Canonical and noncanonical roles of Hop1 are crucial for meiotic prophase in the fungus Sordaria macrospora.

We show here that in the fungus Sordaria macrospora, the meiosis-specific HORMA-domain protein Hop1 is not essential for the basic early events of chromosome axis development, recombination initiation, or recombination-mediated homolog coalignment/pairing. In striking contrast, Hop1 plays a critical role at the leptotene/zygotene transition which is defined by transition from pairing to synaptonemal complex (SC) formation. During this transition, Hop1 is required for maintenance of normal axis structure, formation of SC from telomere to telomere, and development of recombination foci. These hop1Δ mutant defects are DSB dependent and require Sme4/Zip1-mediated progression of the interhomolog interaction program, potentially via a pre-SC role. The same phenotype occurs not only in hop1Δ but also in absence of the cohesin Rec8 and in spo76-1, a non-null mutant of cohesin-associated Spo76/Pds5. Thus, Hop1 and cohesins collaborate at this crucial step of meiotic prophase. In addition, analysis of 4 non-null mutants that lack this transition defect reveals that Hop1 also plays important roles in modulation of axis length, homolog-axis juxtaposition, interlock resolution, and spreading of the crossover interference signal. Finally, unexpected variations in crossover density point to the existence of effects that both enhance and limit crossover formation. Links to previously described roles of the protein in other organisms are discussed.

RNAi-Related Dicer and Argonaute Proteins Play Critical Roles for Meiocyte Formation, Chromosome-Axes Lengths and Crossover Patterning in the Fungus Sordaria macrospora.

RNA interference (RNAi) is a cellular process involving small RNAs that target and regulate complementary RNA transcripts. This phenomenon has well-characterized roles in regulating gene and transposon expression. In addition, Dicer and Argonaute proteins, which are key players of RNAi, also have functions unrelated to gene repression. We show here that in the filamentous Ascomycete Sordaria macrospora, genes encoding the two Dicer (Dcl1 and Dcl2) and the two Argonaute (Sms2 and Qde2) proteins are dispensable for vegetative growth. However, we identified roles for all four proteins in the sexual cycle. Dcl1 and Sms2 are essential for timely and successful ascus/meiocyte formation. During meiosis per se, Dcl1, Dcl2, and Qde2 modulate, more or less severely, chromosome axis length and crossover numbers, patterning and interference. Additionally, Sms2 is necessary both for correct synaptonemal complex formation and loading of the pro-crossover E3 ligase-protein Hei10. Moreover, meiocyte formation, and thus meiotic induction, is completely blocked in the dcl1 dcl2 and dcl1 sms2 null double mutants. These results indicate complex roles of the RNAi machinery during major steps of the meiotic process with newly uncovered roles for chromosomes-axis length modulation and crossover patterning regulation.

Genome editing in the yeast Nakaseomyces delphensis and description of its complete sexual cycle.

The environmental yeast Nakaseomyces delphensis is, phylogenetically, the closest known species to Candida glabrata, a major fungal pathogen of humans. C. glabrata is haploid and described as asexual, while N. delphensis is also haploid, but has been described as competent for mating and meiosis. Both genomes contain homologues of all the genes necessary for sexual reproduction and also the genes for Ho-dependent mating-type switching, like Saccharomyces cerevisiae. We first report the construction of genetically engineered strains of N. delphensis, including by CRISPR-Cas 9 gene editing. We also report the description of the sexual cycle of N. delphensis. We show that it undergoes Ho-dependent mating-type switching in culture and that deletion of the HO gene prevents such switching and allows maintenance of stable, separate, MATa and MATalpha haploid strains. Rare, genetically selected diploids can be obtained through mating of haploid strains, mutated or not for the HO gene. In contrast to HO/HO diploids, which behave as expected, Δho/Δho diploids exhibit unusual profiles in flow cytometry. Both types of diploids can produce recombined haploid cells, which grow like the original haploid-type strain. Our experiments thus allow the genetic manipulation of N. delphensis and the reconstruction, in the laboratory, of its entire life cycle.

