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

Abstract

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. The reasons why all the elements of the mating-type switching system have been conserved in some Ascomycotina, that do not show a sexual cycle nor mating-type switching, remain 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. cerevisiaes 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. cerevisiaes 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. cerevisiaes 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 demonstrate 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. cerevisiaes Ho. Author summaryMating-type switching is one of the strategies developed by fungi to promote crossing, sexual reproduction and propagation. This mechanism enables one haploid cell to give rise to a cell of the opposite mating-type so that they can mate together. It has been extensively studied in the model yeast S. cerevisiae in which it relies on a programmed double-strand break performed by the Ho endonuclease at the MAT locus which encodes the key regulators of sexual identity. Little is known about why the mating-type switching components have been conserved in species like C.glabrata, in which neither sexual reproduction nor mating-type switching is observed. We have previously shown that mating-type switching can be triggered, in C. glabrata, by expression of the HO gene from S. cerevisiae but this leads to massive cell death. We report here evidence toward a degeneration of the mating-type switching system in C. glabrata. We demonstrate that the DSB at MAT is only lethal when the Ho endonuclease performs the break, a situation unique to C. glabrata. Finally, we show that mating-type switching in C. glabrata can be triggered by CRISPR-Cas9 and without any high lethality.