Whole Genome Duplication and Enrichment of Metal Cation Transporters Revealed by De Novo Genome Sequencing of Extremely Halotolerant Black Yeast Hortaea werneckii

Hortaea werneckii, ascomycetous yeast from the order Capnodiales, shows an exceptional adaptability to osmotically stressful conditions. To investigate this unusual phenotype we obtained a draft genomic sequence of a H. werneckii strain isolated from hypersaline water of solar saltern. Two of its most striking characteristics that may be associated with a halotolerant lifestyle are the large genetic redundancy and the expansion of genes encoding metal cation transporters. Although no sexual state of H. werneckii has yet been described, a mating locus with characteristics of heterothallic fungi was found. The total assembly size of the genome is 51.6 Mb, larger than most phylogenetically related fungi, coding for almost twice the usual number of predicted genes (23333). The genome appears to have experienced a relatively recent whole genome duplication, and contains two highly identical gene copies of almost every protein. This is consistent with some previous studies that reported increases in genomic DNA content triggered by exposure to salt stress. In hypersaline conditions transmembrane ion transport is of utmost importance. The analysis of predicted metal cation transporters showed that most types of transporters experienced several gene duplications at various points during their evolution. Consequently they are present in much higher numbers than expected. The resulting diversity of transporters presents interesting biotechnological opportunities for improvement of halotolerance of salt-sensitive species. The involvement of plasma P-type H+ ATPases in adaptation to different concentrations of salt was indicated by their salt dependent transcription. This was not the case with vacuolar H+ ATPases, which were transcribed constitutively. The availability of this genomic sequence is expected to promote the research of H. werneckii. Studying its extreme halotolerance will not only contribute to our understanding of life in hypersaline environments, but should also identify targets for improving the salt- and osmotolerance of economically important plants and microorganisms.


Introduction
Salinization of soil as a form of land degradation is rendering large areas of arable lands useless for crop cultivation and is thus an increasingly important cause of agricultural losses [1,2]. High concentrations of salt decrease the performance of plants by disrupting all the major processes required for their growth, including photosynthesis and energy metabolism [3]. Attempts at breeding salt-tolerant lines or cultivars of crops have failed to overcome this problem and genetic engineering has yet to yield the desired results [4]. The majority of genes that have been used for this purpose to date have originated from salt-sensitive donors. Since novel sources of genes are still much needed [5,6], more attention should be focused on organisms from natural hypersaline environments. Among these, halotolerant or halophilic fungal species are promising candidates [7]. Working with them, however, demands considerably more effort compared to conventional model organisms with sequenced genomes. Accordingly, the draft genome sequence of one such fungus, Hortaea werneckii, can open new avenues for experimental exploitation of its genetic resources. These may prove to be useful not only for crop improvement, but also for industrial microorganisms. For example Saccharomyces cerevisiae with increased tolerance to osmotic stress would be greatly beneficial in the ethanol production industry [8]. In addition to the obvious biotechnological applications, this work represents the first published genome sequence of an extremely halotolerant fungus and is thus an important step toward better understanding of eukaryotic halotolerance.
Hortaea werneckii is a melanised yeast-like fungus, belonging to the ascomycetous order Capnodiales. It was primarily known as a causative agent of tinea nigra, a superficial mycotic infection of the human skin mainly affecting the palms [9]. It was also found on salty food [10] and other low-water-activity substrates such as arid inorganic and organic surfaces [11], seawater [12] and beach soil [13]. The primary environmental ecological habitat of H. werneckii is probably hypersaline water in evaporite ponds of solar eutrophic salterns [14,15] but in the salterns it was also found on wood immersed in brine [16], in biofilms on the surface of hypersaline waters, in the soil in dry evaporite ponds and in the saltern microbial mats [14]. It is one of the most salt-adaptable species known among eukaryotes as it can cope with solutions of NaCl ranging from 0% to almost saturating concentrations [15]. High adaptability and salt tolerance are properties that make H. werneckii a very appropriate model system for studying salt tolerance in eukaryotes.
So far, however, most attempts at improving salt tolerance of plants have focused on either improving osmotic adjustment or Na + exclusion, but targeting the K + homeostasis was mostly neglected (reviewed in [36]). In this sense, the cation transporters of H. werneckii as possible transgenes for crop improvement represent an opportunity that is still completely unexplored. The cell transporter systems are of key importance in survival of high salinity environments. Systems ensuring efficient uptake and efflux of K + and/or Na + are highly conserved [37]. Reports describe several cases of successful expression of plant cation transporters in yeast [38][39][40] and vice versa [41,42]. Various transporters are most extensively studied in the model organism S. cerevisiae. When yeast cells are exposed to high salinities, they expend energy accumulating sufficient amounts of intracellular K + , which is required for many physiological functions; and for maintaining low cytosolic Na + , which binds to and inhibits several enzymes involved in key metabolic processes in the cytoplasm [43].
At the plasma membrane the potassium uptake systems Trk1 and Trk2 [44], the potassium channel Tok1 [45], the P i -Na + symporter Pho89 [46] and the efflux systems Ena (Na + -ATPases) [47][48][49] and Nha1 (Na + /H + antiporter) [50] maintain the sufficient intracellular K + amounts and the K + homeostasis, exclude toxic Na + , preserve the membrane potential, keep the positive turgor inside the cell and cope with osmotic stress [43]. Intracellular cation/H + antiporters, like vacuolar Vnx1 [51], endosomal Nhx1 [52], Golgi apparatus Kha1 [53] and mitochondrial K + /H + exchanger system (Mdm38, Mrs7, Ydl183c) [54,55] similarly serve to regulate the cytosolic and intraorganellar cation homeostasis and pH and modulate protein trafficking through the endosomal pathway [43]. The energy needed for the functioning of these systems comes from the plasma membrane (Pma1, Pma2) and vacuolar (Vma1) H + -ATPases [43]. Pma1 is the most abundant plasma membrane protein, and is responsible for establishing the electrochemical gradient of protons across the membrane that is used by secondary active symporters and antiporters [56,57]. Vma1, on the other hand, is a multi-subunit protein complex that has an important role in energising the organellar cation/H + antiporters [58]. Both are also involved in maintaining the pH homeostasis and are functionally interconnected [43]. Additional K + influx systems, like K + -H + symporter (Hak), K + -Na + P-type ATPase (Acu), and K + efflux channel Tok, were identified in the nonconventional yeasts (reviewed in [59,60]), and were later shown to be widely present in several fungal species [61]. Importantly, fungi and plants show many similarities in the way in which the plasma membrane is energised, and K + and Na + are transported [36,62], but the Na + -ATPases commonly present in fungi are absent in higher plants [63,64].
The extreme conditions that define the natural habitats of H. werneckii demand efficient cellular mechanisms to combat all the problems that are caused by high concentrations of toxic inorganic salt ions. The remarkable ability of H. werneckii to not only thrive in hypersaline conditions that are lethal to a majority of other microorganisms, but also survive and grow without salt (which is not the case with, for example, halophilic Archaea), indicates the existence of unique adaptations. The aim of our study was to unravel such adaptations by making an inventory of metal cation transport systems in H. werneckii, and interpret them in light of its extremely adaptable and halotolerant character. Interesting gene targets for improvement of plant salt tolerance were also identified and discussed. For this purpose the genome of H. werneckii was sequenced, de novo assembled and annotated.