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.

Efficient Mating-Type Switching in Candida glabrata Induces Cell Death.

Candida glabrata is an apparently asexual haploid yeast that is phylogenetically closer to Saccharomyces cerevisiae than to Candida albicans. Its genome contains three MAT-like cassettes, MAT, which encodes either MATa or MATalpha information in different strains, and the additional loci, HML and HMR. The genome also contains an HO gene homolog, but this yeast has never been shown to switch mating-types spontaneously, as S. cerevisiae does. We have recently sequenced the genomes of the five species that, together with C. glabrata, make up the Nakaseomyces clade. All contain MAT-like cassettes and an HO gene homolog. In this work, we express the HO gene of all Nakaseomyces and of S. cerevisiae in C. glabrata. All can induce mating-type switching, but, despite the larger phylogenetic distance, the most efficient endonuclease is the one from S. cerevisiae. Efficient mating-type switching in C. glabrata is accompanied by a high cell mortality, and sometimes results in conversion of the additional cassette HML. Mortality probably results from the cutting of the HO recognition sites that are present, in HML and possibly HMR, contrary to what happens naturally in S. cerevisiae. This has implications in the life-cycle of C. glabrata, as we show that efficient MAT switching is lethal for most cells, induces chromosomal rearrangements in survivors, and that the endogenous HO is probably rarely active indeed.

Comparative genomics of emerging pathogens in the Candida glabrata clade.

BACKGROUND: Candida glabrata follows C. albicans as the second or third most prevalent cause of candidemia worldwide. These two pathogenic yeasts are distantly related, C. glabrata being part of the Nakaseomyces, a group more closely related to Saccharomyces cerevisiae. Although C. glabrata was thought to be the only pathogenic Nakaseomyces, two new pathogens have recently been described within this group: C. nivariensis and C. bracarensis. To gain insight into the genomic changes underlying the emergence of virulence, we sequenced the genomes of these two, and three other non-pathogenic Nakaseomyces, and compared them to other sequenced yeasts. RESULTS: Our results indicate that the two new pathogens are more closely related to the non-pathogenic N. delphensis than to C. glabrata. We uncover duplications and accelerated evolution that specifically affected genes in the lineage preceding the group containing N. delphensis and the three pathogens, which may provide clues to the higher propensity of this group to infect humans. Finally, the number of Epa-like adhesins is specifically enriched in the pathogens, particularly in C. glabrata. CONCLUSIONS: Remarkably, some features thought to be the result of adaptation of C. glabrata to a pathogenic lifestyle, are present throughout the Nakaseomyces, indicating these are rather ancient adaptations to other environments. Phylogeny suggests that human pathogenesis evolved several times, independently within the clade. The expansion of the EPA gene family in pathogens establishes an evolutionary link between adhesion and virulence phenotypes. Our analyses thus shed light onto the relationships between virulence and the recent genomic changes that occurred within the Nakaseomyces. SEQUENCE ACCESSION NUMBERS: Nakaseomyces delphensis: CAPT01000001 to CAPT01000179Candida bracarensis: CAPU01000001 to CAPU01000251Candida nivariensis: CAPV01000001 to CAPV01000123Candida castellii: CAPW01000001 to CAPW01000101Nakaseomyces bacillisporus: CAPX01000001 to CAPX01000186.

A comparative proteomic approach to better define Deinococcus nucleoid specificities.

Compared to radiation-sensitive bacteria, the nucleoids of radiation-resistant Deinococcus species show a higher degree of compaction. Such a condensed nucleoid may contribute to the extreme radiation resistance of Deinococcus by limiting dispersion of radiation-induced DNA fragments. Architectural proteins may play a role in this high degree of nucleoid compaction, but comparative genomics revealed only a limited number of Deinococcus homologs of known nucleoid-associated proteins (NAPs) from other species such as Escherichia coli. A comparative proteomic approach was used to identify potentially novel proteins from isolated nucleoids of Deinococcus radiodurans and Deinococcus deserti. Proteins in nucleoid enriched fractions were identified and semi-quantified by shotgun proteomics. Based on normalized spectral counts, the histone-like DNA-binding protein HU appeared to be the most abundant among candidate NAPs from both micro-organisms. By immunofluorescence microscopy, D. radiodurans HU and both DNA gyrase subunits were shown to be distributed throughout the nucleoid structure and absent from the cytoplasm. Taken together, our results suggest that D. radiodurans and D. deserti bacteria contain a very low diversity of NAPs, with HU and DNA gyrase being the main proteins involved in the organization of the Deinococcus nucleoids.

