A single Ho-induced double-strand break at theMATlocus is lethal inCandida glabrata

Abstract

AbstractMating-type switching is a complex mechanism that promotes sexual reproduction in Ascomycotina. In the model speciesSaccharomyces cerevisiae, mating-type switching is initiated by the Ho endonuclease that performs a site-specific double-strand break (DSB) atMAT, repaired by homologous recombination (HR) using one of the two silent mating type cassettes,HMLalphaandHMRa. 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 yeastCandida glabrata, phylogenetically close toS. cerevisiae,and for which no spontaneous and efficient mating-type switching has been observed. We have previously shown that expression ofS. cerevisiae’sHOgene triggers mating-type switching inC. glabrata, but this leads to massive cell death. In addition, we unexpectedly found, that not onlyMATbut alsoHMLwas cut in this species, suggesting the formation of multiple chromosomal DSBs uponHOinduction.We now report thatHMRis also cut byS. cerevisiae’s Ho in wild-type strains ofC. glabrata.To understand the link between mating-type switching and cell death inC. glabrata, we constructed strains mutated precisely at the Ho recognition sites. By mimickingS. cerevisiae’s situation, in whichHMLandHMRare protected from the cut, we unexpectedly find that one DSB atMATis sufficient to induce cell death. We demonstrate that mating-type switching inC. glabratacan be triggered using CRISPR-Cas9, without high lethality. We also show that switching is Rad51-dependent, as inS. cerevisiaebut that donor preference is not conserved inC. glabrata.Altogether, these results suggest that a DSB atMATcan be repaired by HR inC. glabrata, but that it is prevented byS. cerevisiae’s 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 yeastS. cerevisiaein which it relies on a programmed double-strand break performed by the Ho endonuclease at theMATlocus which encodes the key regulators of sexual identity. Little is known about why the mating-type switching components have been conserved in species likeC.glabrata,in which neither sexual reproduction nor mating-type switching is observed. We have previously shown that mating-type switching can be triggered, inC. glabrata, by expression of theHOgene fromS. cerevisiaebut this leads to massive cell death. We report here evidence toward a degeneration of the mating-type switching system inC. glabrata. We demonstrate that the DSB atMATis only lethal when the Ho endonuclease performs the break, a situation unique toC. glabrata.Finally, we show that mating-type switching inC. glabratacan be triggered by CRISPR-Cas9 and without any high lethality.