Strain and DNA/RNA Preparation
The halophilic black yeast-like fungus H. werneckii (strain EXF-2000) was isolated from marine solar salterns on the Adriatic coast (Slovenia) [15]. It is maintained in the Ex Culture Collection of the Department of Biology, Biotechnical Faculty, University of Ljubljana (Infrastructural Centre Mycosmo, MRIC UL, Slovenia) and in the CBS culture collection (Centraalbureau voor Schimmelcultures, the Netherlands) as strain CBS 100457. H. werneckii cells were grown in supplemented synthetic defined yeast nitrogen base (YNB) liquid medium (ForMedium, UK): 1.7 g YNB medium, 0.8 g complete supplement mixture (CSM), 5 g (NH 4 ) 2 SO 4 , and 20 g glucose, per litre of deionised water. The medium was adjusted to pH 7.0 and to NaCl concentrations of 0%, 5%, 10%, 17% and 25% (w/v). Incubations were performed at 28uC in 500 ml Erlenmeyer flasks on a rotary shaker at 180 rpm. Inoculum cultures were grown in 25 ml YNB at the appropriate NaCl concentrations to the mid-exponential phase. Growth was monitored spectrophotometrically by optical density at 600 nm (OD 600 ), cells were grown to mid-exponential phase (OD 600 = 0.8-1.0) and harvested by centrifugation (40006g; 10 min).
For DNA isolation, H. werneckii was grown in the YNB liquid medium without NaCl and harvested by centrifugation in the midexponential growth phase. The pellet was frozen in liquid nitrogen and homogenised using a mortar and pestle. The DNA was then isolated according to the protocol described by Rozman and Komel [65]. The integrity, purity, and quantity of the DNA were evaluated with Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and spectrophotometrically with NanoDrop 2000 (Thermo Fisher Scientific, USA).
For RNA isolation, H. werneckii was grown in the YNB liquid medium with different amounts of NaCl added (0%, 5%, 10%, 17% and 25%; w/v), and harvested by centrifugation in the midexponential growth phase. RNA was isolated using TRI REAGENT TM (Sigma, Germany) according to the manufacturer instructions. Possible DNA contaminations were degraded with deoxyribonuclease I (Thermo Fisher Scientific -Fermentas, Lithuania) and the RNA was additionally cleaned with Qiagen RNeasy MinElute Clean up Kit (Qiagen, USA). The integrity and purity of the RNA was evaluated with Agilent 2100 Bioanalyzer (Agilent Technologies, USA).

Genome Sequencing and Assembly
A single sequencing library with an input fragment size of 400bp was constructed using the NEBNext DNA sample prep kit (New England Biolabs Ltd). Paired-end 75-bp reads were generated on an Illumina GAIIx DNA sequencer (Illumina Inc). One lane of Illumina GAIIx 75-bp paired-end reads, yielding 5.7 Gbp of sequence and an estimated 110-fold coverage of the genome, was assembled using ABySS 1.2.1. To assemble reads in low-coverage regions, the reads were first assembled setting the de Bruijn graph parameterk to a small value, k = 25. The reads were then reassembled at a larger value of k, k = 60, including the k = 25 contigs as additional sequence. The paired-end assembly parameters of ABySS were set to s = 150 and n = 10. All other parameters were set to their default values. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AIJO00000000. The version described in this paper is the first version, AIJO01000000. The raw short reads are accessible in the NCBI Sequence Read Archive under the accession number SRR866616.

Gene Prediction and Annotation
Automatic prediction of the genes was made by first determining the putative open reading frames (ORFs) with the MAKER genome annotation pipeline version 2. 25 [6665] on a computer running Biolinux 6 [67,66]. Several sets of data were used as evidence for annotation: all H. werneckii transcripts available in GenBank, transcripts of Aureobasidium pullulans (our unpublished data), Pyrenophora tritici-repentis [68] and Mycosphaerella graminicola [69] as well as all proteins from the UniProtKB/Swiss-Prot database. Pezizomycotina were used as a model for repeat masking. Three gene predictors were used: Augustus (trained for Neurospora crassa), GeneMark (self-trained with GeneMark.hmm-ES) and Snap (trained with 12 sequential outputs of the Maker pipeline). Functional annotation of the ORFs was performed with the Blast2GO software [70]. Basic analyses of the predicted genes and proteins were performed with the EMBOSS suite [71]. Pfam domains of predicted proteins were identified with a stand-alone Pfam Scanner and a database downloaded on 30. 1. 2013 [72]. For comparison purposes the same was done for Saccharomyces cerevisiae and M. graminicola. The results were used to determine the number of proteins with a given domain in each of the proteomes (Table S1).

Gene Duplication Analyses
Gene duplications were detected by two different methods. Predicted proteins were aligned to the genome with Exonerate version 2.2.0 using protein2genome model [73] and limiting the reported hits to those above the certain percent of maximal score obtainable for that query. Additionally, an all-against-all protein sequence similarity search of H. werneckii proteins to a H. werneckii protein database was performed by blastp included in the BLAST 2.2.25+ [74]. The number of hits was counted for each query. For comparison purposes the same analysis was also performed for proteomes/genomes of Mycosphaerella graminicola [69] and Saccharomyces cerevisiae (SGD project. http://www.yeastgenome.org/ download-data/ (22. 5. 2012)). The numbers of shared and unique proteins between H. werneckii, M. graminicola and S. cerevisiae were determined by all-against-all blast of their whole proteomes for all possible pairs of species.

Manually Curated Gene Annotations
To identify all alkali metal cation transporters coded in the H. werneckii genome, the databases of automatically annotated H. werneckii ORFs were searched for homologues of the already identified S. cerevisiae transporters or transporters identified in unconventional yeasts [43], using blastn and blastx algorithms, respectively. From the list of results, only hits with e-values lower than 1e 26 were analysed further. The analysis retrieved not only proteins from the same transporter group as the query, but also a significant amount of transporters from other groups; however, to avoid missing possible highly divergent transporter homologues the cut-off e-value was not lowered. Proteins that were retrieved more than once were kept only in one copy for further analyses. ORFs were then re-aligned to the genomic sequence and, where necessary, the translation start and stop sites were manually corrected according to the comparison with the N-terminal and Cterminal ends of homologues from other fungi. The position of introns was confirmed by manually identifying conserved intron donor and acceptor sites. Where we encountered two or more contiguous ORFs that could be determined as parts of the same gene with large certainty, these fragments were merged into a single entry. The same protocol was also used for the analysis of the H. werneckii MAT locus, where S. cerevisiae and M. graminicola Mat1-1-1 and Mat1-2-1 proteins were used as sequence queries.