The deinococcal DdrB protein is involved in an early step of DNA double strand break repair and in plasmid transformation through its single-strand annealing activity.

The Deinococcus radiodurans bacterium exhibits an extreme resistance to ionizing radiation. Here, we investigated the in vivo role of DdrB, a radiation-induced Deinococcus specific protein that was previously shown to exhibit some in vitro properties akin to those of SSB protein from Escherichia coli but also to promote annealing of single stranded DNA. First we report that the deletion of the C-terminal motif of the DdrB protein, which is similar to the SSB C-terminal motif involved in recruitment to DNA of repair proteins, did neither affect cell radioresistance nor DNA binding properties of purified DdrB protein. We show that, in spite of their different quaternary structure, DdrB and SSB occlude the same amount of ssDNA in vitro. We also show that DdrB is recruited early and transiently after irradiation into the nucleoid to form discrete foci. Absence of DdrB increased the lag phase of the extended synthesis-dependent strand annealing (ESDSA) process, affecting neither the rate of DNA synthesis nor the efficiency of fragment reassembly, as indicated by monitoring DNA synthesis and genome reconstitution in cells exposed to a sub-lethal ionizing radiation dose. Moreover, cells devoid of DdrB were affected in the establishment of plasmid DNA during natural transformation, a process that requires pairing of internalized plasmid single stranded DNA fragments, whereas they were proficient in transformation by a chromosomal DNA marker that integrates into the host chromosome through homologous recombination. Our data are consistent with a model in which DdrB participates in an early step of DNA double strand break repair in cells exposed to very high radiation doses. DdrB might facilitate the accurate assembly of the myriad of small fragments generated by extreme radiation exposure through a single strand annealing (SSA) process to generate suitable substrates for subsequent ESDSA-promoted genome reconstitution.

H2O2 activates the nuclear localization of Msn2 and Maf1 through thioredoxins in Saccharomyces cerevisiae.

The cellular response to hydrogen peroxide (H(2)O(2)) is characterized by a repression of growth-related processes and an enhanced expression of genes important for cell defense. In budding yeast, this response requires the activation of a set of transcriptional effectors. Some of them, such as the transcriptional activator Yap1, are specific to oxidative stress, and others, such as the transcriptional activators Msn2/4 and the negative regulator Maf1, are activated by a wide spectrum of stress conditions. How these general effectors are activated in response to oxidative stress remains an open question. In this study, we demonstrate that the two cytoplasmic thioredoxins, Trx1 and Trx2, are essential to trigger the nuclear accumulation of Msn2/4 and Maf1, specifically under H(2)O(2) treatment. Contrary to the case with many stress conditions previously described for yeast, the H(2)O(2)-induced nuclear accumulation of Msn2 and Maf1 does not correlate with the downregulation of PKA kinase activity. Nevertheless, we show that PP2A phosphatase activity is essential for driving Maf1 dephosphorylation and its subsequent nuclear accumulation in response to H(2)O(2) treatment. Interestingly, under this condition, the lack of PP2A activity has no impact on the subcellular localization of Msn2, demonstrating that the H(2)O(2) signaling pathways share a common route through the thioredoxin system and then diverge to activate Msn2 and Maf1, the final integrators of these pathways.

Peroxisomal ABC transporters and beta-oxidation during the life cycle of the filamentous fungus Podospora anserina.

