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 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.

Introduction

In eukaryotes, sexual reproduction is a nearly ubiquitous feature and implies fundamental conserved processes such as gamete fusion, zygote formation and meiosis [1]. Sexual reproduction leads to genetic recombination between organisms and thus enables them to purge their genomes from deleterious mutations, as well as to increase their genetic diversity. It is in the fungal kingdom that the greatest diversity of sexual reproduction is found [1]. Particularly, 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. It has been shown that the yeast Candida albicans can perform a parasexual cycle by mating of two diploid cells, forming a tetraploid, that can undergo chromosome loss [4]. The more distant filamentous opportunistic pathogen, Aspergillus fumigatus exhibits a sexual cycle but only mates after spending 6–12 months in the dark [5]. Altogether, this suggests that, in most fungi, performing genetic exchange is crucial, even in well-adapted human pathogens.

In fungi, sexual reproduction can occur through three mechanisms [1]: heterothallism (requiring two compatible partners for mating to occur), homothallism (self-fertility), and pseudo-homothallism (where a single individual can go through a complete sexual cycle but mating only occurs between two compatible partners). Pseudo-homothallism has mainly been described in ascomycete yeasts where it occurs through a programmed differentiation process called mating-type switching [6]. This mechanism enables one haploid cell to give rise to a cell of the opposite mating-type so that they can mate. In all cases studied so far, it implies a genomic DNA rearrangement of the mating-type locus (MAT, encoding the key regulators of sexual identity) and species have evolved very different molecular pathways for the same aim. In the fission yeast Schizosaccharomyces pombe, an imprint at mat1 (it is unknown whether the imprint is an epigenetic mark or a single nick) is introduced, that leads to a DSB during DNA replication [7,8]. Repair occurs with one of the two silent copies of mat1, called mat2 and mat3. In the ascomycete Kluyveromyces lactis, mating-type switching involves a DSB at MAT but it is performed by two specific nucleases depending on the mating-type of the cell [9,10]. Mating-type switching has been extensively studied in the model yeast S. cerevisiae and has notably allowed a better understanding of cell identity, DSB repair and silencing mechanisms [11].

In S. cerevisiae, haploid cells can be of either mating-type, MATalpha or MATa, which encodes “alpha” or “a” information, respectively, at the Y sequence of the MAT locus [12] (Fig 1). Mating-type switching relies on a programmed DSB at the MAT locus performed by the Ho endonuclease at its 24-bp recognition site (Fig 1). DSBs are highly toxic DNA lesions, and thus have to be efficiently repaired to ensure cell viability. This can be achieved through two major pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR) in the presence of a repair template. The DSB at MAT is repaired ~90% of the time by HR [11], probably because of efficient resection of the DSB that has been shown to prevent NHEJ [13]. The Ho cut at the MAT locus generates 4 bp, 3′-overhanging ends and its repair involves the following steps: the DSB ends are processed by several 5′ to 3′ exonucleases to create long 3′-ended tails [14]; single-strand tails are then converted to Rad51-coated nucleoprotein filaments, which search for homology and promote homologous template invasion [11]; once the homologous donor is found, the MAT locus is repaired by gene conversion. The homologous donor is one of the two silent loci located on the same chromosome as MAT: HML carrying the “alpha” information or HMR carrying the “a” information. The “alpha” or “a” sequence from HML or HMR respectively, replaces the original Y MAT sequence whereas HML and HMR remain unchanged. Despite the fact that HML and HMR contain the Ho recognition site, both are resistant to Ho cleavage, being located in heterochromatic regions [15]. It must be noted that the Ho recognition site is quite different between the "a" and "alpha" versions, one side is unchanged since it is located in the identical Z sequence, while the other side, corresponding to the end of the Y fragment, is different between the two mating-types (Fig 1). There is no measurable difference in the efficacy of the cut between the two sequences, nor between efficiency of mating-type switching from one to another [16,17]. This illustrates the fact that Ho is part of the family of meganucleases, that do not function like type II restriction endonucleases, but recognize large (larger than 12bp), degenerate, non-palindromic cut-sites.

Fig 1

Arrangement of HML, MAT and HMR in S. cerevisiae and in C. glabrata and size of sequences.

(A) Arrangement of the HML, MAT, HMR loci in S. cerevisiae and in C. glabrata. The Ho site sequence of each locus and species is shown in yellow. (B) Size of sequences forming the HML, MAT, HMR loci in S. cerevisiae and in C. glabrata.

In S. cerevisiae, a "donor preference" mechanism ensures an efficient mating-type switching at MAT by promoting the use of the silent locus from the opposite mating-type (MATa is preferentially repaired by HMLalpha and MATalpha by HMRa). This donor preference depends on both the “a” or “alpha” information at the MAT locus and the presence of a specific sequence, the recombination enhancer (RE), located between HML and MAT [18]. It must be noted that mating-type switching occurs only once per cell-cycle, in G1, and that this is thought to be regulated through the control of the expression of HO [12], but experiments of overexpression of HO under the control of a galactose-inducible promoter have shown that the switch can be induced in any part of the cell cycle [19]. Intriguingly, there is no report of switching back and forth between the two mating-types in such overexpression experiments, leaving open the possibility that another mechanism than HO gene expression, is responsible for the "unswitchability" of newly (i.e., unreplicated) switched cells.

C. glabrata is an opportunistic pathogenic yeast, phylogenetically close to S. cerevisiae [20]. Its genome has retained the three-locus system, with homologs of HML, MATa/alpha, and HMR, called Mating-Type Like (MTL) loci (Fig 1). The three loci display a structure comparable to S. cerevisiae’s, the main difference being that HMR is located on a different chromosome from HML and MAT [20]. Despite these similarities, added to the fact that both MATa and MATalpha cells are found naturally and that they maintain some mating-type identity [21-23], C. glabrata is unable to switch mating-type spontaneously at an efficient level, even though rare signs of mating-type switching are observed in culture [24] and in populations [25]. We have previously shown that the expression of the HO gene from S. cerevisiae can trigger mating-type switching in C. glabrata, and that over 99% of C. glabrata cells are unable to survive the expression of S. cerevisiae’s Ho (ScHo) [26]. We have also observed gene conversion events at the HML locus in survivors, revealing that, unlike S. cerevisiae, HML is not protected from the Ho cut. We suggested that the lethality was due to multiple chromosomal DSBs, which would prevent homologous recombination with an intact template in most cells.

In this work, we investigate the reasons for the lethality associated with mating-type switching induced by ScHo. For this purpose, we constructed a series of inconvertible (Inc) C. glabrata strains, mutated precisely at the Ho recognition site, allowing us to control the number and position of DNA breaks during induction of ScHo, as well as to track which donor sequence is used as template. We analyzed two aspects: viability, that reflects both the efficiency of the cut and the success of repair; and molecular structure of repaired loci, in wild-type and mutant strains, in order to reveal which repair pathways were used. We now show that HMR is also cut by Ho in wild-type strains of C. glabrata. In addition, by mimicking S. cerevisiae’s situation, in which HML and HMR are protected from the cut, we unexpectedly find that one DSB at the MAT locus is sufficient to induce cell death, whereas one at HML or HMR is not. Finally, the use of the CRISPR-Cas9 technology enables us to show that mating-type switching can be induced independently of the Ho protein in C. glabrata, and that such switching is efficient and not lethal. Thus, we show for the first time that a chromosomal DSB is repaired by HR efficiently in C. glabrata, at HML and HMR (Ho-cut) and at MAT (Cas9-cut), indicating that, in principle, MAT switching could occur in this species. The fact that an Ho endonuclease, able to induce switching, also induces cell death may be evidence for degeneration of the three MTLs/Ho system in this species, in accordance with the observed asexuality.

Results

All three sites are cut by Ho in C. glabrata, including the one at HMR

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]. As previously described, expression of ScHO in wild-type strains of C. glabrata, leads to the death of about 99.9% of cells and we found that both MAT and HML are efficiently cut [26]. We further analyzed surviving colonies of HM100 (HMLalpha MATalpha HMRa, Table 1) by determining the mating-type at each MTL locus by PCR and we found that nearly all present switching at HMR, indicative of cutting (Table 2).

Table 1

Strains used in this work.

Strains	Parent	Genotype	Reference
C. glabrata strains with wild-type Ho sites
CBS138		HMLalpha MATalpha HMRa	[27]
BG2		HMLalpha MATa HMRa	[28]
BG14	BG2	HMalpha MATa HMRa ura3Δ::Tn903 G418R	[28]
BG87	BG14	HMLalpha MATa HMRa ura3::NeoR his3Δ	[29]
HM100	CBS138	HMLalpha MATalpha HMRa ura3Δ::KANMX	[21]
HM100 Δrad51	HM100	HMLalpha MATalpha HMRa Δrad51 ura3Δ::KANMX	This work.
C. glabrata strains with mutated Ho sites
YL01	HM100	HMLalpha MATalpha HMRa-inc ura3Δ::KANMX	This work.
YL02	HM100	HMLalpha-inc MATalpha HMRa ura3Δ::KANMX	This work.
YL03-MATalpha	YL02	HMLalpha-inc MATalpha HMRa-inc ura3Δ::KANMX	This work.
YL03-MATa	YL03-MATalpha	HMLalpha-inc MATa HMRa-inc ura3Δ::KANMX	This work.
YL04	YL07	HMLalpha MATalpha-inc HMRa ura3Δ::KANMX	This work.
YL05	YL09	HMLalpha MATa-inc HMRa-inc ura3Δ::KANMX	This work.
YL07	YL02	HMLalpha-inc MATalpha-inc HMRa ura3Δ::KANMX	This work.
YL09	YL01	HMLa-inc MATa-inc HMRa-inc ura3Δ::KANMX	This work.
YL10	YL07	HMLalpha-inc MATalpha-inc HMRalpha-inc ura3Δ::KANMX	This work.
SL09	BG87	HMLa-inc MATa-inc HMRa-inc ura3::NeoR his3Δ	This work.
SL0A	YL01	HMLalpha MATalpha-inc HMRa-inc ura3Δ::KANMX	This work.
SL0B	YL04	HMLa-inc MATalpha-inc HMRa ura3Δ::KANMX	This work.
C. glabrata strains with mutated Ho sites and/or deletion(s) of HML, MAT or HMR
CGM460	BG14	Δhml MATalpha HMRa ura3Δ::Tn903 G418R	[22]
CGM390	BG14	HMLalpha Δmat HMRa ura3Δ::Tn903 G418R	[22]
SL-CG1	CGM390	HMLalpha Δmat Δhmr ura3Δ::Tn903 G418R	This work.
CGM498	BG14	Δhml Δmat HMRa ura3Δ::Tn903 G418R	[22]
SL01	BG87	HMLalpha MATa Δhmr ura3::NeoR his3Δ	This work.
SL-CG8	SL01	HMLalpha-inc MATa Δhmr ura3::NeoR his3Δ	This work.
SL-CG9	CGM460	Δhml MATa HMRalpha-inc ura3Δ::Tn903 G418R	This work.
SL-CG10	SL01	HMLa-inc MATa-inc Δhmr ura3::NeoR his3Δ	This work.
SL-CG12	CGM390	HMLalpha-inc Δmat HMRalpha-inc ura3Δ::Tn903 G418R	This work.
SL-CG14	CGM460	Δhml MATalpha-inc HMRalpha-inc ura3Δ::Tn903 G418R	This work.