Gene Phylogeny Reconstruction
Amino acid sequences of the manually curated H. werneckii homologues of the S. cerevisiae alkali metal cation transporters together with the S. cerevisiae, M. graminicola and Crypotococcus neoformans homologous transporters, were used to build phylogenetic trees. The same analysis was performed with proteins that were identified by blastp (e-value cut-off 10 26 ) in the predicted proteome of H. werneckii using the homologues of all known P-type ATPases from S. cerevisiae as queries. Homologues from Ajellomyces dermatitidis, Leptosphaeria maculans, Mycosphaerella graminicola, and Paracoccidioides brasiliensis were also included in the phylogenetic analysis. Protein sequences were aligned using the L-INS-i method in the MAFFT software [75]. ProtTest 3.2.1 [76] was used to estimate the most appropriate model of protein evolution. The gene trees were generated with the PhyML 3.0 software [77] with aLRT implementation, for the calculation of branch supports as Chi2 based support. The analyses were run using the VT model of evolution for a Nhx proteins and all P-type membrane transporters and LG model for all the rest. ProtTest estimate of alpha parameter of gamma distribution of six substitution rate categories was used, and in case of Ena and Nha proteins, also the determined proportion of invariable sites. For comparison, a second set of trees (not shown) was generated by applying a maximum parsimony method as implemented in the Mega software version 5.05 [78].

Gene Transcription Analyses
First strand cDNA was synthesized from 1 mg of total H. werneckii RNAs using RevertAid TM H Minus First Strand cDNA Synthesis Kit and random hexamer primer (Thermo Fisher Scientific -Fermentas, Lithuania) according to the manufacturer instructions. cDNA concentration was measured spectrophotometrically with NanoDrop 2000 (Thermo Fisher Scientific, USA). Approximately 100 ng of cDNA (10 ng for reference gene 28S rRNA) was used as a template for quantitative reverse transcription PCR with oligonucleotides specific for the genes under investigation. The primer sequences are given in the Table S2. The thermal profile of the reaction was as follows: 10 min at 95uC, 45 cycles consisting of 15 s at 95uC, 30 s at 50-60uC and 15 s at 72uC, followed by a dissociation curve (15 s at 95uC, then 60 s at 60uC and 15 s at 95uC, ramping at 0.3uC/sec). The reaction mix was prepared using the Power SYBR Green PCR Master Mix (Life Technologies, USA), according to the manufacturer instructions, in 10 ml of total reaction volume, primer concentration was 300 nM. The reactions were performed in a StepOnePlus Real-Time PCR System (Life Technologies, USA), and analyzed with StepOne v2.2.2 software (Life Technologies, USA) using the standard curve method. Relative standard curves (PCR reaction efficiency) were determined by amplifying five 10-fold serial dilutions of control cDNA. Quantification cycle (C q ) values for our genes of interest were normalised to the quantification cycle of 28S rRNA fragment (reference gene), the transcription of which remains unchanged under different environment conditions [18]. The difference in C q values representing relative mRNA level values between the target gene and the reference gene was calculated, and these values of the different samples were compared directly.

Sequencing and Assembly of the Hortaea werneckii Genome
In the present study, we have sequenced the genome of the extremely halotolerant black yeast Hortaea werneckii using an Illumina GAIIx sequencer. A similar sequencing strategy was reported to be sufficient to produce an assembly covering most of the protein-coding genome for the fungus Sordaria macrospora (genome size 40 Mb) [79]. The total assembly size of the H. werneckii genome is 51.6 Mb ( Table 1). The average genome coverage was 1106 and the assembly process yielded 12620 contigs. The assembled sequence was deposited in the public genome database DDBJ/EMBL/GenBank under the accession number AIJO01000000. The genomic G+C content is relatively high at 54% and is even higher in the coding regions (56%). Of eighteen plant-associated fungi also belonging to Dothideomycetes (Figure 1), which were compared by Ohm et al. [68], only Cladosporium fulvum and Mycosphaerella fijiensis genomes exceed this size (61.11 Mb and 74.14 Mb, respectively), largely due to a substantial amount of repetitive sequences (44.44% and 39.50% of the genomes, respectively). Even in the seven species belonging to the same order as H. werneckii (Capnodiales) the genome sizes are very variable (from 21.88 to 74.12 Mb). In H. werneckii, however, the proportion of repetitive sequences remains low at only 1.02%, despite its large genome size. The average size of sixteen other genomes studied by Ohm et al. [68] is considerably smaller (35.33 Mb).
Despite the great variability of genome sizes in the 18 above mentioned fungi, the differences in the number of predicted genes were much smaller. On average each species contained 11955 genes (minimum 9739, maximum 14127) [68], but in H. werneckii this was almost twice as large (23333). Of these relatively few (18.6%) had no blast hits in the proteomes of Saccharomyces cerevisiae or Mycosphaerella graminicola (one of the phylogenetically closest species with sequenced genomes [69]) or both ( Figure 2A) and 14.0% had no hits in the GenBank non-redundant database (e-value cut-off 10 26 ). This abundance of genes could be explained by large scale genomic duplications, but the relatively poor assembly of the H. werneckii genome could also significantly contribute to the overestimation. The large number of contigs could result in fragmented genes, later falsely identified as individual proteins. However, our manual analysis of a subset of transporter genes strongly suggested that the large number of predicted genes was not primarily the consequence of the assembly quality, as only a small subset of genes were fragmented. Instead, the large number of genes appears to be the result of a relatively recent whole genome duplication (WGD), yielding two nearly identical copies of almost every protein-encoding gene in H. werneckii. The large number of obtained contigs was therefore a consequence rather than the cause of the apparent duplications (due to difficulties in assembling the reads across a large number of highly similar stretches of DNA).

Evidence for Whole-gene Duplication
To investigate if the large number of predicted proteins in H. werneckii is due to large-scale gene duplication, we analyzed its proteome with all-against-all blastp and aligning the proteins back to the genome ( Figure 2B). Using the same parameters of analyses that detected less than 10% of duplicates in the genomes of S. cerevisiae and M. graminicola, we determined that nearly 90% of H. werneckii genes exist in at least two copies. Even for the relatively small fraction of single-copy proteins, we found a disproportionately large number of falsely predicted proteins: only 46.8% of them had blast hits in the non-redundant GenBank database (evalue cut-off 10 26 ), compared to 91.2% of other genes. This discovery was not entirely surprising, since previous studies of several individual genes from H. werneckii already noted that a majority were present in two copies [80]. In most cases, the transcription of both gene copies was salt dependent, but the transcription profiles differed [21,29]. As a consequence of this WGD H. werneckii can benefit from the potential advantages of a large genetic redundancy even though it is formally haploid, as determined by the present study. At this point it is difficult to assess the adaptive value of this phenomenon, and whether it contributed to the ability of the fungus to adapt to such a wide range of environmental salinities.
Studies on other fungi indicate that this may well be the case. WGDs have been observed in several other fungal species and have been extensively studied in S. cerevisiae [81]. An ancient polyploidisation event occurred in the phylogenetic lineage leading to this yeast [82], yet in its contemporary natural population a large portion of triploid and tetraploid strains co-exists with diploids [83]. Experimental evolution studies have reported a very fluid nature of ploidy levels in this and other species [84,85]. Strains with less common genome sizes tend to return to their usual ploidy over time even if this appears to be counterproductive. In S. cerevisiae for example, haploid strains theoretically suffer from the lowest mutation load and have also been shown to adapt faster to stressful concentrations of salt and several other conditions [86,87]. Nevertheless it is its diploid state that appears to have fine-tuned for optimal fitness over evolutionary time. Altered gene expression patterns and altered cell geometries that are associated with other ploidy levels [88] may simply be suboptimal and therefore selected against. In addition, in the short term, diploid individuals are better able to mask deleterious mutations, which is another possible reason why they prevail over haploids in both normal and even faster in stressful conditions [86]. Even in the case of tetraploid S. cerevisiae strains, which normally tend to decrease in genome size toward a diploid state, this process is slower in a salt-stressed compared to a normal medium [84]. Another experimental evolution study suggested that adaptation of S. cerevisiae to stressful concentrations of salt is, among other changes, associated with increases in genome size [89]. Ploidity increase in S. cerevisiae has also been described to act as protection to ultraviolet radiation [90]. We cannot rule out this possibility even in the case of H. werneckii, since in shallow ponds of hypersaline water, its presumed natural habitat, it is exposed to substantial amounts of solar radiation.

