WO2008095826A1 - Method to obtain salt tolerance in eukaryotic cells - Google Patents

Abstract

The present invention relates to a method to obtain salt tolerance in eukaryotic cells, preferably fungal cells or plant cells. More specifically, the invention relates to the use of a HAL2/MET22 homologue comprising a salt resistant META sequence to obtain salt tolerance in eukaryotic cells.

Description

METHOD TO OBTAIN SALT TOLERANCE IN EUKARYOTIC CELLS

The present invention relates to a method to obtain salt tolerance in eukaryotic cells, preferably fungal cells or plant cells. More specifically, the invention relates to the use of a HAL2/MET22 homologue comprising a salt resistant META sequence to obtain salt tolerance in eukaryotic cells.

Salt stress is an important threat to the future agriculture in many productive areas. In some countries such as Australia, Pakistan, California and the Mediterranean region, the hypersalinization is already a national concern (Serrano et al., 2002). To bridge the gap between salt toxicity and applicative agricultural research, the nature of the targets at the cellular and molecular level should be understood. As an important step in this direction is to understand the mechanisms of salt tolerance in those eukaryotic organisms which are salt tolerant by nature. Indeed, the knowledge of the mechanisms involved in adapting to osmotic and ionic stress in eukaryotic cells in general, and in plants and yeast cells in particular have made a significant advance with the identification and isolation of several genes in halotolerant species that are potentially involved in the process of salt tolerance.

Of special interest is the genetic manipulation of crops to include individual transgenes from halotolerant species in order to produce improvement in halotolerance levels. Particularly interesting targets are gene products which are modulated by ion influx in high salinity environment. Among them are sodium and lithium sensitive phosphatases.

3'-phosphoadenosine-5'-phosphatase catalyzes the removal of 3' phosphate from 3'- phosphoadenosine-5'-phosphate (PAP) or 3'-phosphoadenosine-5'-phosphosulphate (PAPS) to form adenosine-5'-phosphatase or adenosine-5'-phosphosulphate, respectively. It belongs to the enzyme family of phosphomonoesterases which are all magnesium- dependent lithium- inhibited phosphatases In Saccharomyces cerevisiae, the gene HAL2/MET22 encodes a sodium and lithium sensitive phosphatase Hal2 protein (Hal2p), which is an important determinant of halotolerance in this yeast (Glaser et al., 1993). This gene is essential for sulphur assimilation and hence the methionine biosynthesis in yeast. PAPS serves as a sulphate donor and the resulting PAP is subsequently hydrolyzed to adenosine-5'-phosphate and inorganic phosphate by Hal2p (Murguia et al., 1996). During salt stress, the enzyme activity of Hal2p is inhibited by lithium (50% inhibitory concentration, 0.1 mM) or sodium (50% inhibitory concentration, 20 mM) and PAP accumulates inside the cell (Murguia et al., 1995). Elevated PAP concentrations are very toxic for the cell as it inhibits sulphotransferase (Albert et al., 2000), RNA processing enzymes, such as Xrn1 protein in yeast (Dichtl et al., 1997), and nucleoside diphosphate kinase (Schneider et al., 1998). Therefore Hal2p catalyzes a key metabolic reaction that is limited under high salt stress conditions (Glaser et al., 1993; Serrano, 1996). Overexpression of yeast HAL2 gene was reported to improve salt tolerance in yeast (Glaser et al., 1993) and in plants (Arrillaga et al., 1998). Recently, additional improvements in halotolerance have been shown with overexpression of the DHAL2 gene from the halotolerant yeast Debaryomyces hansenii (Aggarwal et al., 2005). Overexpression of this gene in S. cerevisiae resulted in tolerance up to 1.2 M NaCI. Another strategy to improve halotolerance has been to use a protein from the halophilic fungus Eurotium herbariorum isolated from the Dead Sea (Jin et al., 2005). A mitogen-activated protein kinase EhHog from the high-osmolarity glycerol (HOG) pathway was over-expressed, and this improved the halotolerance in yeast up to 1 M NaCI and 0.3 M LiCI. However, this strategy seems to be less promising for halotolerance transfer into plants, since there is no evidence for the HOG signaling pathway in plants (Kultz, 1998).

In search for better halotolerance determinants for yeasts and plants, a HAL2 homologue from extremely halotolerant black yeast Hortaea werneckii (Ascomycota, Dothideales), first isolated from the hypersaline waters of marine salterns on the Adriatic coast of Slovenia (Gunde- Cimerman et al., 2000), was identified. H. werneckii was later found elsewhere as a predominant species among the group of halophilic and halotolerant melanized yeast-like fungi, also named black yeasts. It can actively grow at salinities ranging from 0 M to almost saturated solution of NaCI (5.2 M). This ability is presumed to reside with the molecular mechanisms designed to cope with the extreme variation in ion concentration in its natural habitat. Two uncommon 3'-phosphoadenosine-5'-phosphatase genes were identified and characterized in the genome of H. werneckii. The expression of the genes were then studied at both mRNA and protein levels.

