Genome-wide quantification of contributions to sexual fitness identifies genes required for spore viability and health in fission yeast

Abstract

Numerous genes required for sexual reproduction remain to be identified even in simple model species like Schizosaccharomyces pombe. To address this, we developed an assay in S. pombe that couples transposon mutagenesis with high-throughput sequencing (TN-seq) to quantitatively measure the fitness contribution of nonessential genes across the genome to sexual reproduction. This approach identified 532 genes that contribute to sex, including more than 200 that were not previously annotated to be involved in the process, of which more than 150 have orthologs in vertebrates. Among our verified hits was an uncharacterized gene, ifs1 (important for sex), that is required for spore viability. In two other hits, plb1 and alg9, we observed a novel mutant phenotype of poor spore health wherein viable spores are produced, but the spores exhibit low fitness and are rapidly outcompeted by wild type. Finally, we fortuitously discovered that a gene previously thought to be essential, sdg1 (social distancing gene), is instead required for growth at low cell densities and can be rescued by conditioned medium. Our assay will be valuable in further studies of sexual reproduction in S. pombe and identifies multiple candidate genes that could contribute to sexual reproduction in other eukaryotes, including humans.

Laboratory horror stories: Poison in the agars

“…Fission yeast can be cultured as a haploid or as a diploid and these two states can be easily interconverted by mating and meiosis (Kawamukai, 2024;Ohtsuka et al, 2022). This supports powerful genetic approaches that are complemented by equally powerful molecular tools [e.g., (Billmyre et al, 2022;Gao et al, 2014;Ishikawa & Saitoh, 2023;Storey et al, 2019;Torres-Garcia et al, 2020)], which has made fission yeast an eminent model to study a variety of broadly conserved eukaryotic processes [see (Harris et al, 2022;Hoffman et al, 2015;Nurse, 2020;Rutherford et al, 2024) and refs therein]. For example, we use this model organism to discover mechanisms by which specific DNA sequences, their binding proteins, and chromatin remodeling factors control the positioning of meiotic recombination throughout the genome (Mukiza et al, 2019;Protacio et al, 2022;Storey et al, 2018).…”

Going fishing: how to get what you want from a fungal genetic screen

“…Another major focus was on mutants that showed altered morphology, as multiple aggregating strains have been isolated from patients ( 3 – 5 ); screening for altered morphology was the focus of two recent insertional mutagenesis screens ( 6 , 7 ). In other fungi, the interesting biological phenomena and topics of screens range from identifying regulators of sex and meiosis ( 8 ), defining essential genes ( 9 – 11 ), identifying kinases that regulate morphogenesis ( 12 ), determining modulators of host immune responses ( 13 – 15 ), or identifying new potential avenues for antifungal therapies ( 16 – 19 ), to name a few. The critical task is to define what the question is and to spend the time to justify why this is the right question to ask using a screening approach.…”

“…To validate our screen of adherence regulators in C. auris , we used binding to fluorescent polystyrene microspheres and flow cytometry, which both increased the dynamic range of the assay and showed the generalizability of the phenotype across surfaces ( 2 ). For the TnSeq screen to identify sexual regulators in S. pombe, Billmyre and colleagues turned to a viable spore yield assay as a secondary measure of fertility, as well as individual microscopy of spore structure and morphology ( 8 ). These tests were important as they helped define the specific defect of the plb1∆ and alg1∆ mutant strains in gamete health rather than initial formation of spores.…”

“…As a nice example of this, Billmyre and colleagues walk through multiple alternate explanations for their hits, including insertion io essential genes with long-lived transcripts that had not fully been eliminated during mutant pool generation or those with non-sexual-related growth defects ( 8 ). By rapidly ruling out these hits, they were able to focus specifically on those with interesting biology and a specific defect in sexual reproduction.…”

PomBase: a Global Core Biodata Resource—growth, collaboration, and sustainability

“…2022 ), mitotic spindle dynamics ( Lera-Ramirez et al . 2022 ), identification of novel genes involved in sexual reproduction ( Billmyre et al . 2022 ), and defining contractile ring components ( Snider et al .…”