A cross-species rescue by mating method to interrogate gene essentiality across the Saccharomyces genus

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Wang , Abigail Keller , Omar Kunjo , View ORCID Profile Maitreya J Dunham doi: https://doi.org/10.1101/2025.06.02.657496 Taylor K. Wang 1 Department of Genome Sciences, University of Washington , Seattle, WA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abigail Keller 1 Department of Genome Sciences, University of Washington , Seattle, WA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Omar Kunjo 1 Department of Genome Sciences, University of Washington , Seattle, WA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maitreya J Dunham 1 Department of Genome Sciences, University of Washington , Seattle, WA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maitreya J Dunham For correspondence: maitreya{at}uw.edu Abstract Full Text Info/History Metrics Preview PDF Abstract The field of budding yeast research has long been empowered by the vast array of genetic tools and community resources available. Rescue by mating is one useful tool that entails mating of meiotic spores directly post-germination. This minimal cell division mating process facilitates mating based screens studying the effects of otherwise haploid lethal gene deletions. In this study, we describe the successful application of rescue by mating across two different Saccharomyces species: S. cerevisiae and S. uvarum . This novel inter-species tool enables studies on the evolution of essential genes within the broader Saccharomyces genus. Description Performing genetic experiments with mutations in essential genes requires special approaches that avoid passing through lethal intermediates. Such techniques include the use of conditional mutations, transient rescue plasmids, and hypomorphic alleles. One lesser known but potentially useful approach is rescue by mating, in which a lethal mutation in a haploid is temporarily supplemented by inheritance of protein from a heterozygous diploid progenitor and can mate before death. Rescue by mating was described previously in a study elucidating the mechanisms of spore number control and spore mating in Saccharomyces cerevisiae , but references to the phenomenon go even further back ( Taxis et al. 2005 ; Cox 1971 ). Capable of growing as a haploid or diploid, S. cerevisiae often exist in the wild as diploids due to a variety of reasons ( Mortimer 2000 ; Peter et al. 2018 ; Goddard, Godfray, and Burt 2005 ; Herskowitz 1988 ). Upon encountering starvation conditions, diploid yeast will trigger sporulation ( Freese, Chu, and Freese 1982 ), during which the cell undergoes meiosis to produce four haploid spores within a protective double membrane called the ascus ( Esposito and Esposito 1969 ). Before germination, the four post-meiotic spores within the ascus remain dormant in nutrient poor conditions. After encountering rich media, spores enter the germination cell program and begin dividing ( Palleroni 1961 ). The expectation for a spore which has inherited an essential gene deletion is cell death upon growth ( Hartwell, Culotti, and Reid 1970 ; Giaever et al. 2002 ; Winzeler et al. 1999 ). Working with diploid S. cerevisiae strains heterozygous for an essential gene deletion, Taxis et al. discovered that spores which inherited the essential gene deletion from the mother cell were in fact capable of mating immediately after germination ( Taxis et al. 2005 ). Spores with essential gene deletions can often undergo several cell divisions and form a microcolony before cells become incapable of further mitotic growth and undergo cell death (Rodriguez-Peña et al. 1998; Liu et al. 2015 ). This growth before cell death is understood to stem from the presence of sufficient maternal factors within the spore ( Haarer et al. 2011 ) which are capable of supporting germination and limited growth before cell death. Placement of a suitable mating partner next to the germinating cell can thus result in mating and generation of a viable diploid that is heterozygous for the essential gene of interest. Rescue by mating represents both interesting biology as well as a valuable tool. It has enabled work such as mass-screening studies of complex haploinsufficiency that relies on mating haploid essential gene deletion strains ( Haarer et al. 2011 ). However, thus far work in the field has focused on applying this technique exclusively to S. cerevisiae strains. S. cerevisiae is only a single