Table 2

Molecular structure of MTLs in surviving colonies after ScHo induction.

Strain	Locus screened	PCR results	Percentage of switch
C. glabrata strains with wild-type Ho sites
HM100 wt (HMLalpha MATalpha HMRa)	HML	25/34 mixed HMLalpha/a9/34 pure HMLalpha	74 ± 6%
	MAT	26/34 mixed MATalpha/a8/34 pure MATalpha	76 ± 6%
	HMR	30/34 mixed HMRa/alpha2/34 pure HMRalpha2/34 pure HMRa	94 ± 6%
HM100 Δrad51 (HMLalpha MATalpha HMRa)	HML	39/39 pure HMLalpha	0%
	MAT	39/39 pure MATalpha	0%
	HMR	39/39 pure HMRa	0%
C. glabrata strains in which MAT is protected from the Ho cut
YL05 (HMLalpha MATa-inc HMRa-inc)	HML	60/60 pure HMLa-inc	100 ± 8%
YL07 (HMlalpha-inc MATalpha-inc HMRa)	HMR	34/36 mixed HMRa/alpha-inc2/36 pure HMRalpha-inc	100 ± 6%
C. glabrata strains in which MAT can undergo the Ho cut
SL-CG8 (HMLalpha-inc MATa Δhmr)	MAT	36/36 pure MATalpha-inc	100 ± 6%
SL-CG9 (Δhml MATa HMRalpha-inc)	MAT	36/36 pure MATalpha-inc	100 ± 6%

Colonies surviving ScHo induction are screened by PCR at each locus that can be cut by ScHo. Percentage of switch is calculated as the ratio of the total number of pure and mixed colonies exhibiting mating-type switching divided by the total number of surviving colonies tested, expressed as percentage. The square root of the number of surviving colonies screened is used as standard error in last column.

In order to formally confirm that mating-type switching at each MTL depends on HR, we inactivated RAD51 (CAGL0I05544g) in the wild-type strain HM100 (Table 1). Inducing the Ho DSB in this strain leads to an even higher lethality than in the wild-type strain (Fig 2), and no mating-type switching is detected at any MTL locus (Table 2), confirming that switching relies on HR in C. glabrata.

Fig 2

Survival to ScHo induction of strains bearing combinations of wild-type and/or mutated Ho sites.

On the left, diagram of MTL configuration of strains is shown with the corresponding survival histogram to ScHo induction on the right. The blue box represents the Ya, the red box Yalpha, the yellow bar wild-type Ho site and the crossed circle mutated Ho site (Inc loci) (not to scale). On the histogram, black bars are for strains from the BG87 background, grey bars are for strains from the HM100 background. Results for strains HM100 and BG87 are from (26). Values from, at least, four experiments were averaged, the SEM used as estimate of the error and the P-value was calculated using the Wilcoxon test. ***: P-value<0.001. ns: non-significant.

As we hypothesized in our previous work [26], Ho-induced lethality in C. glabrata could be due to concomitant induction of multiple DSBs, in contrast to the situation in S. cerevisiae where HML and HMR are protected from the cut. These unrepairable cuts would lead to death by cell cycle arrest, or because cut and possibly degraded chromosomes segregating into daughter cells lack essential genes. Alternatively, we had mentioned the possibility that switching leads to the repair of the Ho-cut locus by an intact Ho-site containing locus would cause never-ending cycles of cutting and repair that could also lead to cell death in our conditions of continuous induction on plates. We had dismissed this eventuality as unlikely, since the percentage of lethality and switched survivors is highly reproducible in our experiments. Even though the situation is the same in S. cerevisiae when overexpressing Ho with a galactose-inducible promoter, and no switching back and forth between the two mating-types has been reported in S. cerevisiae [14,19], we had no experimental proof that this did not happen in C. glabrata, in our experimental conditions. We thus decided to design experiments which would address both the question of the number of cut loci, and the question of the number of cuts per locus. For this, as explained below, we have used strains containing inconvertible Ho sites (Inc) and/or deletions of MTLs and we have performed a time-course experiment of induction (see Material and methods).

Cleavage at HML and HMR is not an important contributor to lethality

As soon as we started performing experiments using strains with inconvertible MTLs, we noticed difference of behavior between the MAT locus and HML and HMR. We first used strains YL05 (HMLalpha MATa-inc HMRa-inc) and YL07 (HMLalpha-inc MATalpha-inc HMRa), Table 1). In these strains, we mutated the Ho sites in the region known to be essential for Ho cutting in S. cerevisiae [30], yielding loci MAT-inc, HML-inc, HMR-inc (S1 Fig), in configurations where only either the HML or HMR locus can be cut and repaired by the two non-cleavable donors MAT-inc, and HMR-inc or HML-inc.

Upon Ho induction, cell viability drastically increases to 35 to 55%, depending on the strain (Fig 2). Survival does not reach 100% but is more than 2.700 times higher than in the wild-type isogenic strain, HM100 (HM100 vs YL05 P-value<10−4 and HM100 vs YL07 P-value<10−9, Wilcoxon tests) (Fig 2). We then expressed HO in a strain in which only MAT is protected from the cut, while both HML and HMR can be cleaved by Ho (Strain YL04 HMLalpha MATalpha-inc HMRa, Table 1). In this strain, cell viability reaches ~20% which is 2.000 times higher than in the wild-type isogenic strain, HM100 (P-value<0.001, Wilcoxon test) (Fig 2).

We analyzed the molecular structure of the HML and HMR loci in surviving colonies of strains YL05 and YL07 respectively, by PCR using primers specific of the mating-type carried by the MTLs (“alpha” or”a”, wt or inc, S1 Table and S2 Fig). This allows the distinction of the original HML or HMR locus from the repaired locus that has become resistant to cutting. We found that 100% of surviving colonies showed mating-type switching of HML or HMR (Table 2). As mating-type switching reflects the efficiency of the Ho-cut, this suggests that both HML and HMR are efficiently cut by ScHo and that, even though we found some mixed colonies for HMR in strain YL07, MTLs they are repaired by HR.

In order to confirm this, we induced expression of ScHo in strains in which either HML or HMR can be cut by Ho in absence of any repair template, the two other loci being completely deleted (strain SL-CG1, HMLalpha Δmat Δhmr and strain CGM498, Δhml Δmat HMRa, Table 1) (Fig 2). Upon Ho induction, we found that survival rate does not exceed 0.2% in both strains, suggesting that the Ho-cut is efficient at both HML and HMR and that the Ho-DSB at HML and HMR causes lethality when it cannot be repaired by HR. To explore this further, we induced ScHo in a strain in which both HML and HMR can be cut and MAT is deleted (strain CGM390 HMLalpha Δmat HMRa, Table 1) (Fig 2). Once again, survival does not exceed 0.2%, suggesting that HML and HMR are cut concomitantly and thus cannot serve as templates for one another. This reinforces the hypothesis that simultaneous DSBs, happening in wild-type strains, participate in the high lethality observed.

Altogether, these results suggest that the efficient Ho-cut at HML and HMR is not an important contributor to lethality in all configurations where they can be repaired by HR. In the absence of HR, no other mechanism such as NHEJ is able to take over the repair of the Ho-cut, and thus cell survival remains low. Results also show that protecting the MAT locus from the Ho-cut significantly increases survival. This is also confirmed by the fact that strains containing no Ho-site at any MTL locus survive the Ho induction at around 50% (strains YL10, YL09 and SL09, Table 1) (Fig 2).

A single ScHo-DSB at MAT is sufficient to induce cell death in C. glabrata

In order to measure the impact of the Ho cleavage at MAT on cell survival, we mimicked the situation in S. cerevisiae, where a single recipient of the Ho-induced DSB, the MAT locus, can be repaired by the two non-cleavable donors HML and HMR, i.e. strains YL03-MATalpha (HMLalpha-inc MATalpha HMRa-inc) and YL03-MATa (HMLalpha-inc MATa HMRa-inc) (Table 1). Expression of ScHO in those strains leads to a lethality similar to the one obtained in wild-type strains HM100 and BG87 (Fig 3) and all surviving colonies have switched, whatever the mating-type at MAT (Table 2).

Fig 3

Survival to ScHo induction of strains in which only the MAT locus can be cut.

On the left, diagram of MTL configuration of strains is shown with the corresponding survival histogram to ScHo induction on the right. The blue box represents the Ya, the red box Yalpha, the yellow bar wild-type Ho site and the crossed circle mutated Ho site (Inc loci) (not to scale). On the histogram, black bars are for strains from the BG87 background, grey bars are for strains from the HM100 background. Values from, at least, four experiments were averaged, the SEM used as estimate of the error and the P-value was calculated using the Wilcoxon test. ns: non-significant.

Thus, a single Ho-induced DSB at MAT, whatever its mating-type, is sufficient to induce massive cell death in C. glabrata. Furthermore, this experiment allows us to reconsider the question of lethality due to never-ending cycles of cutting and repair. Indeed, if this was the reason for mortality of cells when cutting at MAT, then, since repairing with an Inc locus leads to an unswitchable locus, the mortality should be decreased in these strains upon induction.

Lethality is not due to toxic recombinational repair intermediates

Since we know that the mating-type switching system in C. glabrata is not very efficient, it is legitimate to wonder whether the degeneration of the mating-type switching mechanism could lead to abnormal HR intermediates. We asked whether such repair intermediates could be toxic and cause death. For example, the two ends of the broken MAT locus could invade both HML and HMR, leading to non-resolvable structures. The fact that HMR is not on the same chromosome as HML and MAT could be an additional problem, if, for example, repair of MAT occurs principally with HMR and this leads to lethal rearrangements.

