A single Ho-induced double-strand break at the MAT locus is lethal in Candida glabrata

Mating-type switching is a complex mechanism that promotes sexual reproduction in Ascomycotina. In the model species Saccharomyces cerevisiae, mating-type switching is initiated by the Ho endonuclease that performs a site-specific double-strand break (DSB) at MAT, repaired by homologous recombination (HR) using one of the two silent mating type cassettes, HMLalpha and HMRa. Why have all the elements of the mating-type switching system been conserved in some Ascomycotina, that do not show a sexual cycle nor mating-type switching remains unknown. To gain insight on this phenomenon, we used the opportunistic pathogenic yeast Candida glabrata, phylogenetically close to S. cerevisiae, and for which no spontaneous and efficient mating-type switching has been observed. We have previously shown that expression of S. cerevisiae9s HO gene triggers mating-type switching in C. glabrata, but this leads to massive cell death. In addition, we unexpectedly found, that not only MAT but also HML was cut in this species, suggesting the formation of multiple chromosomal DSBs upon HO induction. We now report that HMR is also cut by S. cerevisiae9s Ho in wild-type strains of C. glabrata. To understand the link between mating-type switching and cell death in C. glabrata, we constructed strains mutated precisely at the Ho recognition sites. By mimicking S. cerevisiae9s situation, in which HML and HMR are protected from the cut, we unexpectedly find that one DSB at MAT is sufficient to induce cell death. We also show for the first time that mating-type switching in C. glabrata can be triggered using CRISPR-Cas9, without high lethality. We also show that switching is Rad51-dependent, as in S. cerevisiae but that donor preference is not conserved in C. glabrata. Altogether, these results suggest that a DSB at MAT can be repaired by HR in C. glabrata, but that it is prevented by S. cerevisiae9s Ho.