MAT Loci of the Heterothallic Hortaea werneckii
H. werneckii has a complex asexual cycle, where changes in nutrition, cell number or temperature mediate conversion between the yeast and hyphae cell morphology [91]. Still, no sexual cycle has been described so far. The H. werneckii draft genome sequence therefore offered the opportunity to gain insight into the genetic information on the mating type/types and on the mating strategy. Previous investigations of the ascomycete sexual reproduction have established that this process is determined by the presence of different arrangements of mating-type (MAT) genes at one or more MAT loci [92]. Two mating types exist, the idiomorph MAT1-1, which contains the MAT1-1-1 gene encoding a protein with an alpha1 domain; and MAT1-2 idiomorph, which contains the MAT1-2-1 gene encoding the high mobility group (HMG) domain protein [92]. The H. werneckii related genus Mycosphaerella contains numerous self-incompatible (heterothallic) species, coding only for one of the idiomorphs; and self-compatible (homothallic) species, coding for both mating types [93]. We used M. graminicola alpha1 domain (Mat1-1-1, XP_003847598.1) and HMG domain (Mat1-2-1, ABH04241.1) containing proteins to screen the H. werneckii genome for existing mating type loci with tblastn. We identified two genes, both encoding a homologue of the M. graminicola MAT1-1-1 gene (Figure 3), and designated them HwMAT1-1-1A (KC961394) and HwMAT1-1-1B (KC961395). HwMAT1-1-1A gene is 1196 bp long and contains two introns, the 58 bp long intron and the 69 bp long intron, which lies inside the alpha1 domain ( Figure 3A). The HwMAT1-1-1B gene is 88.7% identical to HwMAT1-1-1A in the nucleotide sequence, and also contains two introns, 58 bp and 56 bp long, the last one again located inside the alpha domain ( Figure 3A). Both HwMAT1-1-1 homologues translate into 358 aa long proteins (HwMat1-1-1A and HwMat1-1-1B) with 141 aa long alpha1 domain (PF04769), and have an overall amino acid (aa) sequence identity of 87.5% ( Figure 3B). When compared to MgMat1-1-1, the overall aa sequence identity of HwMat1-1-1 homologues was only around 29%, but the identity increased to around 43% if comparing only the alpha1 domains. Importantly, no homologues of the M. graminicola Mat1-2-1 protein were found in H. werneckii, indicating that this species is heterothallic, and needs to mate with the strain coding for the opposite Mat1-2-1 homologue for sexual reproduction.
Interestingly, both HwMAT1-1 and MgMAT1-1 containing genome fragments show strong conservation with respect to the relative positions of genes and orientation of transcription, whereas the gene sequence is not conserved ( Figure 3A). The only two exceptions are the APN2 and the MAT1-1-1 gene homologues, where also the sequence is conserved to some extent. A similar arrangement was also shown for the heterothallic Neurospora species [95].

Alkali Metal Cation Transporters in Hortaea werneckii
H. werneckii natural habitats are thalassohaline hypersaline environments, which originated by the evaporation of seawater [34]. They are characterized by high and dynamically changing concentrations of dominating sodium and chloride ions, and the pH is near neutral to slightly alkaline. These extreme environments are frequently accompanied by other types of stress, such as high temperatures, high UV radiation, low oxygen concentrations, or generally low nutrient availability with occasional peaks in abundance [34,96,97]. A typical example of such environments is the solar saltern, where seawater is evaporated to produce sea salt and the hypersaline brine. Brines derived from seawater contain relatively high concentrations of Ca 2+ and can remain approximately neutral even after extensive evaporation because the molarity of Ca 2+ always exceeds that of CO 3 22 , which is part of the carbonate buffer system of the aquatic environment [98].
For organisms living in such environments, maintaining intracellular cation homeostasis, i.e., maintaining rather high and stable K + content and eliminating toxic Na + ions [36,43], is crucial for survival. H. werneckii maintains very low amounts of internal Na + , even when grown in environments with high NaCl concentrations; although the K + /Na + ratio does fall with increasing salinities [25]. Altogether 2208 (9.6%) of H. werneckii predicted proteins were assigned the GO term ''transport'' (biological processes) ( Figure S1), similarly to what was shown for Hemiascomecete yeasts (10%) [37]. To get an insight into the transport systems responsible for maintaining cation homeostasis in extremely halotolerant H. werneckii, we searched the databases of predicted H. werneckii ORFs and proteins for homologues of metal cation transporters from S. cerevisiae and nonconventional yeasts [43,59]. The predicted H. werneckii transporters were assigned standard names of the closest orthologues from S. cerevisiae according to the Saccharomyces Genome Database (SGD) [99]. The prefix ''Hw'' for Hortaea werneckii was added, the genes were consecutively numbered, and in case of paralogues resulting from a recent (WGD) duplication, this was indicated by a suffix of letters ''A'' or ''B''. Similarly, we searched the proteomes of the M. graminicola [100] and the Cryptococcus neoformans var. neoformans JEC21 [101], using the same list of transporter proteins for query sequences as for H. werneckii.
We identified homologues of all characterized S. cerevisiae plasma membrane (Trk1, Trk2, Tok1, Nha1, Ena proteins, Pho89) and intracellular cation transporters (Kha1, Mrs7, Vnx1, Nhx1) ( Table 2) as well as homologues of the H + -ATPases (Pma paralogues, V-ATPase complex) important for supplying the energy to the secondary transporters (Table 3). Interestingly, no homologues of the Hak1 and Acu1 transporters, found in nonconventional yeasts [59,60] and in many other Ascomycete fungi [61], were identified in the predicted H. werneckii proteome. On the other hand, these two transporters are present in the closely related M. graminicola, in addition to all above mentioned S. cerevisiae transporters.