ATP-binding cassette transporters are ubiquitous proteins that facilitate transport of diverse substances across a membrane. However, their exact role remains poorly understood. In order to test their function in a fungus life cycle, we deleted the two Podospora anserina peroxisomal ABC transporter pABC1 and pABC2 genes as well as the three genes involved in peroxisomal (fox2) and mitochondrial (scdA and echA) beta-oxidation. Analysis of the single and double mutants shows that fatty acid beta-oxidation occurs in both organelles. Furthermore, the peroxisomal and mitochondrial fatty acid beta-oxidation pathways are both dispensable for vegetative and sexual development. They are, however, differently required for ascospore pigmentation and germination, this latter defect being restored in a DeltapABC1 and DeltapABC2 background. We report also that lack of peroxisomal ABC transporters does not prevent peroxisomal long-chain fatty acid oxidation, suggesting the existence of another pathway for their import into peroxisomes. Finally, we show that some aspects of fatty acid degradation are clearly fungus species specific.

Combination of different molecular mechanisms leading to fluconazole resistance in a Candida lusitaniae clinical isolate.

We report on the underlying molecular mechanisms likely responsible for the high-level fluconazole resistance in a Candida lusitaniae clinical isolate. Fluconazole resistance correlated with overexpression of ERG11 and of several efflux pump genes, in particular, the orthologs of the Candida albicans MDR1, PDR16, CDR1, CDR2, and YOR1.

Role of Sho1p adaptor in the pseudohyphal development, drugs sensitivity, osmotolerance and oxidant stress adaptation in the opportunistic yeast Candida lusitaniae.

In yeast, external signals such as high osmolarity or oxidant conditions activate the high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) cascade pathway, which consists of two upstream branches, i.e. Sho1p and Sln1p and common downstream elements, including the Pbs2p MAPK kinase and the Hog1p MAPK. We recently showed that the Candida lusitaniae SLN1 gene, potentially encoding a histidine kinase receptor, is crucial for oxidative stress adaptation when the fungus grows as budding yeast and during the early steps of pseudohyphal development. In the current study, we characterized the SHO1 gene of this opportunistic fungus. Complete loss of SHO1 function causes profound defects in pseudohyphal differentiation, especially in high osmolarity and oxidative stress conditions, suggesting a crucial role of SHO1 in the pseudohyphae morphogenetic transitions. Moreover, when grown as budding yeast, the sho1Delta mutant revealed a sensitivity to compounds that interfere with the cell wall assembly, pointing to a potential role of Sho1p in cell wall biogenesis. However, the sho1Delta mutant does not display evident cell-wall architecture modifications, such as aggregation phenotypes. Although not hypersusceptible to antifungals of clinical relevance, the sho1Delta mutants are susceptible to the filamentous fungi-specific antifungals dicarboximides and phenylpyrroles. Finally, our findings highlight some significant phenotypic differences when the C. lusitaniae sho1Delta mutant is compared with the corresponding mutants described in Saccharomyces cerevisiae, Candida albicans and Aspergillus fumigatus.

Insight into the role of HOG pathway components Ssk2p, Pbs2p, and Hog1p in the opportunistic yeast Candida lusitaniae.

In the present study, we have investigated the role of SSK2, PBS2, and HOG1, encoding modules of the high-osmolarity-glycerol mitogen-activated protein kinase pathway in Candida lusitaniae. Functional analysis of mutants indicated that Ssk2p, Pbs2p, and Hog1p are involved in osmotolerance, drug sensitivity, and heavy metal tolerance but not in oxidant stress adaptation.

Contributions of the response regulators Ssk1p and Skn7p in the pseudohyphal development, stress adaptation, and drug sensitivity of the opportunistic yeast Candida lusitaniae.

We recently characterized the histidine kinase receptor genes of Candida lusitaniae. For the present study, we have further investigated the role of SSK1 and SKN7, encoding response regulators. The results of functional analysis of mutants indicated that Ssk1p is involved in osmotolerance and pseudohyphal development, whereas Skn7p appears crucial for oxidative stress adaptation.

The peroxisomal import proteins PEX2, PEX5 and PEX7 are differently involved in Podospora anserina sexual cycle.