member of the greater Saccharomyces genus, which contains multiple species representing varying evolutionary distances ( Borneman and Pretorius 2015 ). The greater Saccharomyces genus is of interest due to phenotypic diversity and the adaptability of S. cerevisiae genomic tools to other Saccharomyces species ( Caudy et al. 2013 ; Bleuven et al. 2019 ). In addition, the mating competent Saccharomyces species are all capable of forming cross-species hybrids ( Alix et al. 2017 ; Belloch et al. 2008 ; Bellon et al. 2013 ; 2015; Bernardes, Stelkens, and Greig 2017 ; da Silva et al. 2015 ). In this study we attempted to perform rescue by mating between haploid essential gene deletion S. cerevisiae strains and a wt S. uvarum mating partner—one of two most distantly related Saccharomyces species to S. cerevisiae [ Figure 1A ]. Download figure Open in new tab Figure 1. (A) Schematic depicting the S. cerevisiae and S. uvarum parent strain genotypes and the rescue by mating protocol steps. (B) Results of tetrad dissection for each of the three heterozygous knockout parent strains proving gene essentiality for AME1, BRR6 , and ORC6 via 2:0 viability segregation pattern. Segregation of inviability with KanMX cassette on selection was also confirmed. (C) Recorded number of PCR and Sanger sequence verified S. cerevisiae x S. uvarum hybrid colonies recovered from rescue from mating protocol for each candidate gene. Studying the comparative biology of S. cerevisiae and S. uvarum is of particular interest. Previous work from our lab unveiled a significant number of orthologous genes which are essential only in S. cerevisiae , or only S. uvarum ( Sanchez et al. 2019 ). We leveraged rescue by mating as a tool to study candidate genes which are essential only in S. cerevisiae . The differentially essential genes studied to date can functionally complement across species. Thus, a heterozygous essential gene knockout S. cerevisiae strain could theoretically be used to generate gene knockout spores that could mate with a wild type S. uvarum partner within the rescue by mating protocol. The S. uvarum ortholog of the S. cerevisiae essential gene of interest will rescue the lethality of the essential gene knockout upon spore mating. The differentially essential candidate genes studied here include AME1, BRR6 , and ORC6 ( Sanchez et al. 2019 ). The identities of these specific strains were verified by PCR and sequencing of the barcodes from the original deletion collection construction ( Shoemaker et al. 1996 ). The essentiality of each candidate was confirmed via tetrad dissection of each respective heterozygous knockout parental strain. [ Figure 1B ]. We followed previous mass haploid progeny recovery methods developed to selectively destroy unsporulated diploid cells in a sporulation culture [ Figure 1A ] ( Samsonova et al. 1985 ). These treatments also result in the removal of the ascus surrounding the spores. In brief, sporulation cultures of heterozygous essential gene knockouts were treated with an array of chemical and mechanical stresses as described in the Methods. The post treatment spores were split into two pools. One pool was mixed with a MATa ‘wild type’ S. uvarum partner, while the other was mated to a MATα S. uvarum partner. Performing rescue by mating with both mating types maximizes the number of potential rescue by mating events since both mating types of S. cerevisiae spores containing the essential gene knockout will have a mating partner available. These mating cultures were incubated in a roller drum overnight and surviving cells were plated onto +G418 -ura media to select for diploid hybrids and against unmated haploids: a KanMX cassette was present at the essential gene locus from the S. cerevisiae segregant while the S. uvarum partners were sensitive to G418, and a functional URA3 was only present in the S. uvarum mating partner. Colonies on selection plates were verified as hybrids via PCR and Sanger sequencing. Final counts of recovered hybrid colonies for each mating were recorded [ Figure 1C ]. A small number of colonies were recovered with the correct genetic markers but that did not pass PCR validation due to the presence of both the deletion and wt S. cerevisiae alleles; these false positives appeared to be caused by S. cerevisiae segregants with aneuploidy for the chromosome carrying the essential gene locus. We found that rescue by mating between S. cerevisiae and S. uvarum partners was possible, despite previously observed low