In order to test this, we constructed two strains in which MAT can be cut by Ho and can only be repaired either by HML or by HMR (SL-CG8, HMLalpha-inc MATa Δhmr, and SL-CG9, Δhml MATa HMRalpha-inc, respectively, Table 1). Expression of ScHO in both strains leads to a high lethality (Fig 3), similar to the ones of the wild-type or YL03 strains (HMLalpha-inc MATalpha or MATa HMRa-inc, Table 1). We analyzed the molecular structure in surviving colonies (Table 2). All have switched, whatever the location of the repair template (HML in strain SL-CG8 and HMR in strain SL-CG9).

Thus, forcing MAT to repair solely on HML or HMR results in the same lethality as in wild-type strains. From this, we conclude that the cut MAT locus DNA ends probably do not interact with both HML and HMR in such a way that it is toxic to cells, and that there is no specific problem due to the fact that HMR is on another chromosome than MAT. It, therefore, seems unlikely that lethality could be due to non-resolvable HR intermediates.

Time course experiments reveal that growth arrest is quick and irreversible leading to cell death

In order to shed light on whether the toxic effect leading to cell death could be reversible and if not, whether the effect operates rapidly or not, we performed a time course experiment in which Ho is induced in liquid medium and its expression is repressed, at different time points, by plating cells on repressive medium. The survival can thus be calculated by the ratio of colonies obtained on repressive medium to the theoretical number of cells plated.

In order to easily follow events, we used again strain SL-CG9 (Δhml MATa HMRalpha-inc, Table 1), where only the MAT locus can be cut and a single inconvertible template for HR is present. This allows to follow a single Ho-cut and a single repair event at the MAT locus. We scored the survival along the time-course experiment as well as the percentage of survivors that have undergone switching. As shown on Fig 4, two hours upon induction of a single Ho-DSB at MAT, survival drastically drops to less than 2%. From T = 4 hrs to T = 15 hrs, survival remains very low. Molecular analysis shows that mating-type switching in survivors reaches its maximum very rapidly after four hours of induction, at around 90%. All screened colonies display a pure genotype in PCR just as on solid medium (see Table 2). The last two points of our experiment, T = 19 hrs and T = 38 hrs show that survivors have invaded the liquid culture, thus giving rise to many colonies on repressive medium plates. These survivors thus consist of 90% of switched inconvertible clones, and around 10% of cells that have escaped Ho-induction probably by rearranging or mutating the plasmid. These results show that the toxic effect of inducing an Ho-cut at the MAT locus is irreversible even after only two hours in induction medium.

Fig 4

Time-course experiment of ScHo induction in liquid culture.

Strains SL-CG9 (blue curve) and HM100 (green curve) were followed during a time-course experiment of induction of ScHo in liquid culture. Survival is shown on the Y-axis as a curve and is calculated by comparing the number of colony-forming units on SC-Rep with the number of cells plated, as estimated by counting. This is normalized by dividing it by the survival rate of the control strain, i.e., the strain transformed by pYR32, grown in the same conditions. Values from four experiments were averaged and the SEM is used as an estimate of the error. Percentage of switched colonies is also shown for strain SL-CG9 as a histogram. This is expressed as a percentage of colonies showing switching by PCR at MAT on the total number of colonies screened (this was not performed at T = 19hrs). At each time-point, except T = 19hrs, 36 colonies over the four experiments were screened and the square root of 36 is used as an estimate of the error.

We next wanted to check whether mortality was as quick and as irreversible in wild-type strains. Induction in HM100 leads to the same pattern as SL-CG9 (Fig 4). This confirms that the lethality in HM100 is not due to never-ending cycles of cutting and repair since it cannot happen in SL-CG9 and the lethality occurs at the same rate.

The lethality induced by the DSB at MAT is specific to ScHo

We wanted to investigate whether the lethality is caused by the DSB at MAT per se or by the specific combination of MAT with ScHo. Since we have previously shown that inducing other Ho endonucleases from the Nakaseomyces, including C. glabrata’s own gene, does not result in high lethality nor efficient mating-type switching [26], we decided to use the CRISPR-Cas9 system from [31]. This system relies on a unique URA3 plasmid, pCGLM1, in which the CAS9 gene is under the control of the inducible MET3 promoter, as is the case for the HO gene in the p7.1 plasmid used in the experiments above. This allows us to induce a DSB at MAT with Cas9, in the same conditions as with ScHo.

In order to allow a full comparison between ScHo and Cas9 induction, we wanted to generate a single DSB at the same locus, here MAT, whatever the endonuclease used. Indeed, the Ho site between MTLa and MTLalpha is very different while the Cas9-cut is directed with a specific gRNA, we used SL-CG8 and SL-CG9 (HMLalpha-inc MATa Δhmr, and Δhml MATa HMRalpha-inc, respectively, Table 1) with a gRNA that targets only the MAT locus, since it is directed to the Ya sequence (Fig 5A). This gRNA allows to target the Cas9-cut from 14 pb from the Ho cut (Fig 5A).

Fig 5

Survival upon Cas9 induction and gRNAs used.

(A) gRNA Ya2 targeting the MATa locus of C. glabrata. Sequence shown is a segment of the MATa locus of BG87, including the gRNA in bold and the PAM sequence in red. Plain double arrow indicates the Ho cleavage site and dashed double arrow the Cas9 cleavage site. (B) Survival of strains SL-CG8 and SL-CG9 upon Cas9-induced DSB at MAT. Induction is performed on solid medium. Results for strains SL-CG8 and SL-CG9 upon ScHo induction are from Fig 3. Values from four experiments were averaged, the SEM used as estimate of the error and the P-value was calculated using the Wilcoxon test. ***: P-value<0.001. (C) Induction was performed in liquid during a time-course experiment for strain SL-CG9 expressing Cas9 (harboring pCGLM1-Ya2). The Y-axis represents both the survival (curve) expressed as a percentage, and the percentage of switched colonies (histogram). Survival is calculated by comparing the number of colony-forming units on SC-Rep with the number of cells plated, as estimated by counting; and is normalized by dividing it by the survival rate of the control strain, i.e., strain SL-CG9 transformed by pCGLM1 for Cas9 induction, grown in the same conditions. For survival rate, values from four experiments were averaged and the SEM is used as estimate of the error. For the percentage of switched colonies, the square root of the number of surviving colonies screened is used, i.e., sqrt of 36. For time-course experiments, at points T = 17 and T = 21 hrs, no PCR of surviving colonies was performed.

Fig 5B shows that induction of the Cas9-cut in both SL-CG8 and SL-CG9 does not lead to any lethality. We wondered whether Cas9 had indeed cut the MAT locus by screening mating-type switching of surviving colonies by PCR (Table 3).

Table 3

Molecular structure of the MAT locus in colonies after Cas9 induction.

Strain	Locus screened	gRNA used	PCR results of surviving colonies	PCR results of sub-clones	Percentage of switch
SL-CG8 (HMLalpha-inc MATa Δhmr)	MAT	Ya2	36/40 mixed MATalpha-inc/a4/40 pure MATalpha-inc	40/48 MATalpha-inc8/48 MATa	85%
SL-CG9 (Δhml MATa HMRalpha-inc)	MAT	Ya2	32/45 mixed MATalpha-inc/a7/45 pure MATalpha-inc6/45 pure MATa	38/48 MATalpha-inc10/48 MATa	72%

After Cas9 induction, colonies are screened by PCR at MAT. When PCR revealed mixed colonies, at least four individual colonies are sub-cloned in order to get the ratio of each mating-type; percentage of switch is calculated as follows: the ratio of the number of pure switched colonies on the total number is added to the ratio of the number of mixed colonies on the total number screened multiplied by the ratio of the number of pure switched sub-clones on the total number of sub-clones screened, expressed as percentage. An example for the molecular analysis of SL-CG8 using gRNA Ya2, is: (4/40 + (36/40 x 40/48)) x 100 = 85%.

Results show that the Cas9-induced DSB leads to mating-type switching as efficiently as the Ho protein (Table 2 and Table 3). Indeed, 72 to 85% of the colonies tested presented “alpha-inc” information at MAT, confirming the cut of this locus by Cas9 and induction of mating-type switching (Table 3). This gRNA is adjacent to an optimal NGG PAM sequence and targets a site very close to the Ho site, 14 pb away from the Ho cut.

We then asked whether we could detect a transient lethality, reflecting the Cas9-cut. Indeed, previous experiments, using the same system for inducing a Cas9-cut, but at the unrelated ADE2 locus, have shown that a transient lethality occurred in liquid culture in a time-course experiment, whereas no apparent lethality was detected on induction plates [31]. Thus, we performed a time-course experiment with Cas9 in strain SL-CG9. Fig 5D shows that Cas9 induction never leads to a sharp increase in lethality at any time point and survival remains between 65 and 92% for the whole experiment. Surprisingly, contrary to what we observe in induction on plates, screening of mating-type switching at MAT reveals that only ~20 to 36% of surviving colonies have switched mating-type (see Discussion).

These results show for the first time that mating-type switching can be induced without any lethality in C. glabrata using the CRISPR-Cas9 system. In conclusion, the fact that the MAT locus can be cut and repaired by HR without any accompanying high lethality demonstrates that it is ScHo cutting specifically at the MAT locus that is highly lethal in C. glabrata.

Choice of repair template reveals a complex interplay between the MTL loci

As the three MTLs are efficiently cut by ScHo and as the cut at MAT is the only one to lead to a high lethality, we decided to study these differences in C. glabrata by asking how each locus interacts with the two other templates.

In wild-type strains of S. cerevisiae, only the MAT locus is cut, and the mechanism of mating-type switching is productive thanks to control by the sexual identity of the cell. Indeed, the use of the donor locus of the opposite mating-type to repair the DSB at MAT is promoted (“donor preference”) [18]. Since it has been shown that sexual identity of cells is maintained in C. glabrata, at least in MATa cells (i.e., MATa cells express MATa identity, but MATalpha cells have no mating-type specific expression of key genes [21,22]), we asked whether this “donor preference” from S. cerevisiae is conserved in C. glabrata at the MAT locus, and also whether HML and HMR use a preferential template for repair. For this, we constructed strains that carry different and inconvertible mating-types in various combinations. First, for the MAT locus, we used strains YL03-MATalpha and YL03-MATa (HMLalpha-inc MATalpha HMRa-inc and HMLalpha-inc MATa HMRa-inc, respectively, Table 1). Analysis of surviving colonies from strain YL03-MATa shows that 78% display only the alpha-inc information at MAT, the remainder exhibiting various genotypes, pure a-inc for 3%, and mixed alpha-inc and a-inc for 19% (Table 4). The overwhelming percentage of pure colonies displaying only the alpha-inc information at MAT demonstrates that HML is the preferred template for repair of the MATa locus.

