Allodiploidization is a fundamental yet evolutionarily poorly characterized event, which impacts genome evolution and heredity, controlling organismal development and polyploid cell-types. In this study, we investigated the sex determination system in the allodiploid and sterile ATCC 42981 yeast, a member of the Zygosaccharomyces rouxii species complex, and used it to study how a chimeric mating-type gene repertoire contributes to hybrid reproductive isolation. We found that ATCC 42981 has 7 MAT-like (MTL) loci, 3 of which encode α-idiomorph and 4 encode a-idiomorph. Two phylogenetically divergent MAT expression loci were identified on different chromosomes, accounting for a hybrid a/α genotype. Furthermore, extra a-idimorph-encoding loci (termed MTLa copies 1 to 3) were recognized, which shared the same MATa1 ORFs but diverged for MATa2 genes. Each MAT expression locus was linked to a HML silent cassette, while the corresponding HMR loci were located on another chromosome. Two putative parental sex chromosome pairs contributed to this unusual genomic architecture: one came from an as-yet-undescribed taxon, which has the NCYC 3042 strain as a unique representative, while the other did not match any MAT-HML and HMR organizations previously described in Z. rouxii species. This chimeric rearrangement produces two copies of the HO gene, which encode for putatively functional endonucleases essential for mating-type switching. Although both a and α coding sequences, which are required to obtain a functional cell-type a1-α2 regulator, were present in the allodiploid ATCC 42981 genome, the transcriptional circuit, which regulates entry into meiosis in response to meiosis-inducing salt stress, appeared to be turned off. Furthermore, haploid and α-specific genes, such as MATα1 and HO, were observed to be actively transcribed and up-regulated under hypersaline stress. Overall, these evidences demonstrate that ATCC 42981 is unable to repress haploid α-specific genes and to activate meiosis in response to stress. We argue that sequence divergence within the chimeric a1-α2 heterodimer could be involved in the generation of negative epistasis, contributing to the allodiploid sterility and the dysregulation of cell identity.
Here, we report draft genome sequences of the halotolerant and allodiploid strains Zygosaccharomyces rouxii ATCC 42981 and Zygosaccharomyces sapae ABT301T. Illumina and Oxford Nanopore MinION sequencing revealed genome sizes of 20.9 and 24.7 Mb, respectively.
The pre-whole genome duplication (WGD) Zygosaccharomyces clade comprises several allodiploid strain/species with industrially interesting traits. The salt-tolerant yeast ATCC42981 is a sterile and allodiploid strain which contains two subgenomes, one of them resembling the haploid parental species Z. rouxii . Recently, different mating-type-like ( MTL ) loci repertoires were reported for ATCC42981 and the Japanese strain JCM22060, which are considered two stocks of the same strain. MTL reconstruction by direct sequencing approach is challenging due to gene redundancy, structure complexities, and allodiploid nature of ATCC42981. Here, DBG2OLC and MaSuRCA hybrid de novo assemblies of ONT and Illumina reads were combined with in vitro long PCR to definitively solve these incongruences. ATCC42981 exhibits several chimeric MTL loci resulting from reciprocal translocation between parental haplotypes and retains two MAT a/ MAT α expression loci, in contrast to MAT α in JCM22060. Consistently to these reconstructions, JCM22060, but not ATCC42981, undergoes mating and meiosis. To ascertain whether the damage of one allele at the MAT locus regains the complete sexual cycle in ATCC42981, we removed the MAT α expressed locus by gene deletion. The resulting MAT a/- hemizygous mutants did not show any evidence of sporulation, as well as of self- and out-crossing fertility, probably because incomplete silencing at the chimeric HML α cassette masks the loss of heterozygosity at the MAT locus. We also found that MAT α deletion switched off a2 transcription, an activator of a-specific genes in pre-WGD species. These findings suggest that regulatory scheme of cell identity needs to be further investigated in Z. rouxii protoploid yeast.
In haploid Saccharomyces cerevisiae, a complex recombination system regulates mating-type switching and requires one MAT expression locus, two donor cassettes (HML and HMR) and the HO endonuclease that catalyses gene conversion. Zygosaccharomyces rouxii is the most distant species from S. cerevisiae with a functional HO, but with a poorly understood mating-type switching. Here, we described that two subcultures of the type strain CBS 732T underwent the α to a genotype switching leading to mixed MATα and MATa populations. Remarkably, during this event the donor cassette was copied into the MAT locus, except for its own 3΄ end, resulting in a new MATa2 gene copy different from the silenced HMRa2. Moreover, CBS 732T cells bypassed the cell-cycle control, which oversees HO transcription in S. cerevisiae, and expressed HO at the stationary phase. Despite HO dysregulation, mating-type switching seemed to occur rarely or belatedly during CBS 732T colony formation in most of the tested conditions. When morphology and mating behaviour were analysed, two subcultures displayed distinct outcross fertility responses. Overall, our data support that mating-type switching causes genotype instability and phenotypic novelties in CBS 732T, and open the question whether this mechanism is shared by other Z. rouxii haploid homothallic strains.
The so‐called nonconventional yeasts are becoming increasingly attractive in food and industrial biotechnology. Among them, Zygosaccharomyces rouxii is known to be halotolerant, osmotolerant, petite negative, and poorly Crabtree positive. These traits and the high fermentative vigour make this species very appealing for industrial and food applications. Nevertheless, the biotechnological exploitation of Z. rouxii has been biased by the low availability of genetic engineering tools and the recalcitrance of this yeast towards the most conventional transformation procedures. Centromeric and episomal Z. rouxii plasmids have been successfully constructed with prototrophic markers, which limited their usage to auxotrophic strains, mainly derived from the Z. rouxii haploid type strain Centraalbureau voor Schimmelcultures (CBS) 732T. By contrast, the majority of industrially promising Z. rouxii yeasts are prototrophic and allodiploid/aneuploid strains. In order to expand the genetic tools for manipulating these strains, we developed two centromeric and two episomal vectors harbouring KanMXR and ClonNATR as dominant drug resistance markers, respectively. We also constructed the plasmid pGRCRE that allows the Cre recombinase‐mediated marker recycling during multiple gene deletions. As proof of concept, pGRCRE was successfully used to rescue the kanMX–loxP module in Z. rouxii ATCC 42981 G418‐resistant mutants previously constructed by replacing the MATαP expression locus with the loxP–kanMX–loxP cassette.
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