Cell cycle-dependent and independent mating blocks ensure fungal zygote survival and ploidy maintenance

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly 2 haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins postfusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 not only imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least 2 mechanisms where the zygotic fate imposed by Mei2 and the cell cycle reentry triggered by Mei3 synergize to prevent zygotic mating.

mCherry is expresed from the strong p act1 promoter (822bp upstream START codon) integrated at the ade6 locus. PmeI linearized pAV0614 was used to obtain blasticidin-S resistant transformants.
The ade6 locus is linked to the construct for cytosolic sfGFP expression from the M-gamete specific p mam1 * promoter (1751bp upstream START codon, single nucleotide mutated), the bsdMX selection cassette and the construct for mCherry expression from the P-gamete specific p map3 promoter (2063bp upstream START codon).
Mei3 expression is regulated by the strong, constitutive p tdh1 promoter (1000bp upstream START codon) and the budding yeast transcriptional terminator linked to the ade6 locus. The eGFP is fused to the N-terminus of the S-phase marker Pcn1 and regulated by the pcn1 promoter and the 3'UTR followed by the nmt1 terminator. The construct is integrated at the his5 locus and linked with the bleMX selection marker. sfGFP is expressed from the strong p tdh1 promoter, the construct linked to kanMX selection cassette and integrated in the intergenic region between aha1 and SPBC1711.09c loci.
lys3+::p map3 :mCherry::natMX SpeI linearized pAV0543 was used to obtain nourseothricin resistant transformants. mCherry is expressed from the P-gamete specific p map3 promoter (2063bp upstream START codon), the construct integrated at the lys3 locus and linked with the natMX selection cassette. The GFP is placed under the regulation of the M-cell specific p mam2 promoter (438bp upstrem the START codon) and integrated at the mam2 genomic locus.
Native rlc1 is fused with C-terminal sfGFP and linked with natMX resistance cassete.
Native scd2 is fussed with C-terminal eGFP and linked with the patMX selection cassette.
Native uch2 is fused to C-terminal mCherry and linked with natMX resistance cassete.
Mei2 is expressed from the zygote-specific p mei3 promoter (1054bp upstream START codon), linked with hphMX selection cassete and integarted at the ura4 locus.
The eGFP is fused to the N-terminus of the S-phase marker Pcn1 and regulated by the pcn1 promoter and the 3'UTR followed by the nmt1 terminator. The construct is integrated at the ura4 locus and linked with the bsdMX selection cassette. AfeI linearized pAV0620 was used to obtain blasticidin-S resistant transformants.
The eGFP is fused to the N-terminus of the S-phase marker Pcn1 and regulated by the pcn1 promoter and the 3'UTR followed by the nmt1 terminator. The construct is integrated at the ura4 locus and linked with the natMX selection cassette. AfeI linearized pAV0728 was transformed into ura4-D18 mutant followed by selection for uracil prototrophs The DegGreen construct. The N-terminus of E3 ligase Pof1 (a.a. 1-261) is fused to the GFP-binding protein (GBP) and expresssed under the regulation of the strong p tdh1 promoter (1000bp upstream the START codon) and the budding yeast ADH1 transcriptional terminator. The construct is integrated at the ura4 locus.
Mei3 is under the regulation of the strong, constitutive p tdh1 promoter (896bp upstream START codon) and integrated at the ura4 locus.
ura4+::p tdh1* :sfGFP:terminator tdh1 AfeI linearized pAV0569 was transformed into ura4-D18 mutant followed by selection for uracil prototrophs sfGFP is is under the regulation of the strong p tdh1* promoter (896bp upstream START codon) and integrated at the ura4 locus.