Table 4

Analysis of template choice for repair of Ho cut at MTLs.

Strain	Locus screened	PCR results	Use of each locus
YL03-MATa (HMLalpha-inc MATa HMRa-inc)	MAT	25/32 pure MATalpha-inc6/32 mixed MATalpha-inc/a-inc1/32 pure MATa-inc	HML: 96%HMR: 4%
YL03-MATalpha (HMLalpha-inc MATalpha HMRa-inc)	MAT	42/50 pure MATalpha-inc5/50 mixed MATalpha-inc/a-inc2/50 pure MATa-inc1/50 pure MATalpha	HML: 95%HMR: 5%
SL0A (HMLalpha MATalpha-inc HMRa-inc)	HML	33/69 pure HMLalpha-inc20/69 mixed HMLalpha-inc/a-inc9/69 pure HMLa-inc purs7/69 mixed HMLalpha/alpha-inc/a-inc	MAT: 79%HMR: 21%
SL0B (HMLa-inc MATalpha-inc HMRa)	HMR	38/45 mixed HMRa/a-inc4/45 pure HMRa-inc3/45 pure HMRa	HML: ~100%MAT: ~0%

In this experiment, strains are chosen so that template choice for ScHo-cut can be analyzed. As before, colonies are screened by PCR at the single locus that can be cut by ScHo. In the first three strains, since the percentage of pure colonies alone is sufficient to know which template is preferentially used for repair, the percentage of the use of each locus is calculated by only taking into account the number of pure switched surviving colonies (i.e., we did not sub-clone mixed colonies and we also omitted the pure unswitched colony obtained in strain YL03-MATalpha). The use of each locus is calculated as the ratio of the number of colonies showing the use of that locus on the total number of pure switched surviving colonies screened, expressed as percentage. In the case of strain SL0B, we fail to detect by PCR the use of MATalpha-inc to repair HMR, thus we estimate that in switched colonies, the use of HML is of the order of 100%.

In strain YL03-MATalpha: 84% of tested surviving colonies display only the MATalpha-inc genotype, the others displaying various mixed or pure genotypes at MAT (Table 4). Thus, in contrast to S. cerevisiae, HML is preferentially used as template for repair in C. glabrata, whatever the mating-type at MAT.

In order to know by which template HML is preferentially repaired, we performed a molecular analysis of the HML locus, upon ScHo induction, in a strain that carries different and inconvertible mating-types at MAT and HMR (strain SL0A, HMLalpha MATalpha-inc HMRa-inc, Table 1). Analysis of surviving colonies shows that 47% display pure alpha-inc information, 40% are alpha-inc and a-inc in mixed colonies and 13% are pure a-inc (Table 4). This indicates that HML preferentially repairs the DSB using MAT over HMR.

In the same way, molecular analysis of the HMR locus was performed in strain SL0B (HMLa-inc MATalpha-inc HMRa, Table 1). Table 4 shows that, 9% of surviving colonies are pure a-inc and 84% display both a and a-inc information We observe no repair event where MATalpha-inc was used as template. HML is thus the preferred template for the repair of HMR.

Overall, these results show that MAT and HML preferentially repair on each other and that HMR is preferentially repaired by HML.

Exploring the residual lethality in the absence of MTL-cuts

When cuts are non-repairable by HR; i.e., the three strains containing wild-type Ho sites: HM100 Δrad51 (HMLalpha MATalpha HMRa), SL-CG1 (HMLalpha Δmat Δhmr) and CGM498 (Δhml Δmat HMRa) and inexplicably, the YL03 strains (HMLalpha-inc MATa or alpha HMRa-inc) (Table 1), 99.9% of the cells die. As shown above, in other strains where the Ho-cut is repaired by HR (at HML and HMR) and in strains where there is no Ho cut (Y09, YL10 and SL09, Table 1) survival varies between 20% and 61%, never reaching 100%. This residual lethality can be explained by a general toxic effect of the expression of a heterologous protein in C. glabrata or by of the existence of (a) cryptic unrepairable Ho site(s) elsewhere in the genome. We thus decided to test this residual lethality in other strains where no cutting occurs. For this, we constructed strains where one MTL is deleted and two are inconvertible, SL-CG10 (HMLa-inc MATa-inc Δhmr), SL-CG12 (HMLalpha-inc Δmat HMRalpha-inc) and SL-CG14 (Δhml MATalpha-inc HMRalpha-inc) (Table 1). Upon Ho induction, survival in these three strains is around ~80%, whatever the MTL deleted (Fig 6). Comparing the survival of SL-CG10, SL-CG12 and SL-CG14 to SL09 (HMLa-inc MATa-inc HMRa-inc) (same genetic background and highest survival in our previous experiment, i.e., 60%) show that the increase in survival is significant (P-value<0.05 in each pairwise combination of SL09 with SL-CG10, SL-CG12 and SL-CG14, Wilcoxon tests). If the residual lethality observed was due to a general toxic effect or to (an) extra Ho site(s), the lethality would be the same in those strains as in strains that are inconvertible for the three MTLs (YL10, YL09 and SL09) (Fig 3) since the constructions do not modify the context outside of the MTL loci.

Fig 6

Survival of strains without wild-type Ho sites and containing deletion of one MTL.

Blue box represents Ya, red box Yalpha and crossed circle mutated Ho site (Inc loci) (not to scale). Histogram shows survival of strains with corresponding MTL configuration. Results for strains YL10, YL09 and SL09 are from Fig 2. Values from, at least, four experiments were averaged, the SEM used as estimate of the error and the P-value was calculated using the Wilcoxon test. *: P-value<0.05. ns: non-significant.

Since the only difference between those strains is the number of inconvertible sites and thus potential binding sites for ScHo, we believe this may explain residual lethality by binding of ScHo (see Discussion).

Discussion

Mating-type switching is a highly regulated mechanism that relies on a chromosomal DSB. DSBs are a major threat for genome integrity [32]. Repair of such damage is thus essential and can be achieved through Rad51-dependent HR which involves many steps in order to succeed: search for homology involving Rad51 and Rad52 in S. cerevisiae, copy on the donor locus and displacement and resolution of the double Holliday junction [33]. In S. cerevisiae, the DSB at the MAT locus is repaired by HR using HMR or HML as template, depending on the original mating-type of the cell. C. glabrata does not switch mating types spontaneously at high frequency [24]. We have previously shown that overexpression of HO genes from C. glabrata and related species fails to induce efficient mating-type switching and that switching can be efficiently induced by expressing ScHO, but that it is lethal to most cells [26]. Our previous work also showed that the HML locus is cut in C. glabrata; something that never happens in wild-type strains of S. cerevisiae [26]. In this work, we aimed at understanding the link between mating-type switching and cell death in C. glabrata. First, we show that HMR is also cut in wild-type strains of C. glabrata, overexpressing ScHO. Next, we constructed strains with mutated uncuttable Ho sites (inconvertible, Inc) and with deleted MTL loci, in order to examine survival to individual DSBs at the different MTL loci as well as knowing which MTL has been used as template for repair.

In S. cerevisiae, the donor preference mechanism ensures an efficient mating-type switching at MAT by promoting the use of the template from the opposite mating-type, in repair [18]. We found, in C. glabrata, that whatever the mating-type at MAT, HML is preferentially used as template for repair. Thus, donor preference from S. cerevisiae seems not to be conserved in C. glabrata. In its absence, it also shows that the length of the sequence homology does not influence the use of the donor for repair. Indeed, the Ya and Yalpha segments, determining mating-type, share no homology and we observe that MATa, containing Ya, repairs preferentially using HMLalpha, containing Yalpha. We also demonstrate that HML is preferentially repaired using MAT and that HMR is preferentially repaired using HML, revealing a complex interplay between those different chromosomal segments. Loss of donor preference at MAT, along with the fact that the C. glabrata endogenous Ho protein fails to induce efficient mating-type switching [26], point to the possible degeneration of the mating-type switching system in C. glabrata. Thus, rules of DSB repair by HR observed in our inductions may reflect true preferences, independently of the MAT/Ho switching system. When the DSB can be repaired by HR using a template on the same chromosome or another chromosome, the intra-chromosomal template will be mostly chosen over the other (HML repairs preferentially with MAT and vice versa). When the DSB can only be repaired by an extra-chromosomal template, the sub-telomeric HMR was repaired preferentially using the sub-telomeric template HML. We can speculate that HR using a template on a same chromosome is preferred for DSB repair and in the absence of such a template, the sub-telomeric loci are repaired using other sub-telomeric loci.

In our previous work, we hypothesized that multiple DSBs at the MTL loci would be unrepairable and that this was the cause for lethality when mating-type switching is induced. As cited above, to mimic S. cerevisiae’s situation, in which MAT is the only recipient of the Ho cut, we mutated the Ho site at both HML and HMR. We are now able to demonstrate that one ScHo-DSB at the MAT locus is sufficient to induce cell death at a similar level to wild-type cells, thus invalidating our previous hypothesis. This means that, even in the presence of two intact homologous sequences, the MAT locus is not able to repair the break. More surprisingly, the DSB at MAT is only lethal when it is performed by the ScHo protein. We show, for the first time, that mating-type switching can be triggered efficiently by CRISPR-Cas9, thus independently of the Ho protein, in C. glabrata. This has been shown only recently in the model species S. cerevisiae [34]. No lethality is observed after a Cas9-DSB at MAT on plates. In liquid induction experiments, we observe a transient lethality of ~35% and a lower efficiency of switch (~30%). In both plate and liquid inductions, the Cas9 expression plasmid is constantly under selection pressure. We believe that observed discrepancies can be explained if induction is less efficient on individual cells in liquid medium than on plates, implying that cells in liquid medium can mutate the plasmid before switching (in the CAS9 gene or its promoter in such way that CAS9 is not expressed anymore, or in the gRNA sequence). These cells will never switch and can invade the culture. In contrast, some cells undergoing switching may not recover (~35% of lethality) but the ones that have survived the switch become “immune” to the Cas9-DSB, thus alleviating the need for mutating the Cas9 plasmid. Nonetheless, these cells may grow more slowly because of the maintenance of CAS9’s expression and are thus less likely to be in excess in the culture. Thus, these MAT-switched cells will be diluted and less represented on repressive plates.

