis of multicellularity can help us understand the environmental and genetic factors driving the evolutionary transition from single cell to multicellular organisms.