PEX5, PEX7 and PEX2 are involved in the peroxisomal matrix protein import machinery. PEX5 and PEX7 are the receptors for the proteins harbouring, respectively, a PTS1 and a PTS2 peroxisomal targeting sequence and cycle between the cytoplasm and the peroxisome. PEX2 belongs to the RING-finger complex located in the peroxisomal membrane and acts in protein import downstream of PEX5 and PEX7; it is therefore required for the import of both PTS1 and PTS2 proteins. We have shown previously that PEX2 deficiency leads to an impairment of meiotic commitment in the filamentous fungus Podospora anserina. Here we report that both PEX5 and PEX7 receptors are dispensable for this commitment but are needed for normal sexual cycle. Data suggest also a new role of PEX2 and/or the RING-finger complex in addition to their role in PTS1 and PTS2 import. Strikingly, Deltapex5 and Deltapex7 single and double knockout strains analyses indicate that Deltapex7 acts as a partial suppressor of Deltapex5 life cycle deficiencies. Moreover, contrary to pex2 mutants, Deltapex5 and Deltapex7 show mitochondrial morphological abnormalities.

Comparative study of class 1 integron and Vibrio cholerae superintegron integrase activities.

Superintegrons (SIs) and multiresistant integrons (MRIs) have two main structural differences: (i) the SI platform is sedentary, while the MRI platform is commonly associated with mobile DNA elements and (ii) the recombination sites (attC) of SI gene cassette clusters are highly homogeneous, while those of MRI cassette arrays are highly variable in length and sequence. In order to determine if the latter difference was correlated with a dissimilarity in the recombination activities, we conducted a comparative study of the integron integrases of the class 1 MRI (IntI1) and the Vibrio cholerae SI (VchIntIA). We developed two assays that allowed us to independently measure the frequencies of cassette deletion and integration at the cognate attI sites. We demonstrated that the range of attC sites efficiently recombined by VchIntIA is narrower than the range of attC sites efficiently recombined by IntI1. Introduction of mutations into the V. cholerae repeats (VCRs), the attC sites of the V. cholerae SI cassettes, allowed us to map positions that affected the VchIntIA and IntI1 activities to different extents. Using a cointegration assay, we established that in E. coli, attI1-x-VCR recombination catalyzed by IntI1 was 2,600-fold more efficient than attIVch-x-VCR recombination catalyzed by VchIntIA. We performed the same experiments in V. cholerae and established that the attIVch-x-VCR recombination catalyzed by VchIntIA was 2,000-fold greater than the recombination measured in E. coli. Taken together, our results indicate that in the V. cholerae SI, the substrate recognition and recombination reactions mediated by VchIntIA might differ from the class 1 MRI paradigm.

Overexpression of a human and a fungal ABC transporter similarly suppresses the differentiation defects of a fungal peroxisomal mutant but introduces pleiotropic cellular effects.

Among the peroxisome membrane proteins, some are required for peroxisome biogenesis (e.g. PEX2) while others are not, e.g. ABC (ATP-binding cassette) transporters. Unexpectedly, overproduction of the peroxisomal ABC transporter PMP70 was found to be able to restore peroxisome biogenesis in mammalian pex2 mutant cell lines. In the filamentous fungus Podospora anserina, pex2 mutations not only impair peroxisome biogenesis but also cause a precise cell differentiation defect. Here, we show that both defects are partially suppressed by expression of the human cDNA encoding PMP70. In addition, PMP70 expression causes new developmental defects, different from those induced by pex2 mutations. We also show that overexpression of the P. anserina pABC1 gene, which encodes a peroxisomal ABC transporter, leads to similar effects. Taken together, our results demonstrate that: (i) the genetic relationship between PEX2 and PMP70, initially observed in mammals, has been conserved through evolution; (ii) the cell differentiation defect observed in the P. anserina pex2 mutants is indeed linked to impairment in peroxisome biogenesis; and (iii) unexpected detrimental cellular defects result from overproduction of peroxisomal ABC transporters.