To go back to the lethality induced by ScHo, unless the difference in the lethality with Cas9 is due to the 14 bp shift in cutting, which seems highly unlikely, these results suggest that the ScHo protein prevents DSB repair specifically at the MAT locus of C. glabrata, in such a way that 99.9% of the cells die. We have recently published that, in heterothallic strains of N. delphensis, overexpression of ScHo with the same plasmid as in this work leads to efficient switching, without lethality [35]. It is surprising that ScHo could have a deleterious effect in a locus- and species-specific manner. As in all three-loci-based mating-type switching systems, the three MTL loci of C. glabrata share identical sequences and only differ by the mating-type carried and/or their location in the genome [36]. We know that mating-type does not influence lethality in any of our experiments. Thus, only the location of the MAT locus could explain the specificity of lethality induced by ScHo. The MAT locus is located in a central region on chromosome B whereas HML and HMR are positioned in sub-telomeric regions on chromosome B and E, respectively [36]. Thus, the ScHo specificity for MAT could only be achieved either through the structure of the chromatin or through the flanking sequences of the MAT locus. If the ScHo protein causes lethality by preventing repair at MAT, it is perhaps because it gets stuck at MAT, after performing the DSB, preventing recruitment of recombination proteins and thus repair of the locus. In S. cerevisiae, it is possible to follow the fate of repairable and non-repairable ScHo-cuts at MAT by Southern-blot analysis from a time-course experiment of the wild-type strains, overexpressing ScHo [37,38]. S3 Fig shows that, upon Ho induction, we are not able to visualize cut chromosomal arms at MAT. This could be explained if ScHo stays bound on the DNA end(s) and prevents HR at this locus: continuous resection will degrade DNA ends so that the probe cannot hybridize anymore. Indeed, in S. cerevisiae, unrepairable ScHo-cuts are more extensively degraded than repairable ones [37]. Nonetheless, we can note that we clearly observe the parental uncut band before induction and that this band disappears completely as soon as we start the experiment, thus confirming that the MAT locus is efficiently cut in our system.

Outside of its role in inducing a lethal DSB at MAT, ScHo displays further toxicity in C. glabrata. Strains that are inconvertible for the three MTLs exhibit a survival of 61% at the most, and lethality is strongly reduced by the deletion of one MTL, whatever its position, HML, MAT or HMR. The difference between those two types of strains is the number of inconvertible (Inc) Ho sites present at the MTL loci. One explanation for this would be that the ScHo protein binds the Inc Ho sites and gets stuck there, in a way that is toxic for C. glabrata cells. ScHo probably has a high affinity for the C. glabrata Ho sites since we know that they are cut very efficiently and rapidly. In the case of an Inc site, the protein may be stuck there because the substrate is not transformed into a product. Indeed, we hypothesize that the cut releases the endonuclease and as we speculated above, this is prevented at the MAT locus, even after cutting, possibly by the chromatin structure. On the Inc sites, the binding of ScHo does not induce massive cell death but could, for example, physically hinder replication forks and thus disrupt DNA replication and cell division. Performing a ChIP-PCR on the Ho protein to examine its binding on the three MTL loci would allow us to better explore this aspect.

To put our results back into an evolutionary perspective and explore the link between switching and sexuality, we can turn to the other Nakaseomyces species. This group comprises C. glabrata, two other pathogens, Candida nivariensis and Candida bracarensis and three environmental species, N. delphensis, Candida castelli, Nakaseomyces bacillisporus. This clade contains species both varied in their lifestyle and in their lifecycle; all Candida species are described as asexual and haploid and in the two sexual species, N. delphensis is an obligate haploid while N. bacillisporus is an diploid [27]. N. delphensis undergoes natural Ho-induced mating-type switching, just like S. cerevisiae [35]. It is striking that none of the asexual species exhibit switching, thereby reinforcing the notion that switching is a mechanism that favours sexual reproduction. It is also remarkable, in such a case, that all asexual species have conserved the three MTL and a highly similar HO gene [25,36,39] while switching is supposed to be a mechanism that favours sexual reproduction. It is understandable that such a dangerous mechanism, involving a chromosomal DSB, would be lost if it is not essential. It has been hypothesized that C. glabrata sometimes undergoes switching [40,41]. These events could indeed be the result of very rare Ho-induced cuts or could also be fortuitous gene conversion events independent of the Ho protein (replication accident, repair of accidental DSBs etc). If we accept that this system is largely non-functional in C. glabrata and possibly other asexual species, the question of why they have kept both HO and the MTL remains open. In addition, it has been reported that the HO gene is under purifying selection in the population of C. glabrata strains analysed [25]. This may suggest that both Ho and the MTLs have acquired another function in the asexual Nakaseomyces. In S. cerevisiae, the MTL loci are one of the very few structured regions of the genome [42] and there remains an intriguing possibility that the conserved function of these MTLs and the Ho protein would be in structuring the chromosomes bearing them. Further studies of the 3D structure of the C. glabrata nucleus would shed light on this point.

Materials and methods

Strains, cultures, and transformation

C. glabrata strains used in this study are listed in Table 1. Strains are grown in broth or on plates at 28°C in YDP (non-selective, 1% Yeast Extract, 1% Peptone, 2% glucose), in Synthetic Complete medium lacking uracil (SC-Ura, 0.34% Yeast Nitrogen Base without amino acids, 0.7% ammonium sulfate, 2% glucose, supplemented with adenine and all amino acids except uracil) or in Synthetic Complete medium lacking uracil, methionine, and cysteine (induction conditions for the MET3 promoter, SC-Ind, 0.34% Yeast Nitrogen Base without amino acids, 0.7% ammonium sulfate, 2% glucose, supplemented with adenine and all amino acids except methionine and cysteine). For selection of transformants of the Ho plasmid or Cas9 plasmid and maintenance in repressive conditions for the MET3 promoter, strains are grown in SC-Ind supplemented with 2 mM each of methionine and cysteine (SC-rep) and in YPD supplemented with 2 mM each of methionine and cysteine (YDP-Rep) when repression but no selection is needed. For SC-Rep, medium is buffered by 10 mL of Na2HPO4 0.05 M and NaH2PO4 0.95 M per liter. For URA3 marker counter-selection, yeast strains are grown on 5-FOA medium (SC-Ura supplemented with 1 g/L of 5-fluoroorotic acid (5-FOA) and 50 mg/L of uracil).

Transformation is done according to the “one-step” lithium acetate transformation protocol from [43].

Induction of mating-type switching by ScHo

The HO gene from S. cerevisiae is cloned into the pCU-MET3 plasmid under the MET3 promoter (p7.1, S2 Table) [44] and protocol for solid induction is detailed in [26]. For time-course of induction in liquid medium, transformants are grown overnight in liquid SC-Rep medium, counted, washed and resuspended in sterile water at 4.107 cells/mL. 100 μL is used to inoculate 40 mL of liquid SC-Ind medium and the culture is placed at 28°C with agitation. For each time point, a sample of the culture is counted under the microscope, diluted and plated on SC-Rep plates. Each strain that was analysed in a time-course, were also transformed with the plasmid that does not contain ScHO, pYR32 plasmid, cells were diluted, counted and plated, allowing the normalization of the survival in Ho induction.

Induction of mating-type switching by CRISPR-Cas9

We used the inducible CRISPR-Cas9 system for C. glabrata from [31] using plasmid pCGLM1. We cloned into pCGLM1 a sequence corresponding to a guide RNA (gRNA) targeting the Ya sequence (S1 Table), giving rise to plasmids pCGLM1-Ya2.

Induction of Cas9-DSB was then performed as inductions of the ScHO gene done with p7.1 (see above).

Cell viability estimation

Different dilutions of cultures, containing between 200 to 106 cells, are spread on both inductive and repressive media. When the survival rate is over 20%, cell viability is determined directly as the ratio of the number of colonies counted on inductive medium to the number of colonies counted on repressive medium, for the same dilution. When the survival rate is under 1%, colonies are confluent on repressive medium at the same dilution where several colonies can be observed on induction medium. Thus, survival rate is measured by first comparing the number of colony-forming units (CFU) on inductive medium with the theoretical number of cells plated, as estimated by counting on a Thoma counting chamber. This is then corrected by the ratio of CFU to the number of cells counted, estimated by plating 200 cells on repressive medium. All the values were obtained from at least four independent transformants. Colonies number from a minimum of 2 to a maximum 746 was counted on plates. Numerical data used for drawing graphs is shown in S3 Table.

Determining the genotype at MTL loci

The genotype at each MTL locus is determined by PCR, when needed, directly on colonies [26] using specific primers: the forward primer is located upstream of the locus (ensuring specificity of the locus screened; HML, MAT or HMR) and a reverse primer located precisely on the Ho site (ensuring specificity of the information carried by the locus; alpha or a and wt or inc) (S1 Table, S2 Fig). In most induction experiments, we did not check switching at Inc loci since preliminary experiments showed that there was no switching, indicating that the alpha-inc and a-inc Ho sites are not cut after Ho induction. This genotyping is performed on surviving colonies directly on induction plates (for solid induction) and on repression plates (for liquid induction). As previously shown, PCRs often reveal that most surviving colonies are mixed for genotypes at MTLs [26]. In the case of Ho inductions, we have already shown that sub-cloning of such mixed colonies yields more than 80% of switched pure clones [26]. Therefore, sub-cloning has not been done on any Ho induction in this work. In the case of Cas9 induction, since we had never used this system for switching MTLs, and we have decided to sub-clone mixed colonies in order to assess the true efficiency of mating-type switching.

Construction of strains

We mutated the Ho sites in the region known to be essential for Ho cutting in S. cerevisiae [30], as shown on S1 Fig, yielding loci HML-inc, MAT-inc and HMR-inc. Modification of HML, MAT, or HMR loci was realized either by marker selection (pop-in/pop-out) [45] or by mating-type switching upon HO gene expression or by use of CRISPR-Cas9. The three methods are detailed below. Primers and plasmids are listed in S1 and S2 Tables, respectively. Method used to construct each strain is listed in S4 Table.

Construction of PCR fragments and plasmids for pop-in

In order to integrate the URA3 marker at the targeted locus (pop-in), we amplified the URA3 gene from S. cerevisiae under its own promoter by PCR using primers Sc-URA3-F and Sc-URA3-R and, YEp352 as template. The PCR fragment was cloned into the EcoRV-digested pBlueScript, giving rise to pURA (S2 Table).