Plasma membrane cation transporters. Fungi in general, including H. werneckii, live in environments with highly variable potassium concentrations. Nevertheless, K + is present in all cells at relatively high concentrations, compared to other cations, and it is crucial for several basic physiological functions, like osmotic regulation, protein synthesis and enzyme activation [102]. Another consequence of regulated K + uptake and efflux across the membrane is also the maintenance of the plasma membrane potential [103]. High sodium concentrations in the environment disturb K + homeostasis, as Na + intrudes into the cell and causes lowering of K + intracellular concentration, because cells have to maintain electroneutrality. While extracellular sodium causes severe osmotic stress, high intracellular concentrations of Na + interfere with growth by inhibiting many important enzymatic functions [43]. Keeping a low intracellular sodium concentration and a high intracellular K + /Na + ratio is therefore crucial for functioning of the organism [36]. In S. cerevisiae, transporters maintaining high intracellular K + concentrations are high affinity K + channels Trk1 and Trk2, involved in potassium uptake [44], and three different transporters involved in potassium efflux: membrane depolarization activated K + channel Tok1 [45], Ena1-5 ATPases [47][48][49] and Na + /K + antiporter Nha1, which were first identified as Na + efflux systems [50]. Ena P-type ATPases couple ATP hydrolysis to export Na + (or K + ) from the cells at alkaline pH, whereas antiporter Nha1 uses an H + gradient to energise the efflux at acidic external pH values. There is no single specific uptake transporter for sodium in S. cerevisiae, so it is proposed that besides K + , Trk1 also transports Na + , although with a much lower affinity. Additionally, at alkaline pH, symporter Pho89 catalyzes a sodiumdependent phosphate uptake (reviewed in [43]).
In H. werneckii we identified 8 homologues of the Trk1 and Trk2 K + channels (Table 2), each containing the conserved TrkH domain (PF02386) typical for cation transport proteins. In general, they show low similarity to Trk1 protein, but the amino acid (aa) sequence identity increases in the TrkH domain, the value falling between 37.4% and 49.6%, depending on the H. werneckii homologue. The expansion of the Trk channels in H. werneckii was also confirmed by the comparative analyses of the PFAM domains (Table S1), as PF02386 domain in H. werneckii was enriched 86relative to M. graminicola and 46relative to S. cerevisiae. Phylogeny of Trk proteins from H. werneckii, M. graminicola and S. cerevisiae, rooted with C. neoformans homologue ( Figure 4A) indicates that two duplications of H. werneckii Trk channels happened before the separation of the S. cerevisiae and H. werneckii ancestors, but one gene copy was later lost in S. cerevisiae and M. graminicola. An additional duplication occurred later, but before the separation of the H. werneckii and M. graminicola lineages. Here, again, one of the gene copies appears to have been lost in M. graminicola. The recent duplication of all H. werneckii genes presumably results from a whole genome duplication event that occurred after the M. graminicola/H. werneckii split.
Interestingly, we also observed expansion of the Tok channels in H. werneckii (Table S1), since Ion_trans_2 domain (PF07885) in H. werneckii was enriched 46 relative to M. graminicola and to S. cerevisiae. We identified 4 homologues of the Tok1 K + channels ( Table 2), each containing two conserved transmembrane helices (PF07885 domain) typical of the ion channel family. Again, sequence identity to the Tok1 protein is low, but the identity is high in both PF07885 domains, with values from 33% to 45.2%, depending on the H. werneckii homologue. According to the phylogenetic analysis ( Figure 4A) one duplication of Tok genes occured after the separation of H. werneckii and S. cerevisiae ancestors, but before the separation of H. werneckii from M. graminicola. In the latter one copy was later lost.
We identified 8 homologues of the Nha1 Na + /K + , H + antiporter (Table 2), each containing a transmembrane region (PF00999) at the N-terminal, which is conserved through the Na + / K + , H + exchanger family, and only two of them additionally containing the C-terminal cytoplasmic region (PF08619), with little similarity across the family. Similar to previous transporters, sequence identity to the Nha1 is low, but the aa identity in the conserved PF00999 domain is high, reaching up to 64% in some H. werneckii homologues. Several ancient duplications can be seen in the inferred phylogeny of Nha proteins, of which only one copy has been preserved in the genome of S. cerevisiae ( Figure 4A). This was also confirmed by the comparative analyses of the PFAM domains (Table S1), where PF00999 domain is enriched 56 in H. werneckii relative to S. cerevisiae, whereas the 26 enrichment of H. werneckii relative to M. graminicola can be explained by the WGD event.
In contrast with the above transporter families, only 4 homologues of three S. cerevisiae Ena Na + P-type ATPases were identified in H. werneckii (Table 2). Each homologue contained all 4 conserved domains found in S. cerevisiae Ena proteins: the Cation_ATPase_N (PF00690) and Cation_ATPase_C (PF00689) domains, representing the conserved N-terminal and C-terminal region found in H + , Na + , Ca 2+ , Na + /K + and H + /K + transporting P-type ATPases; the E1-E2ATPase domain (PF00122), representing the actuator domain and some transmembrane helices found in P-type ATPases; and the Hydrolase_like2 domain (PF13246), a putative hydrolase of sodium-potassium ATPase alpha subunit. The aa sequence identity of the four H. werneckii homologues to Ena1 was highest in the PF00122 domain, with values between 44.2% to 52.9%. S. cerevisiae Ena1, Ena2 and Ena5 result from recent duplications, while Ena proteins in H. werneckii and M. graminicola have duplicated much earlier and diverged substantially ( Figure 4A). The recent duplication of H. werneckii genes is the consequence of the WGD, as in all other cases. Extensive expansion was also observed for the Pho89 homologues in H. werneckii, since the PHO4 domain (PF01384), typical for the phosphate transporter family, was enriched 56 relative to M. graminicola and 106 relative to S. cerevisiae (Table S1). We identified 6 homologues of the Pho89 Na + , P i symporter in H. werneckii (Table 2), each homologue containing at least one PHO4 domain (up to two). The aa identity in the N-terminal PF01384 domain was from 44.2 up to 54.7%, depending on the homologue. Pho89 proteins of H. werneckii are the result of several duplications that occurred at various times after the separation of its ancestor from the lineage of S. cerevisiae ( Figure 4A).
Together these observations support the conclusion that for H. werneckii, regulation of transport of K + and Na + across the plasma membrane is of utmost importance, because most homologues of the S. cerevisiae plasma membrane Na + and K + transporters are enriched in this fungus. This is not unexpected in the case of transporters involved in uptake of K + and efflux of Na + (HwTrk, HwNha), which help to maintain a high intracellular K + /Na + ratio together with low concentrations of Na + in hypersaline environments [36]. However, enrichments were also observed for transporters responsible for K + efflux and Na + intake (HwTok, HwPho). One reason for this could be the need of H. werneckii to quickly adapt to highly dynamic concentrations of NaCl (and other salts) typically encountered by the fungus in its natural environment. When NaCl concentrations are high, import of K + and export of Na + are crucial for survival of the organism. Yet if the concentration of NaCl suddenly drops, the competition between K + and Na + for import into cells ceases to exist, and K + could accumulate to excessive intracellular concentrations [104]. To avoid these consequences, cells must export K + quickly, until K + homeostasis is achieved and the plasma membrane potential restored.
Also of interest is the difference in numbers of H. werneckii Nha and Ena homologues. While the first are substantially enriched, this was not observed for the latter. Both proteins export Na + from the cells, but Nha proteins are active at slightly acidic conditions, whereas Ena proteins work in alkaline environments [105]. In laboratory conditions, growth of H. werneckii is accompanied by rapid acidification of the medium; however, pH in the solar salterns is typically neutral to slightly alkaline. Although H. werneckii could potentially acidify its surroundings in some microenvironments, this cannot fully explain the observed HwNha transporter enrichment. The increase in HwNha numbers may therefore be important for the increase of transcript and protein numbers, as NHA gene expression is generally constitutive and very low [105]. Regulation of the transcript numbers of the ENA genes on the other hand, is known to occur at the transcriptional level in S. cerevisiae [48,49,106]. Alkaline pH and increased salinity were shown to be inducers of transcription also in H. werneckii [24], which might reduce the need for regulation of expression with gene copy number, as was observed for the HwNHA. The HwNha enrichment could also provide the material for evolution of some paralogues towards changed specificity (for Na + or K + ) or even specialization for other functions. In S. cerevisiae, Nha1 namely also has a role in regulation of intracellular pH, cell cycle, cell volume and membrane potential [105,[107][108][109].