Surprisingly we found that these genes, HwHAL2A and HwHAL2B, can increase halotolerance in eukaryotic cells such as plant cells and yeast cells. When expressed in S. cerevisiae, halotolerance up to 1.8 M NaCI and up to 0.8 M LiCI was obtained, a level that was never obtained with any other approach. Even more surprisingly, based on homology modeling, a novel domain (called META) has been identified that is essential for the halotolerance. When inserted in a halosensitive Hal2p framework, said domain is necessary and sufficient to make the Hal2p enzyme resistance against salt stress and to confer halotolerance to the host organism upon transformation of the fusion protein into a eukaryotic cell. Optionally, the effect may be enhanced by inserting a second domain (called ANA).

A first aspect of the invention is an artificial recombinant gene, encoding a protein comprising a META sequence as represented by SEQ ID N°1 (DSEPL[T/R/QA/]E[D/G][L/I]). Preferably, said protein is comprising SEQ ID N°2 (DSEPLREGI). Optionally, said protein comprises also the ANA sequence as represented by SEQ ID N°3 (NSQLRD) in addition to SEQ ID N°1 or SEQ ID N°2. Gene, as used here, comprises the coding sequence with, if relevant, it's intron - exon structure, and the regulatory sequences such as the promoter and terminator sequences. A recombinant gene is a gene whereby the sequence of the natural gene has been cloned, and where this sequence, possible after introduction of some changes, is reinserted in the parental strain, or inserted in a different strain. Preferably, the sequence is introduced in a different host strain. An artificial recombinant gene, as used here is a gene whereby the natural occurring sequence of the gene has been changed, e.g. by replacement of the promoter sequence, or by adaptation of the coding sequence, such as codon usage optimization. Preferably, an artificial recombinant gene according to the invention is a gene whereby the sequence encoding SEQ ID N°1 , even more preferably the sequence encoding SEQ ID N°2 has been inserted in the coding sequence. Optionally, also a sequence encoding SEQ ID N°3 is inserted in the coding sequence, in addition to the sequence encoding SEQ ID N°1 or SEQ ID N°2. Preferably said recombinant gene and said artificial recombinant gene encode a Saccharomyces cerevisiae Hal2p homologue, more preferably said gene encodes a protein with 3'-phosphoadenosine-5'- phosphatase activity. A Hal2p homologue, as used here is a protein with at least 30% identities, preferably 35% identities, more preferably 40% identities, even more preferably 45% identities, most preferably 50% identities to the S. cerevisiae Hal2p (genbank accession number CAA51361 ), as determined by a BLAST 2 sequences search for proteins (Tatusova and Madden, 1999). Even more preferably, said recombinant gene and said artificial recombinant gene encode a protein that can complement the S. cerevisiae hal2 (met22) mutation. Another aspect of the invention is an artificial recombinant protein comprising SEQ ID N°1 , preferably an artificial recombinant protein comprising SEQ ID N°2. Optionally, said artificial recombinant protein comprises also SEQ ID N°3 (NSQLRD) in addition to SEQ ID N°1 or SEQ ID N°2. As used here, a recombinant protein is a protein encoded by a recombinant gene as defined above. An artificial recombinant protein is a protein of which the amino acid sequence differs from the natural occurring amino acid sequence. Preferably, said artificial sequence differs from the natural occurring sequence by insertion of SEQ ID N°1 , preferably by the insertion of SEQ ID N°2. Preferably, said artificial recombinant protein is a S. cerevisiae Hal2p homologue as defined above. More preferably, said recombinant artificial protein has 3'- phosphoadenosine-5'-phosphatase activity. Most preferably, said recombinant artificial protein can complement a defect S. cerevisiae Hal2p. Still another aspect of the invention is the use of a recombinant gene encoding a protein comprising SEQ ID N°1 , preferably comprising SEQ ID N°2 to obtain salt tolerance in a eukaryotic cell. Optionally, said protein comprises also SEQ ID N°3 (NSQLRD) in addition to SEQ ID N°1 or SEQ ID N°2. Salt tolerance as used here means that the organism, transformed with said recombinant gene can grown in the presence of sodium- and/or lithium ion concentrations that are significantly higher than for the non transformed organism. Salt tolerance can be obtained cis-genetically by overexpression of the endogenous gene in a recombinant way, or trans-genetically, by expression of a heterologous gene, or by expression of an artificial recombinant gene in a eukaryotic cell. Preferably, said eukaryotic cell is a yeast cell or a plant cell. Preferably, said yeast cell is a S. cerevisiae cell. Halotolerant yeasts can be used in high salt fermentations, such as the preparation of Soy sauce.