To direct integration of the URA3 marker at the targeted locus, here the MTL loci HML, MAT or HMR, the 5’ and 3’ flanking regions was added to the URA3 marker in multiple steps.

First, the Z sequence, shared by the three MTL loci, was amplified by PCR using primers 68/70 and HM100 strain DNA as template (S1 Table). Primers 68 and 70 contain BamHI and EcoRI restriction sites, respectively, to allow cloning of the Z PCR fragment upstream of the URA3 marker into pURA, giving rise to pZU (S2 Table).

Second, Ya and Yalpha sequences were amplified on strain HM100 by PCR, using primers 73/72 and 74/69 respectively (S1 Table). Primers 73 and 72 contain HindIII and SalI restriction sites, respectively, in order to clone the Ya PCR fragment downstream of the URA3 marker into pZU, giving rise to pZUA (S2 Table). The SalI restriction site was added to primer 69 and no restriction site was added to primer 74 as the Yalpha PCR fragment already contains the HindIII restriction site 38 bp from the 5’ of the fragment. Thus, the Yalpha PCR fragment, digested by both SalI and HindIII, was cloned downstream of the URA3 marker into pZU to give rise to pZUAlpha (S2 Table).

Amplification by PCR, using universal primers M13F/M13R, on both pZUA and pZUAlpha plasmids, led to ZUA and ZUAlpha fragments, respectively. These fragments have been used for targeting HML, MAT or HMR loci (S2 Table) and Ura+ transformants were selected on SC-Ura. Correct integration of the fragment was checked by PCR.

Construction of plasmids and PCR fragments for pop-out

The URA3 marker is removed (pop-out) from the target locus by homologous recombination with a DNA fragment derived from the upstream and downstream sequences of that locus (S4 Table).

In order to replace the wild-type Ho site in the different MTL loci, by the inconvertible-mutated Ho site, we constructed two plasmids; pZA-inc and pZalpha-inc (S2 Table). The pZA-inc plasmid (without URA3 gene) results from double digestion of pZUA by EcoRI and HindIII and ligation after Klenow fill-in. The pZAlpha-inc plasmid (without the URA3 gene) was constructed by cloning the BamHI/EcoRI-digested Z fragment and the EcoRI/SalI-digested Yalpha fragment into the pBlueScript double digested by BamHI and SalI. Amplification by PCR using primers M13F/M13R, from both pZA-inc and pZAlpha-inc plasmids, lead to the ZA-inc and ZAlpha-inc fragments that have been used for pop-out. The comparison of wild-type and inconvertible Ho sites is presented in S1 Fig.

In addition to the construction of Inc sites, we have also used strains with deletion of MTLs (Table 1). Strains with deletions of MAT and/or HML were directly obtained from [22] (Table 1) and Inc sites were introduced in those strains when needed (S4 Table). In addition, we constructed deletion of HMR in strains BG87 and CGM390. After pop-in of URA3 at HMR, amplification of upstream and downstream sequences (500 bp each) of HMR was performed on strain BG87, using primers Up-HMR-F/Up-HMR-R and Down-HMR-F/ Down-HMR-R, respectively (S1 Table). Primer Up-HMR-R contains 40 bp of homology to the 5' end of the downstream PCR fragment. These two fragments were then combined by fusion PCR using primers Up-HMR-F and Down-HMR-R, giving rise to the Δhmr fragment (S4 Table).

As shown in S4 Table, other fragments for pop-out experiments were obtained by direct PCR on genomic DNA.

About 1 μg of each pop-out fragment was used to transform Ura+ strains, which were then plated onto YPD, grown for 24 hrs and replica-plated onto 5-FOA plates. Resulting 5-FOAR colonies were checked by PCR for correct removal of the URA3 marker, and the locus sequenced.

Strains obtained by mating-type switching

When possible, we took advantage of the efficient mating-type switching induced by ScHO to transpose the inc-Ho site mutation from one MTL to another, instead of doing pop-in/pop-out transformations as above. For example, an HMLalpha-inc locus can easily be used as template, during gene conversion, to repair either MAT wt or HMR wt. In addition, extra-chromosomal copies of either MATa-inc or MATalpha-inc were also used as templates for mating-type switching of MTL loci, in order to insert inc-Ho sites. These copies were introduced in the p7.1 plasmid, as follows. Plasmid p7.1 [26] was digested by KpnI, and MATa-inc and MATalpha-inc sequences were amplified by PCR using primers Up-Rec-MAT-F/Down-Rec-MAT-R on strains YL09 and YL07, respectively (Table 1 and S1 Table). Both primers share, respectively, 40 bp of homology to the ends of the KpnI-digested plasmid. This allows PCR fragment cloning in p7.1, at the KpnI restriction site, by homologous recombination in E. coli [46]. Correct assembly was confirmed by both analytic colony PCR and restriction digests.

Expression of Ho is induced in strains that are targeted for modification, either from the p7.1 plasmid, when a genomic MTL locus is used as template, or from p7.1-derived plasmids that contain a copy of MATa-inc or MATalpha-inc. Final loci are checked by PCR and sequencing.

Southern-blot analysis

Genomic DNAs were prepared using the Qiagen genomic DNA kit, according to manufacturer's instructions. 2 μg DNA was subjected to enzymatic digestions and protocol for Southern-blot is detailed in [26]. Primers used for probe PCR amplifications are given in S1 Table.

Construction of the Δrad51 mutant using CRISPR-Cas9

The Δrad51 mutant of strain HM100 was constructed with the CRISPR-Cas9 system on plasmid pJH-2972 (kind donation from J. Haber, https://protocolexchange.researchsquare.com/article/nprot-5791/v1). We cloned a sequence corresponding to a gRNA targeting the RAD51 gene into plasmid pJH-2972 (S1 Table), giving rise to plasmid pJH-RAD51.

We amplified upstream and downstream sequences (500 bp each) of the RAD51 CDS (CAGL0I05544g) on strain HM100 by PCR using primers Up-Rad51-F/Up-Rad51-R and Down-Rad51-F/Down-Rad51-R, respectively (S1 Table). Primer Up-Rad51-R contains 40 bp of homology to the 5' end of the downstream PCR fragment. These two fragments are then combined by fusion PCR using primers Up-Rad51-F and Down-Rad51-R, giving rise to the Δrad51 fragment.

The strain was then co-transformed with both 1 μg of pJH-RAD51 and 1 μg of Δrad51 fragment. Ura+ transformants were then selected on SC-Ura and checked for deletion at the RAD51 locus by PCR. Deletion was confirmed by Southern blot analysis (S4 Fig) and by sequencing.

Comparison of wild-type and mutated Ho sites of loci carrying Yalpha (A) or Ya information (B). The wild-type Ho site is shown on top in blue letters, the mutated Ho site is shown below with mutated bp in red and deleted bp as dashes. Arrows indicate the Ho cleavage site.

(TIF)

Click here for additional data file.

Mating-type screened by PCR at MAT in different strains.

All strains are analyzed with primer pairs that are specific to MATa, MATalpha, MATa-inc and MATalpha-inc, respectively GS01/123, GS01/121, GS01/122 and GS01/120. Top left panel: amplification obtained on BG87 (MATa); bottom left panel: amplification obtained on YL05 (MATa-inc); top right panel: amplification obtained on HM100 (MATalpha); bottom right panel: amplification obtained on YL07 (MATalpha-inc). MM: Molecular Marker, GeneRuler 1 kb (Thermo Fisher Scientific Inc).

(TIF)

Click here for additional data file.

Southern-blot analysis of the DSB at the MAT locus in the wild-type HM100 strain.

Left panel: chemiluminescence image of blot of PstI/XhoI digestion hybridized with a probe corresponding to 484 bp located 262 bp away from the first nucleotide of the Ho site. Diagram of probe in top middle panel. The regions are represented with restriction sites and size of expected fragment. Right panel: chemiluminescence image of blot of BglII/EcoRI digestion hybridized with a probe corresponding to 1,013 bp located 2,119 bp away from the first nucleotide of the Ho site. Diagram of probe is in lower middle panel.

(TIF)

Click here for additional data file.

Molecular characterization of Δrad51 mutations in HM100 by Southern blot hybridization.

Left panel: chemiluminescence image of blot of HindIII digestion. Right panel: chemiluminescence image of blot of NdeI/EcoRI digestion. The probe used is 1 kb long and is composed to the 500 bp upstream of the RAD51 ORF fused to the 500 bp downstream of the RAD51 ORF.

(TIF)

Click here for additional data file.

Primers used in this work.

Fw: Forward; Rv: Reverse. The lowercase letters represent sequences with no homology to template DNA and reverse complete regions are indicated in uppercase.

(DOCX)

Click here for additional data file.

Plasmids used in this work.

(DOCX)

Click here for additional data file.

Numerical data for figures.

Tables show percentage of survival calculated for each individual transformant in experiments represented as graphs. ND: Not done. SEM: Standard error of the mean. For Figs 4 and 5C number of colonies counted during time-course experiments is also shown.

(XLSX)

Click here for additional data file.

Methods used for strain construction.

(DOCX)

Click here for additional data file.

9 Mar 2020

Dear Cecile,

Thank you very much for submitting your Research Article entitled 'A single Ho-induced double-strand break at the MAT locus is lethal in Candida glabrata' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers who are all experts in the field. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review again a much-revised version. We cannot, of course, promise publication at that time.  The reviewers have provided ideas about how to restructure the manuscript, how to present the models or hypotheses being tested, and suggested additional experiments.  Given the extent of their reviews, this was on the edge of major revision/rejection.  I am sending to you the decision of major revision to provide an opportunity to address the reviews.  I appreciate that this may take some time, and thus am extending the time available from 60 to 120 days.  A resubmitted manuscript would be reviewed again by the same set of reviewers if available.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

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Gregory P. Copenhaver

Editor-in-Chief

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: This is a careful analysis of the fate of Candida glabrata cells when the Ho endonuclease is expressed in Candida. An earlier publication showed that expression of Ho generated DSBs that were substrates for recombination and mating type switching but that resulted in very drastic lethality. The result shown here by analyzing switching and lethality in mutant strains where Ho sites have been removed from MAT, HML or HMR is that cleavage at MAT alone is sufficient to generate lethality independent of the mating type information at MAT. The data are consistent with the DSB being poorly repaired when generated by Ho, since a DSB generated by CRISPR/CAS show low lethality.