We can only speculate on the role of the Na + , P i symporter enrichment in H. werneckii (HwPho1-3). Pho89 in S. cerevisiae utilizes the Na + gradient for import of P i into the cell [110]. Interestingly, PHO89 gene transcription is not induced only by P i limitation, but is strongly induced also by alkaline pH, even when cells are grown in medium with normal phosphate concentrations [111]. Induction by alkaline pH seems to be largely dependent on calcineurin [112]. We propose that in H. werneckii, in conditions of high salinity and alkaline pH, where H + gradient cannot efficiently energise the P i import, Na + gradient could represent an alternative energy source for transport.
No homologues of the K + , H + symporter Hak1, mediating high affinity K + uptake, or Na + uptake, as is the case in Yarrowia lipolytica [113]; or the P-type ATPase Acu, mediating high affinity K + or  Na + uptake, were found in H. werneckii. Its K + management system therefore differs substantially compared to the closely related M. graminicola, which has both Hak1 and Acu homologues [61]. M. graminicola also has only one Trk and one Tok channel, compared to 8 and 4 copies of each in H. werneckii, respectively. It appears that M. graminicola requires active import of potassium, while in H. werneckii passive transport is more pronounced. The difference in the transporter inventories of closely related H. werneckii and M. graminicola possibly reflect the living styles of these fungi, one being an extremely halotolerant fungi and the other a plant pathogen. Intracellular cation transporters. Maintenance of K + homeostasis and sodium detoxification in the cytosol is connected to the cation transport across organellar membranes, which are important for the regulation of organellar pH and volume [43]. In S. cerevisiae, endosomal Nhx1 [52] and Kha1 from the Golgi apparatus [53] are typical Na + , H + exchangers, similar to the plasma membrane Nha1 [50]. Vacuolar Vnx1 [51] and mitochondrial Mdm38 and Mrs7 [54,55] have similar Na + /K + , H + exchanger functions, but different structures.
Homologues of the Nhx1 and Kha1 are duplicated in the H. werneckii genome, as a consequence of the WGD event (Table 2 and Figure 4B). All homologues contain the Na_H_Exchanger domain (PF00999) typical for the sodium/hydrogen exchanger family, but lack the C-terminal cytoplasmic region (PF08619) found in Nha1. The aa sequence identity between HwNhx1A, HwNhx1B and Nhx1 is especially high in the conserved PF00999 domain (up to 62.2%). Similar relationships were observed for the Kha1 homologues, where identity in the PF00999 domain is around 47%. We identified two homologues of the human LETM1 transporter in H. werneckii, with high sequence identity to the Mrs7 and Mdm38 transporters from S. cerevisiae (Table 2 and Figure 4B). Both H. werneckii Mrs7 (and Mdm38) homologues contain the LETM1 conserved domain (PF07766), which have 50% aa identity when compared to the Mrs7 domain.
Of the intracellular cation transporters, only the homologues of the vacuolar Vnx1 are enriched in H. werneckii relative to the S. cerevisiae (46) (Table S1). We identified 8 homologues of the Vnx1 Na + /K + , H + antiporter (Table 2), each containing two PF01699 domains, otherwise typical for the sodium/calcium exchanger protein family, but in the case of Vnx1 involved in the Na + /K + , H + exchange [51]. The enrichment is the result of an ancient duplication before the separation of H. werneckii and S. cerevisiae ancestors, with one of the lineages, which was lost in S. cerevisiae, leading to three copies in M. graminicola and six in H. werneckii through several additional duplications ( Figure 4B). The sequence identity of the HwVnx proteins compared to Vnx1 is low, but the aa sequence identity in the conserved PF01699 domains is up to 70.2% for the first and up to 55% for the second domain, the value also depending on the HwVnx homologue type. Interestingly, the enrichment of the Vnx1 homologues relative to S. cerevisiae is observed for both, H. werneckii and M. graminicola. The abundance of vacuolar Na + /K + , H + antiporters in H. werneckii could contribute to the fungus highly haloadaptable character, probably by accumulating Na + in the vacuoles and thereby helping in detoxification of the cytosolic Na + . The role of Vnx transporter enrichment in M. graminicola is unkown, but would be interesting to study.
Plasma membrane and vacuolar H + ATPase. The activities of many transporters are essential for maintaining the gradient of protons across membranes, generated in large part by the Pma1 P-type ATPase at the plasma membrane [56,57] and V-type ATPase at the vacuolar membrane [58]. As H. werneckii shows itself to have a complex cation transporter system, we analysed the transporters responsible for supplying the needed energy in further detail.
We identified four homologues of Pma1 in H. werneckii; HwPma1A (46% identity to Pma1), HwPma1B (46.1%), HwPma2A (45.6%) and HwPma2B (45.9%) ( Table 3). Each homologue containes 3 conserved domains also found in S. cerevisiae Pma1 and Pma2 proteins: the Cation_ATPase_N domain (PF00690), the E1-E2ATPase domain (PF00122) and the Hydro-lase_like2 domain (PF13246). Similar domains are also typical for other members of the P-type ATPase family, such as the previously mentioned Ena proteins. Generally, P-type ATPases have a common mechanism of action -hydrolysis of ATP to energise the transport of different ions and other substrates through the membrane; therefore they have a similar structure [114]. Based on the reconstruction of their phylogeny, they have been classified into five families and further into two or more subfamilies [115]. The number of P-type ATPases in different species is highly variable [116]. By searching the predicted H. werneckii proteome with blastp (e-value cut-off 10 26 ) using P-type transporters from S. cerevisiae as queries, we identified 40 members of the P-type family in H. werneckii ( Figure 5). The number of proteins was twice as large in H. werneckii as in S. cerevisiae in the case of groups IB (transport of Cu 2+ , Cd 2+ and other metals), IIB (Ca 2+ transport), IID (Na + transport), and V (pumps with unknown function). The following groups were even more enriched in H. werneckii, group IIA (Ca 2+ transport) having 4 members, group IIIA (proton transport) 6 members and group IV (phospholipid transport) 14 members ( Figure 5).