inter-species mating frequencies. Previous work has suggested the ability of Saccharomyces spores to exhibit mating partner preference both within and between species ( Jacobs, Gorman, and Lew 2022 ; Strauss et al. 2024 ; McClure et al. 2018 ; Murphy and Zeyl 2012 ; Murphy et al. 2006 ; Knop 2006 ; Jackson and Hartwell 1990 ). These studies are supported by work in our group showing the increased time for mating necessary for inter-species S. cerevisiae x S. uvarum matings compared to intra-species matings for either species: about 1 hour for intraspecies and 3 hours for interspecies. The dynamics of this mating partner preference is just one example of the questions that can be evaluated with the tool of rescue by mating across species. This novel finding that rescue by mating can result in inter-species hybrids suggests interesting biology at the level of conservation between germination and spore mating machinery across the two species within the Saccharomyces genus and provides another important tool in the arsenal to ask evolutionary questions across related Saccharomyces species. Materials and Methods Strains All three heterozygous essential knockout S. cerevisiae strains were sourced from the heterozygous deletion collection originally constructed by Winzeler et al. as described there, which then had the SGA marker suite engineered later ( Tong et al. 2001 ). Strain identity was confirmed by PCR and Sanger sequencing of the uptag barcodes. The S. uvarum rescue mating partners were both derived from the CBS7001 strain background. View this table: View inline View popup Sporulation A single colony of each heterozygous essential gene knockout S. cerevisiae strain was inoculated overnight in 3 mLs of YPD. The next day, cultures were back diluted to 0.2 OD in fresh YPD and outgrown for two more hours. 1 mL of each culture was spun down and washed twice with 1 mL of ddH 2 0. These washed cultures were each resuspended in 5 mL sporulation media consisting of 1% potassium acetate, 0.05% dextrose, and 0.1% yeast extract and placed in a roller drum at 30°C for 4-7 days. Rescue by mating 1 mL of sporulation culture was centrifuged for 10 seconds at 13,000xg. The pellet was resuspended in 5 mL of ddH 2 0. To this suspension 100 µL of 100 Unit/mL YLE, and 10 µL of 2- mercaptoethanol were added. Cultures were incubated overnight at 30°C with gentle shaking. The next day 5 mL of 1.5% triton X-100 was added and tubes were then incubated on ice for 15 minutes. Sonication was performed with the microtip function of a MISONIX Ultrasonic Liquid Processor. The sonicator probe was inserted into the 10 mL of spores and sonication performed as followed: microtip power setting Amplitude of 30, with three cycles total of 20 seconds of sonication and 1 minute on ice. Sonicated spores were then spun down for 10 minutes at 1200xg. The pellet was resuspended in 5 mL of 1.5% triton X-100 and vortexed vigorously. These spores were then spun down for 10 minutes at 1200xg and washed once more. After final centrifugation the spores were resuspended in 5 mL of ddH 2 0 and a 10-fold dilution counted by hemacytometer to confirm the absence of any asci or spore clumps. Spores were diluted if necessary to 1 × 10 3 spores/mL. Matings with the S. uvarum rescue partner were carried out by mixing 200 µL of spores with 200 µL of log phase S. cerevisiae haploid mating partner at equivalent cell density. 200 µL of YPD media was added to this mating mixture, and incubated in a roller drum overnight at 30°C. The following day dilutions of the mating mixtures were plated on C -Ura +G418 Funding This work was supported by National Science Foundation grant 1516330 and by R01 HG010378 from the National Human Genome Research Institute at the National Institutes of Health. Author Contributions Taylor Wang: Investigation, Data curation, Methodology, Formal analysis, Writing – 1 st draft Abigail Keller: Investigation, Data curation, Methodology, Writing – review Omar Kunjo: Data curation, Methodology, Writing – review Maitreya Dunham: Conceptualization, Investigation, Funding acquisition, Project administration, Supervision, Writing – review and editing. References ↵ Alix , Karine , Pierre R. Gérard , Trude Schwarzacher , and J. S. (Pat) Heslop-Harrison . 2017 . “ Polyploidy and Interspecific Hybridization: Partners for Adaptation, Speciation and Evolution in Plants .” Annals of Botany 120 ( 2 ): 183 – 94 . doi: 10.1093/aob/mcx079 . 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