The authors nicely show that lethality depends on having a Ho-sensitive MAT allele. Interestingly, the survival does not increase when both HMR and HML are Inc. This suggests that cleavage at these loci is not an important contributor to lethality but also that repair from these loci and then persistent re-cutting is probably not the mechanism of lethality since repairing with an INC substrate would be expected to increase survivability. Rather the data suggest that the cut at MAT is not easily repairable. Cutting at MAT by CRISPR/CAS results in efficient switching but no lethality. In colonies that result, 80-100% of cells have switched and are viable. This suggests that the DSB generated by Ho is specifically lethal.

These data are excellent and the conclusions are compelling and the genetic analysis with the engineered strains is elegant and informative, and the results are surprising. Some additional controls are needed, or if they exist should be shown, to see whether cleavage by Ho is occurring at HMR and HML. If these are actively being cut and gene converted by the other HM locus or by MAT, then the question is why there is no lethality. Are the survivors in YL04 YL05 and YL07 able to repair a DSB, or is the information between HML and HMR non-essential and simply lost, or are these loci not being cleaved at appreciable rates? The manuscript does not present quantitative data to show whether there is cleavage at HML and HMR and whether DSBs there are repaired by homologous recombination. This would help distinguish models for why MAT DSBs are so toxic.

The paper is a little anticlimactic since there is no final conclusion as to why the Ho generated DSB can't be repaired. it would be easily improved by direct assays demonstrating persistence of the DSB. If it is happening in 99% of cells, can the DSB be visualized by Southern, or quantitated by a PCR assay with a primer ligated to the break? If so, comparing DSB persistence at MAT HML and HMR would add significantly to the paper. Can a tagged version of Ho be used for an IP to show presence at the MAT locus and/or the HML and HMR loci?

Reviewer #2: This is an intriguing and well written manuscript that aims to understand the mechanism by which the Saccharomyces cerevisiae (Sc) HO endonuclease causes severe lethality when expressed in Candida glabrata (Cg) cells. It has implications for our understanding of how HO works, and the manuscript provides some interesting indications that HO has a function that goes beyond being a simple endonuclease.

In previous work (Boisnard et al 2015, ref. 22) the authors’ group discovered that expressing Sc HO in C. glabrata causes a high rate of cell death (99.9% of cells die). In that study, they proposed that the lethality was caused by simultaneous cutting of the Cg HMR and HML loci as well as the MAT locus, leading to absence of intact templates for DNA repair. However, in the current manuscript Maroc et al test this hypothesis and reject it. In a careful series of experiments in which they mutated the HO cleavage sites in Cg MAT, HML and/or HMR, they show that HO-cleavage of the MAT locus alone is sufficient to cause lethality, even though most of the few cells that survive HO expression have switched mating type. Maroc et al also show that, in contrast, cleavage of the Cg MAT locus by CRISPR-Cas9 does not cause lethality and leads to mating-type switching. So the lethality does not stem from the existence of a DSB per se, but from the existence of a DSB in association with HO, which is intriguing. Although the manuscript does not succeed in its goal of identifying the molecular cause of the incompatibility between Sc HO and Cg MAT, it goes a long way towards narrowing down the range of possible causes.

Major comment

L368-378 (end of Discussion section): The manuscript has made a substantial step towards understanding the toxic role of S. cerevisiae HO in C. glabrata, but we are left with the possibility that HO might be toxic because it attacks some unknown off-target (non MAT) site in the C. glabrata genome (L372-373). A fairly simple way to address this question experimentally would be to see if the lethality can be moved into S. cerevisiae, by moving the Cg MAT cleavage site into S. cerevisiae. Construct a haploid S. cerevisiae strain that is uncleavable at the Sc MAT locus (MAT-inc or similar), but which also carries the Cg cleavage site (~100 bp spanning the Cg Y/Z junction) somewhere in its genome, and then introduce HO from p7.1 to see if it causes lethality.

Minor comments

L24, L28, The taxonomic group can be called either Ascomycota (phylum) or Saccharomycotina (subphylum), but there is no ‘Ascomycotina’.

L40, add “…, whereas one DSB at HML or HMR is not.”

L44, change to “…, but that repair is prevented by S. cerevisiae’s Ho”.

L86, change to “… mat1, called mat2 and mat3”.

L88, also cite Rajaei et al, PMID 25313032.

L100, change “implies” to “involves”

L114, L221, L305: When referring to the RE, the manuscript cites reference 15 (a review article by Herskowitz 1988) several times. Surely there are more relevant references, such as PMID 8861911, 9335581.

L132-133: change to “a series of inconvertible (inc) C. glabrata strains … DNA breaks during induction of S. cerevisiae Ho … “

L152: I was surprised by the reference to data “not shown” here, because the title of this section is “HMR is cut by Ho …”. The authors do show data in support of HMR cleavage later in the manuscript (SL-CG9 results in Table 2) so it would be preferable to rearrange the order of the sections in Results / or merge some of them / or rephase L152 to say that evidence for cleavage of HMR will be presented in a subsequent section of the paper.

L232: This paragraph should not begin with “Similarly...” but “In contrast,… ” because the frequency of switching in the correct direction changes dramatically from 97% (MATa to MATalpha) to <14% (MATalpha to MATa).

L300: change to “cut by the S. cerevisiae Ho endonuclease”

L311-315: I agree with the authors that the mating-type switching system in C. glabrata is less active/efficient than in S. cerevisiae, and that the word “degenerated” is probably an accurate word to use. However Cg HO is not a pseudogene, which is what we would expect if it truly has no function. One could speculate that HO might have a second function in addition to being the mating-type switching endonuclease, and this second function causes the HO gene to be maintained in C. glabrata. In the population genomics data of Carrete et al (2018), did all the studied C. glabrata stains have an intact HO ORF, and was the nonsynonymous/synonymous substitution ratio for this gene <1, indicating purifying selection ?

L350: Change to “suggest that the S. cerevisiae Ho protein prevents DSB repair specifically at the MAT locus of C. glabrata”

L357-359. I didn’t understand the sentences “We know that mating-type borne by any of the MAT does not influence lethality as both HM100 and BG87 die at 99.99 % (Fig 1). Thus, only the location of the MAT locus could explain the specificity of lethality induced by Ho.” Do you mean “We know that lethality occurs in both MATa and MATalpha cells, because both HM100 and BG87 die at 99.99%”?

L361: ref 36 is the wrong reference.

L368: “Finally, we would like to discuss the toxic role of S. cerevisiae’s Ho, in C. glabrata, outside its role in lethal DSB at MAT.” Is this an hypothesis (you are proposing that it has an unidentified toxic role apart from DSB formation), or is it a statement of fact? If it is a statement of fact, what is the evidence that such a toxic role outside DSB formation exists?

The plasmid name p7.1 appears in the titles of several Figures and Tables, but it is not explained in the text until line 402. It should be introduced on approx. line 180.

Figure 1: Shouldn’t HML be colored red (alpha) in HM100 and HM100 Δrad51 ?

Figure 2: I found the X-axis title “Time of nuclease induction” confusing because it seems to indicate that the nuclease was induced at time T, where T = 0, 2, 4 hrs etc. But I think that you actually mean that the nuclease was induced at T=0 and then repressed at T = 2, 4, hrs etc. Change the title to “Duration of nuclease induction” ?

Figure 3B: The HO cut is shown as a blunt DSB, but it should be a 4-bp 3’ overhang. Also, the Y/Z junction is drawn in the wrong place: it should be between the TTTT and the CGCAACA on the bottom strand (if we permit a 1-bp mismatch in the Z region, CGCAACA in MATa and CGCAGCA in MATalpha).

Tables 2 and 3: For readers like me who are slow at mental arithmetic, it would be easier to relate the colony numbers in the Tables to the percentages in the text, if you showed percentages as well as numbers in the Tables. For example, for the SL-CG9 Cas9 experiment, Line 264 says that 87% of colonies presented MATalpha-inc and cites Table 3, but to find this information in Table 3 I have to calculate that 32/45 + 7/45 = 87%.

Reviewer #3: Mating type switch in Saccharomyces and related yeasts is a well-studied process, but many unanswered questions remain. The authors have addressed the puzzling retention of the mating-type switching apparatus in Candida glabrata, even though it is apparently asexual. Intriguingly, they showed that that they can induce switching in C. glabrata using CRISPR-Cas9, demonstrating that the all the components (apart from the endonuclease) are fully functional. However, when the S. cerevisiae HO endonuclease is overexpressed, cutting at MAT results in cell death. HML and HMR are also cut. These experiments are carefully performed, but the underlying biological mechanism effect is not clear.

1. I think that the CRISPR-Cas9 mediated induction of switching is one of the most interesting parts of this paper. It has been shown in S. cerevisiae, but this is the first time that it is clear that switching is fully functional in C. glabrata (apart from the endonuclease) and that cells can switch without inducing cell death. I suggest re-framing the paper, and starting from here.

2. RAD51 should be inactivated in the Cas9-activated strain, this would show more clearly if switching relies on homologous recombination.

3. The authors show that Cas9 induces switching but not cell death, whereas expression of ScHO induces switching and cell death. They suggest that Ho may remain at the cut ends. What happens if Cas9 and Ho are added/induced at the same time? This might help to work out the mechanism.

4. I think that the observation that expressing ScHO in K. delphensis does not induce cell death is important, and should be shown in the manuscript.

5. There seems to be an error in Fig. 1, the colors for the top two strains are not correct, surely they are HMLalpha?

Minor points

1. The start of the results section is hard to follow. It is assumed that the reader is very familiar with the earlier experiments. More details, for example how ScHO is expressed and how the target sites are changed, would be helpful. Switching at HMR should be shown, PLoS does not allow “data not shown”

2. There are some odd phrases. Line 138 might be better phrased as “ We find that even when HML and HMR are protected, introducing a DSB at MAT is sufficient…”

3. Line 124 has an extra “to”.

4. Line 128: unlike, rather than contrary

5. Line 85: Clarify what an “imprint” at mat1 means.

Reviewer #4: The manuscript ‘A single HO-induced double-strand break at the MAT locus is lethal in Candida glabrata’ seeks to follows up on an interesting result from a previous study that found lethality associated with heterologous S. cerevisiae HO expression in C. glabrata. The authors find that the rare cells that survive ScHO expression can indeed undergo mating type switching, although there is a preference for HML over HMR, regardless of the MAT genotype. Furthermore, mating type switching is dependent on Rad51 and homologous recombination. The authors construct a series of strains in which they disrupt the HO-cleavage sites at the MAT or cryptic mating type loci, HML and HMR and then measure cell lethality when cutting is blocked. Intriguingly, it is cleavage at the MAT locus, but not the cryptic mating type loci that results in a lethal phenotype. Furthermore, MAT-cleavage lethality is specific to HO cutting – the authors induce CAS9 cleavage at the MAT locus and find that lethality is abolished, but the frequency of mating-type switching is reduced compared to HO cleavage. While this is an interesting insight into the degradation of pseudo-homothallism in C. glabrata, the current presentation and organization of this manuscript makes it difficult to follow and does not provide enough context for non-expert readers, and the interpretation of the HO-specific lethality is tenuous, given the limitations of the experiment. Taken together, this work is not of sufficient interest to the broad range of PLoS Genetics readers in its current state. Furthermore, there are several experimental considerations that should be taken into account.