The analysis of H. werneckii Pma-family proton pumps showed that apart from the gene duplications presumably originating from a WGD, one additional duplication occurred in the evolution of Pma pumps, resulting in two lineages, of which only one survived in the genome of M. graminicola ( Figure 6A). To investigate the relevance of the four H. werneckii Pma homologues for the cation homeostasis, we checked the level of transcription of each gene  (Sc). The trees were rooted with homologous proteins from Cryptococcus neoformans and the root location is marked with an arrow. Putative gene duplications leading to the present diversity of these genes in H. werneckii are marked with different symbols: two triangles (duplications that happened before the separation of S. cerevisiae and H. werneckii ancestors), two half-circles (duplications after the separation of S. cerevisiae and H. werneckii ancestors, but before the separation of H. werneckii and M. graminicola), a combination of triangle and half circle (duplications before the separation of H. werneckii and M. graminicola but unclear regarding to separation from S. cerevisiae ancestor) and with circles on the bifurcation (recent duplications presumably resulting from a whole genome duplication). A. Plasma membrane transporters. B. Transporters located on internal membranes. The following S. cerevisiae transporters (Trk1 (YJL129C), Trk2 (YKR050W), Tok1 (YJL093C), Nha1 (YLR138W), Ena1 (YDR040C), Ena2 (YDR039C), Ena5 (YDR038C), Pho89 (YBR296C), Nhx1 (YDR456W), Kha1 (YJL094C), Vnx1 (YNL321W), Mrs7 (YPR125W)) and its H. werneckii (Table 2)  during growth at different NaCl concentrations ( Figure 6A). S. cerevisiae has two copies of the gene, but PMA2 is expressed at such low levels relative to PMA1, that it is considered nonessential with only minor impact on the homeostasis [117]. In H. werneckii the transcription profiles of the PMA1 and PMA2 homologues show responsiveness to different salinity conditions, with the lowest transcription of PMA1 homologues at a salinity of 17% NaCl (w/ v), where the growth starts to slow down [25]. This result corresponds to the findings of Vaupotič and Plemenitaš [33] where the lowest transcription of H. werneckii PMA homologue (sequence corresponding to our gene HwPMA1B) was detected when the H. werneckii cells were grown at 3 M (17.5%) NaCl in comparison to the transcription at 4 M (26%) NaCl. The H. werneckii PMA2 homologues had the lowest transcription at optimal salinity of 5% NaCl and at 25% NaCl. The low PMA2 transcription at 25% NaCl, where the stress becomes more severe, can then be partially replaced by the transcription of PMA1 homologues. Comparisons of the transcription profiles of PMA genes in H. werneckii with those described in S. cerevisiae showed that in S. cerevisiae PMA1 was not induced by salt stress [118]. In H. werneckii both PMA1 and PMA2 manifest salt-regulated transcription. These gene transcription measurements are suggestive of function in halotolerance but do not consider the impact of post-translation modification on activity.
In addition to its critical role(s) in acidification of the vacuolar lumen, the yeast vacuolar ATPase is also important for the proper functioning of other organelles [43]. It has a complex structure, consisting of the peripherally associated catalytic V 1 subcomplex, comprised of proteins coded by 8 different genes (VMA1, VMA2, VMA4, VMA5, VMA7, VMA8, VMA10, VMA13), and a proton translocating membrane V 0 subcomplex comprised of proteins coded by 6 different genes (VPH1, STV1, VMA3, VMA6, VMA11, VMA16) [58]. H. werneckii codes for homologues of all subunits of the S. cerevisiae V-ATPase complex, which are always duplicated as a consequence of the WGD event ( Table 3). The H. werneckii V 1 subunits (Table 3) in general share a lot of similarity with the S. cerevisiae subunits ( Figure 6B), which is not surprising, as their structure and function have been highly conserved through evolution [58]. One of the most conserved subunits are the subunit A (Vma1) and B (Vma2), which form the catalytic and nucleotide-binding subunits [58]. The overall aa sequence identity between H. werneckii subunit A homologues and Vma1 is 48.6%, reaching 73.5% if compared to the sequence of the final product of Vma1, which is lacking the intein sequence. The intein sequence, typical for S. cerevisiae [119], is namely missing in the H. werneckii Vma1 homologue, and also in the related M. graminicola. Interestingly, both H. werneckii (HwVPH1A, HwVPH1B) and S. cerevisiae (VPH1, STV1) code for two homologues of the V 0 subunit a ( Table 3). Vph1 in yeast is a part of the vacuolar membrane V-ATPase; whereas its homologue Stv1 is part of the V-ATPase found on the Golgi or endosome membranes [58]. Vph1 and Stv1 have 49.4% identity in the aa sequence, whereas HwVph1A and HwVph1B have 99.5% aa identity. It is therefore unlikely that HwVph1A and HwVph1B proteins would localise to different cellular locations, as is observed for the S. cerevisiae homologues. It remains to be resolved how H. werneckii specifically localises the ATPase complexes to the vacuoles or to the Golgi or endosome membranes. Although the HwPMAs transcription is salt-regulated, no significant trends were seen for the transcription of VMA homologues under different salinities (Fig. 5B). In contrast in S. cerevisiae salt stress induces the transcription of vacuolar ATPase subunits [118].
Promising Hortaea werneckii genes for improvement of crop salt tolerance. Many cation transporters have been conserved throughout evolution, therefore similar transporters found in the S. cerevisiae, H. werneckii and other fungi are also found in plants. For example, Quintero et al [38] showed that expression of the Arabidopsis thaliana AtNHX1 protein functionally substituted the endosomal Na + /H + antiporter function lacking in the nhx1 S. cerevisiae mutant. Another study similarly showed that the nhx1 S. cerevisiae mutant could be used for selection of AtNHX1 proteins, which were improved for salt tolerance by random mutagenesis [40]. Quintero et al [39] have also successfully reconstituted the A. thaliana SOS signalling pathway in the yeast. This pathway is essential for Na + homeostasis and is composed of the ion transporter SOS1, protein kinase SOS2 and the Ca 2+ sensor SOS3.
Recently, it was shown that cell type specific expression of the Na + transporter AtHKT1 [42] or overexpression of the plasma membrane Na + /H + antiporter SOS1 [41] in Arabidopsis thaliana improved the plant salt tolerance. The above mentioned studies have only focused on plants as the source of transgenes for improvement of crop resistance to high salinity environments. As shown by this study, enrichment in cation transporters important for maintaining K + homeostasis, low Na + content and pH homeostasis, are crucial for H. werneckii survival in environments with rapid changes of NaCl concentrations. Therefore these genes could be interesting novel candidates for improving the halotolerance of plants. Testing new approaches for this purpose is of great importance, since past considerable efforts with various targets (genes encoding enzymes involved in compatible solute synthesis, antioxidants heat-shock and late embryogenesis abundant proteins, and transcription factors for gene regulation), did not yet produce crops with satisfactory improvements in salt tolerance under field conditions [4,120,121].
Sodium is toxic to plant cells, because it competes with K + for binding sites involved in activation of at least 50 cytoplasmic enzymes [122]. Central to salt tolerance is therefore the reduction of Na + toxicity, which can be achieved by restricted Na + uptake, active Na + exclusion or compartmentalization of excessive Na + in the vacuole. Although some improvements of plants were done in this direction [41,42], H. werneckii Nha1 and Vnx1 homologues are very interesting novel targets for plant transgenes. In H. werneckii low amounts of cytosolic Na + were observed over the whole range of salinities [25], indicating very efficient exclusion mechanisms, possibly due to action of export from the cell by HwNha proteins, and effective accumulation into vacuoles by HwVnx proteins. Due to both large copy numbers and great diversification of HwNha and HwVnx proteins, it would be interesting to test them in the nha1 and vnx1 S. cerevisiae mutants to see if any of the homologues have evolved to confer greater specificity towards Na + exclusion and consequently greater halotolerance. Such modification has already been reported for plant transporters. For example, it was suggested that the difference in salt sensitivity between bread wheat and durum wheat is in enhanced K + /Na + discrimination [123]. A similar experiment was done when testing AtNHX1 random mutagenesis mutants in nhx1 strain by Hernández et al [40].
