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

Laetitia Maroc,Stéphanie Boisnard,Cécile Fairhead,Youfang Zhou-Li,Joseph Heitman

doi:10.1371/journal.pgen.1008627

Abstract

Mating-type switching is a complex mechanism that promotes sexual reproduction in Saccharomycotina. 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 loci, HMLalpha and HMRa. The reasons why all the elements of the mating-type switching system have been conserved in some Saccharomycotina, that do not show a sexual cycle nor mating-type switching, remain unknown. To gain insight on this phenomenon, we used the 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. cerevisiae's Ho (ScHo) 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 ScHo 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. We find that even when HML and HMR are protected from the Ho-cut, introducing a DSB at MAT is sufficient to induce cell death, whereas one DSB at HML or HMR is not. 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 repair is prevented by ScHo.

Highlights

In eukaryotes, sexual reproduction is a nearly ubiquitous feature and implies fundamental conserved processes such as gamete fusion, zygote formation and meiosis [1]
This mechanism enables one haploid cell to give rise to a cell of the opposite mating-type so that they can mate. It has been extensively studied in the yeast S. cerevisiae in which it relies on a programmed double-strand break performed by the Ho endonuclease at the MAT locus which determines sexual identity
We expressed S. cerevisiae’s HO gene (ScHO) using the URA3 selectable plasmid p7.1 in which ScHO is under control of the inducible MET3 promoter [26]

Summary

Introduction

Sexual reproduction is a nearly ubiquitous feature and implies fundamental conserved processes such as gamete fusion, zygote formation and meiosis [1]. It is in the fungal kingdom that the greatest diversity of sexual reproduction is found [1]. Sexual reproduction in fungal pathogens of human exhibits a considerable plasticity between species [2,3]. While many were thought to be asexual, several atypical sexual or parasexual cycles have been discovered. The more distant filamentous opportunistic pathogen, Aspergillus fumigatus exhibits a sexual cycle but only mates after spending 6–12 months in the dark [5]. This suggests that, in most fungi, performing genetic exchange is crucial, even in well-adapted human pathogens

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