1) There are currently three main figures – but many aspects of figures 1 and 2 and not discussed until much later in the manuscript. As a reader this was very difficult to discern what was going on. I suggest rearranging the figures. The first part of figure 1 (all the strains that have intact cleave sites) should be presented with figure 2a. The second figure should be the constructed strains that cannot cut at the various loci and their resulting survival. The third figure should be regarding the Cas9-induced cleavage and include figure 2b (which is currently not discussed or referred to in the text).

2) The CAS9-induced cleavage is a very cool experiment, but given that the cleavage site is different, if only by an 18-bp shift, it is hard to directly compare HO- and CAS9- induced cleavage. If the authors designed additional gDNAs that could cut at many different places in the MAT locus this would bolster their argument that lethality is specific to HO. Alternatively, if the HO cleavage site was moved to different locations within the MAT locus and still resulted in lethality, this also would better support that HO-cleavage is distinct from other DSBs and not due to exact location of the cut.

3) The authors refer to cell viability as the survival rate, but my interpretation of the experiment is that this is a frequency of surviving cells relative to the total number in the population, and thus it would be more appropriate to refer to this as a frequency, or simply % survival.

4) I’m a little confused about the ‘mixed’ mating-type analysis – does this suggest that some surviving cells are able to survive because they are delayed in HO cleavage and thus, when HO cuts it can switch using HML or HMR within that colony? Also, the sub-cloning results seemed inconsistent with the original analysis. The original analysis showed that ~20% of colonies were mixed, but then the subcloning indicated that there were no MATa-inc isolated? Clarification regarding this experiment and analysis is needed.

5) I’m not exactly sure what audience this paper was written for. Certainly for an expert audience in yeast mating-type switching, but aside from that, it’s unclear if the scope of the paper is to investigate the molecular mechanism by which switching is lethal in C. glabrata or to investigate the evolutionary trajectory as to why C. glabrata is seemingly asexual. I think it is likely both, but these ideas are not woven together and developed in this manuscript. Most experiments in the results are not introduced with the specific question they are designed to address, which leaves the reader guessing as to what we are trying to learn. Providing more background and context for the motivation for this study (beyond just stating that an earlier study showed heterologous expression of ScHO is lethal in C. glabrata) is needed.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No: Some experiments listed as "not shown"

Reviewer #4: No: NA. No large-scale experiments were discussed in this study. Also, a number of results were indicated as 'data not shown'

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

26 Jun 2020

Submitted filename: Reply to reviewers PLoS Genet v2cf.docx

Click here for additional data file.

17 Jul 2020

Dear Cecile,

Thank you very much for submitting your revised Research Article entitled 'A single Ho-induced double-strand break at the MAT locus is lethal in Candida glabrata' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by the same set of independent peer reviewers who reviewed the original submission. The reviewers appreciated the attention to an important problem, and found the revised manuscript greatly improved.  This said, two of the four still found that additional experiments might be needed to address models, hypotheses advanced, and two others felt the manuscript would benefit from having some data removed and not presented.  Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review again a revised version. This would fall in between minor and major revision.  We appreciate that this isn't the response that authors are hoping to receive, and appreciate your continued interest in publishing these studies in PLOS Genetics.  We hope that you find the reviews and comments helpful and constructive, and look forward to receiving a revised manuscript that addresses the reviews.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

If you decide to revise the manuscript for further consideration at PLOS Genetics, please aim to resubmit within the next 60 to 90 days, unless it will take extra time to address the concerns of the reviewers, in which case we would appreciate an expected resubmission date by email to plosgenetics@plos.org.

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To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see our guidelines.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

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We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Joseph Heitman, MD, PhD

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Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: This revision appropriately addresses many reviewers' concerns. Demonstration of efficient cleavage and repair at HML and HMR adds significantly to the paper. It highlights the the key question of why HO generated breaks at MAT are lethal while cleavage at HML or HMR is non lethal as long as there is a template to repair from.

To limit the reasons for this locus specific lethality, an additional experiment would be helpful.

Replace the entire HML or HMR locus with the entire MAT locus, and determine if cleavage here results in lethality. The MAT/HML/HMR loci are not identical and perhaps sequence differences in the locus are responsible. In any case, it would be worth eliminating this possibility.

This proposed additional experiment is similar to the suggestion from a previous reviewer to test lethality of MAT +ScHO in S. cerevisiae, and eliminates a trivial explanation for the striking difference in lethality for HO-generated cleavage at MAT and HML/HMR

Reviewer #2: I am happy with the changes that have been made to the manuscript. The authors have addressed the point about residual lethality that I raised in my previous review.

The revised manuscript has been restructured and is presented in a clearer way than originally. The experiments and arguments are logical and show that:

ScHO endonuclease is lethal when expressed in C. glabrata.

ScHO cleaves all three Cg MTL loci (MAT, HML and HMR).

The lethality of ScHO is due specifically to it cutting the Cg MAT locus, no HML or HMR.

A CRISPR cut at Cg MAT is not lethal and is repaired by mating-type switching.

One small comment is that Figure 1 would be clearer if the HO sites were marked on the sequences, e.g. by underlining, because they are in different orientations for the two species. Also, please check the sequence shown for the Z1 region at Sc HMRa: it shows a 3-bp insertion compared to the Z1 sequences of Sc HMLalpha and MATalpha, and I think this is an error.

In Figure 5, I am not sure that inclusion of the experiments with additional CRISPR guide RNAs (Ya1, Ya3, Ya4) has added anything to the manuscript and I would suggest dropping these.

Reviewer #3: Many, though not all, of the reviewers' requests have been addressed. The most useful are the addition of data in Table 2 indicating that HML and HMR cutting is efficient, and the experiment in Fig. 6 addressing possible lethality caused by cutting at sites other than MAT.The introduction is also much improved. However, i don't think that adding extra gRNAs (Figure 5) has helped. Only one gRNA (the one described in the original submission) induced cutting. Looking at Fig. 5, this is not surprising. For Ya1 and Ya3, the cut site is some distance from the guide, and there is a lot of evidence that distance reduces efficiency. For Ya4, the PAM site is a very inefficient NAG, and this is part of the gRNA sequence. I don't think Cas9 will cut within a guide sequence. I understand that the authors were trying to address a reviewer's comment, but the added experiments add more confusion rather than clarifying. I suggest reverting to the original.

Reviewer #4: The revised manuscript ‘A single HO-induced double-strand break at the MAT locus is lethal in Candida glabrata’ has adequately addressed the most of the considerations I posed during its initial review. The added discussion regarding the evolution and mechanism of mating type switching is welcome, as is the inclusion of the experimental questions in the results section.

There are three outstanding concerns with the revised manuscript.

1) It is presumed that the C. glabrata HO endonuclease is functional, yet doesn’t result in lethality when expressed, but there is 58% identity (82% similarity) with the S. cerevisiae homolog. Has there been biochemical analysis of its cutting efficiency already reported? If not, an EMSA experiment, could demonstrate whether both endonucleases have the capacity to cut at the same efficiency.

2) The low lethality when CAS9 is expressed – could this also be due to low expression or inefficient cutting? Clearly it is capable of cutting, otherwise you wouldn’t see any switching, but how can one distinguish between low efficiency and strong selection for escaping the cleavage of MAT (such as by mutating the plasmid as the authors suggest)?

3) I still find presentation of the figures to be problematic with the text. For example, in Figure 2 there are two strain that are initially discussed (HM100 and HM100 �rad51; lines 178-182) and the other four strains that are not discussed until after Figure 3 has been presented (lines 222-232). Likewise, the text regarding data presented in Figure requires us to go back to Figure 3 to have a control to compare it. For experts in the field, this may not be an issue, but for those new to the field or still in their scientific training, this is very confusing and can be easily rectified. For example, in the latter case, adding the data from strain SL09 to Figure 6 with a note in the legend that it is the same data as in Figure 3 would suffice and would allow for the statistical significance to be conveyed on the graph in addition to in the text.

Minor notes:

Line 161 is written in the present tense, yet the rest of the section is written in the past tense.

Line 344: ‘inducting’ should be inducing?

Line 363: what is meant by ‘some sexual identity of cells in maintained in C. glabrata’?

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

10 Sep 2020

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

12 Sep 2020

Dear Cecile,

Congratulations!  We are pleased to inform you that your revised manuscript entitled "A single Ho-induced double-strand break at the MAT locus is lethal in Candida glabrata" has been editorially accepted for publication in PLOS Genetics.

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional accept, but your manuscript will not be scheduled for publication until the required changes have been made.

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In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!  We appreciate your careful revision of the manuscript, including additional experimental analysis, and we hope that our review process has been a professional, collegial, and positive experience.  We very much appreciate your entrusting this outstanding manuscript to PLOS Genetics, and look forward to seeing this published.

Yours sincerely,

Joe

Joseph Heitman, MD, PhD

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

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Comments from the reviewers (if applicable):

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Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly:

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-20-00064R2

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8 Oct 2020

PGENETICS-D-20-00064R2

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

Dear Dr Fairhead,

We are pleased to inform you that your manuscript entitled "A single Ho-induced double-strand break at the MAT locus is lethal in Candida glabrata" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript.

Soon after your final files are uploaded, unless you have opted out or your manuscript is a front-matter piece, the early version of your manuscript will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting PLOS Genetics and open-access publishing. We are looking forward to publishing your work!

With kind regards,

Jason Norris

PLOS Genetics

On behalf of:

The PLOS Genetics Team

Carlyle House, Carlyle Road, Cambridge CB4 3DN | United Kingdom

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