Although exclusion of Na + is essential, the key determinant of salt tolerance is the ability to support a high intracellular K + /Na + ratio and not the absolute quantity of Na + in the cell. Environmental Na + competes with K + for uptake sites of the transporters at the plasma membrane, and causes membrane depolarization [36,124]. The functionality of the polarization channels is disturbed and consequently, the passive K + uptake is diminished, whereas K + efflux through the channels is increased [36]. Therefore restoring the K + homeostasis is crucial for the halotolerance of the plant, as was already suggested by Horie et al. [125], since constitutive expression of rice Na + insensitive K + transporter, OsHAK5, in cultured tobacco BY2 cells enhanced the accumulation of K + and conferred increased salt tolerance to the cells. It would be interesting to test how H. werneckii Trk1 uptake and Tok1 efflux channels function in plant cells exposed to high salinity. The absence of transporters for active import of K + (like Hak1 and Acu) suggests that this fungus achieves homeostasis by specialization of some of its numerous homologues of HwTrk and HwTok. An efficient K + management strategy would also be very beneficial in plants. The salt-tolerant Thellungiella halophile, for example, is capable of increasing K + content under saline conditions, in contrast to the decline of K + observed in related A. thaliana [126].
The cellular response to hypersaline stress is energetically very demanding, reducing the ATP pools of the cells. Importantly the H + gradient generated by the plasma membrane and vacuolar H + -ATPase is crucial to energise secondary cation transport systems [43]. Understanding the regulation and functioning of the H. werneckii Pma and vacuolar H + -ATPase homologues will shed light on this crucial adaptation processes. However, because of their diverse roles, these genes are not very suitable candidates for transgenes. H + ATPases in plants are namely involved in many physiological functions, like mineral nutrition in the root, metabolite translocation, regulation of cytoplasmic pH, and cell turgor-related functions, such as organ movement and cellular growth [127].

Conclusions
The Hortaea werneckii genome sequence presented here shows many features consistent with adaptation to its unique lifestyle and saline tolerance. Two features in particular stood out in analysis: its large genetic redundancy, presumably resulting from an evolutionarily recent whole genome duplication and the expansion of families of genes encoding metal cation transporters.
Ploidy levels of fungal species can be very fluid [84,85] and therefore the fact that H. werneckii has undergone a recent WGD is not necessarily unique. The fact that duplication has not yet been followed by selective gene loss is however, of considerable interest from both a biological and a biotechnological perspective. Interestingly, experiments on S. cerevisiae revealed a tendency toward increased genome size as a response to stressful concentrations of salt [84,89]. Such redundancy may be an excellent reservoir of cryptic genetic variability, which is of importance in stressful environments that require good adaptability [80].
In hypersaline environments regulation of the transport of K + and Na + across the plasma membrane is of utmost importance. In H. werneckii this is seen from the fact that most homologues of the S. cerevisiae plasma membrane Na + and K + transporters (e.g. Trk1, Trk2, Tok1, Nha1 and Pho89) are enriched in this fungus. Especially surprising is the enrichment of the Na + , P i symporter (HwPho1-3) in H. werneckii. Since in S. cerevisiae PHO89 gene transcription is induced by alkaline pH regardless of environmental phosphate concentrations [111] we propose that in H. werneckii, in conditions of high salinity and alkaline pH, Na + gradient could energise the P i import instead of the H + gradient. No homologues of the K + , H + symporter Hak1 and the P-type ATPase Acu, important for K + homeostasis in a closely related M. graminicola, were found in H. werneckii. The enrichment in plasma membrane cation transporters is accompanied by transcript enrichment of the plasma P-type H + ATPases in H. werneckii, as transcription of all four HwPMAs is salt dependent. In S. cerevisiae, only PMA1 is transcribed in sufficient quantities to substantially contribute to the generation of the proton gradient [117]. The difference in the transporter inventories of the mesophylic S. cerevisiae, a plant pathogen M. graminicola, and its closely related extremely halotolerant H. werneckii does not only reflect their phylogenetic relations, but in an even greater extent also their diverse life styles. Therefore, the great diversity of H. werneckii cation transporters Figure 6. Proton ATPases. A. Plasma membrane ATPases (Pma). Gene phylogeny (above) of homologues from Hortaea werneckii (HwPma), Mycosphaerella graminicola (MgPma1: XP_003852209.1) and Saccharomyces cerevisiae (Pma1: YGL008C, Pma2: YPL036W), rooted by a homologue from Cryptococcus neoformans (XP_568571.1). Two half-circles mark a duplication after the separation of S. cerevisiae and H. werneckii ancestors, but before the separation of H. werneckii and M. graminicola, black circles on the bifurcation mark recent duplications presumably resulting from a whole genome duplication. Transcription profiles (below) of plasma membrane H + -ATPases of H. werneckii homologues at different concentrations of NaCl (w/v). Quantitative reverse transcription PCR (qRT-PCR) was performed with RNA isolated from cells grown in YNB medium, supplemented with 0, 5, 10, 17, and 25% NaCl (w/v). Quantification cycle (Cq) values for our genes of interest were normalised to the quantification cycle of 28S rRNA fragment (reference gene). The difference in Cq values (relative mRNA level values) between the target gene and the reference gene was calculated, and these values of the different samples were compared directly. Data are means of relative mRNA level values obtained by two qRT-PCR experiments performed with biological triplicates. B. The subunit A of the vacuolar ATPases (Vma1). Gene phylogeny (above) of homologues from Hortaea werneckii (HwVma), Mycosphaerella graminicola (MgVma1: XP_003850333.1) and Saccharomyces cerevisiae (Vma1: YDL185W), rooted by a homologue from Cryptococcus neoformans (XP_570895.1). Black circles on the bifurcation marks a recent duplication presumably resulting from a whole genome duplication. Transcription profiles (below) of vacuolar H + -ATPases of H. werneckii homologues at different concentrations of NaCl (w/ v). Quantitative reverse transcription PCR (qRT-PCR) experiment and the analysis of the data was performed as described above. doi:10.1371/journal.pone.0071328.g006 and their possibly novel adaptations to high concentrations of salt may harbour a great biotechnological potential for improving the halotolerance of salt-sensitive species. In H. werneckii, low amounts of cytosolic Na + were observed over the whole range of salinities [25], indicating very efficient plasma membrane Na + exclusion and vacuole Na + import mechanisms, possibly through action of HwNha and HwVnx proteins. This makes H. werneckii Nha1 and Vnx1 homologues especially interesting targets for plant transgenes.
The genetic redundancy and enrichment of cation transporters seem to be at the core of the extremely halotolerant phenotype of H. werneckii. Thorough understanding of eukaryotic halotolerance is important for alleviating problems such as those caused by soil salinization in agriculture or osmotic stress in the production of bioethanol. H. werneckii is an appropriate model organism for studying an excellent ability for adaptation to almost the whole range of salinities. While working with it in the past was challenging, the availability of the genomic sequence should significantly ease further studies of this exceptional species.