Chimeric Sex-Determining Chromosomal Regions and Dysregulation of Cell-Type Identity in a Sterile Zygosaccharomyces Allodiploid Yeast

Abstract

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.

Fig 1. Phylogenetic analysis of MATα1 proteins.

The neighbour-joining (NJ) tree shows the phylogenetic relationships between the allodiploid strain ATCC 42981 and other hemiascomycetes inferred from MATα1 proteins. Numbers on branches indicate bootstrap support percentages (1,000 pseudoreplicates) higher than 60% from NJ. The red branch indicates the Z. rouxii yeast complex, which includes Z. rouxii MATα1, ATCC 42981 MATα1 copies 1 and 2, Z. sapae MATα1 copies 1, 2, and 3 sequences. The dark dot indicates WGD species, whereas the dark triangle indicates non-WGD species with the HO gene.

Fig 2. Phylogenetic analysis of MATα2 proteins.

The neighbour-joining (NJ) tree shows the phylogenetic relationships between the allodiploid strain ATCC 42981 and other hemiascomycetes inferred from MATα2 proteins. Numbers on branches indicate bootstrap support percentages (1,000 pseudoreplicates) higher than 60% from NJ. The red branch indicates Z. rouxii complex, which includes Z. rouxii MATα2, ATCC 42981 MATα2 copies 1 and 2, and Z. sapae MATα2 copies 1 to 3 sequences. The dark dot indicates WGD species, whereas the dark triangle indicates non-WGD species with the HO gene.

Fig 3. Phylogenetic analysis of MATa2 proteins.

The neighbour-joining (NJ) tree shows the phylogenetic relationships between the allodiploid strain ATCC 42981 and other hemiascomycetes inferred from MATa2 proteins. Numbers on branches indicate bootstrap support percentages (1,000 pseudoreplicates) higher than 60% from NJ. The red branch indicates the Z. rouxii complex, which includes Z. rouxii MATa2, Z. sapae MATa2 and ATCC 42981 MATa2 copies 1 to 3 sequences. The dark dot indicates WGD species, whereas the dark triangle indicates non-WGD species with the HO gene.

Fig 4. Sequence comparison of MATa2 proteins.

Alignment of MATa2 from Z. rouxii (ZrMATa2, GenBank: XP002496430), Z. sapae (ZsMATa2, GenBank: CDM87352), ATCC 42981 MATa copies 1 to 3 and Torulaspora delbrueckii (TdMATa2, GenBank: XP003682598). The MATA HMG domain, which binds the minor groove of DNA, is noted (horizontal black bar). In both alignments, the amino acid identities were coloured according the Clustal X colour scheme and the conservation index at each alignment position were provided by Jalview [50].

Fig 5. Phylogenetic analysis of HO endonucleases.

Neighbor-joining (NJ) tree shows evolutionary relationships between ATCC 42981 strain and other hemiascomycetes as inferred from HO proteins. Numbers on branches indicate bootstrap support (1,000 pseudoreplicates) from NJ. The red branch indicates Z. rouxii complex, which includes ZrHO, ZsHOs and ATCC 42981 HO sequences, whereas the dark triangle designates pre-WGD species.

Fig 6. Inferred genomic organization around MAT-like loci in the ATCC 42981 allodiploid genome.

Two sex homologous/homeologous chromosome pairs are depicted, namely F/F’ and G/G’. Chromosome G bears the MATα copy 2 expression locus, which is linked to the putative silent cassettes HML copy 2, whereas chromosome F’ bears the MATa copy 2 expression locus, which is linked to the putative silent cassette HML copy 1. Chromosomes F and G’ harbour three a-idiomorph HMR loci, which differ for MATa2 genes. The HMR copy 2 locus is on chromosome F, while the HMR copies 1 and 3 loci are on chromosome G’. Blue arrows represent Z. rouxii-like (blue bordered) and Z. sapae-like (light green bordered) α-idiomorph loci. Red arrows indicate a-idiomorph loci with different MATa2 genes: copy 1 (dark red surrounded), copy 2 (dark green), copy 3 (grey), respectively. Chromosomal organization of the three-cassette system in Z. rouxii haploid strain CBS 732^T was reported for comparative purposes according to Souciet et al. [34]. CHA1_L indicates the ZYRO0F18524g locus, while dark green and light blue arrows indicate the ZYRO0F18634g and ZYRO0C18392g loci, respectively at the right side of mating-type loci.

Fig 7. Expression pattern of mating-type, HO and IME4 genes.

Panel A reports positive amplified cDNAs generated with MAT and HO copy variant-specific primers from ATCC 42981 unstressed cells in stationary growth phase. Panel B depicts IME4-specific PCR products generated from CBS 732^T and ATCC 42981 cDNA IME4 antisense (AS-IME4 lncRNA) and sense transcripts (S-IME4 mRNA), respectively. +/- RT indicates addition of reverse transcriptase to the cDNA synthesis reaction. For each RT-PCR reaction gDNA was used as positive control. Abbreviations: AS, anti-sense long non-coding RNA; S, sense mRNA.

Fig 8. Differential expression by quantitative real-time PCR of mating-type and HO genes in ATCC 42981 cells after hyperosmotic stress.

Expression of target genes was normalized on the reference ZrACT1 (GenBank: XM002497273). Fold change was measured by ΔΔCt or PCR Miner methods and reported as the mean (± SEM) of three biological replicates. * indicates significant difference from controls as measured by independent Student's t-tests (*P<0.05, **P<0.01).