The presence of Hal2 homologues in plants is known. As a non-limiting example, the existence of multiple forms of HAL2-\\ke 3'- phosphoadenosine-5'-phosphatase has been reported for Arabidopsis, with three HAL2-\\ke gene members (Gil-Mascarell et al., 1999). Such Hal2p homologues can serve as framework for inserting SEQ ID N°1 or SEQ ID N°2, or one or more of the homologues can be replaced by an artificial recombinant gene according to the invention. Alternatively, said artificial recombinant gene is expressed in parallel with the endogenous Hal2p encoding homologues. Preferably, said plant cell is a corn cell or a rice cell. Indeed both corn (Zea mays) and rice (Oryza sativa) comprise Hal2p homologues without SEQ ID N°1. Therefore, introduction of SEQ ID N°1 , preferably SEQ ID N°2 in the normal framework of the Hal2p homologue, or replacing the Hal2p homologue by an artificial recombinant protein comprising SEQ ID N°1 , preferably comprising SEQ ID N°2, optionally comprising SEQ ID N°3 in addition to SEQ ID N°1 or SEQ ID N°2 will increase the salt tolerance. Alternatively, said artificial recombinant protein according to the invention is expressed in parallel with the endogenous Hal2p homologues.

In one preferred embodiment, said recombinant gene, used to obtain salt tolerance comprises SEQ ID N°4 (HwHAL2A DNA). In another preferred embodiment, said recombinant gene comprises SEQ ID N°6 (HwHAL2B DNA). In an even more preferred embodiment, said recombinant gene is encoding SEQ ID N°5 or SEQ ID N°7 (HwHal2 A and B proteins) Another aspect of the invention is a transgenic plant, comprising an artificial recombinant gene according to the invention, and/or an artificial recombinant protein, according to the invention. Preferably, said plant is a transgenic corn or rice plant.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Hortaea werneckii 3'-phosphoadenosine-5'-phosphatases. (A) Amino acid alignment of ScHal2 (GenBank accession no. X72847), HwHal2A and HwHal2B with ClustalX. Conserved motifs involved in the co-ordination of the phosphate and metal ions or implicated in catalysis are indicated by shaded areas with A, B and C (Atack et al., 1995). The phosphomonoesterase amino acid consensus (York et al., 1995) within motif A is denoted by a black line. Amino acids blocks in HwHal2A and HwHal2B with no homology in ScHal2 are framed as ANA and META sequences. The ATP binding consensus motif found only in HwHal2B is marked as P-loop. Black arrows represent the amino acids that differ between HwHal2A and HwHal2B. (B) Southern blot analysis with [32P]-HwHAL2 probe of the Not\, BamH\, Hind\\\ and BamH\+Hind\\\ digests of H. werneckii genomic DNA. Neither of the restriction enzymes used have a cutting site within the probe sequence. The two bands in the digest represent the two different HwHAL2 genes. (C) Western blot analysis of the cytosol fraction from H. werneckii cells adapted to indicated NaCI concentrations with anti-Hal2 antibodies, β-actin was used as an internal control. (D) Expression of HwHAL2A and HwHAL2B mRNA in salt adapted (ADAPTATION) or NaCI shocked H. werneckii cells (HYPERSALINE STRESS). Hw26SRR represents a housekeeping gene for 26S rRNA used as an internal control. The primer specificity controls for each of HwHAL2 isoforms in pBK- CMV plasmids are denoted as pHAL2A and pHAL2B, respectively. (E) RNAPoI-ChIP analysis of actual transcription rate of HwHAL2A and HwHAL2B genes. Promotor and coding region occupancies by RNA polymerase Il are presented in cells adapted to the indicated salinity. Representatives of at least three independent experiments are presented in PCR analyses.

Figure 2. Functional complementation of HAL2/MET22 and improved halotolerance in S. cerevisiae strains harbouring HwHAL2A, HwHAL2B or ScHAL2. (A) 10-fold serially diluted cultures of transformed cells with empty plasmid pRD53 (vector) as control or vector carrying indicated inserts were plated on YNB-Ura+Gal plates without salt (No salt) or without methionine (No Met) or containing indicated concentration of salts (NaCI or LiCI). (B) Growth curves of different hal2 transformants in medium with 1.6 M NaCI. For the reason of clearness, curves of the other strains were omitted. (C) PAP phosphatase activities and the level of Hal2 proteins in hal2 transformants were comparable. The results of the enzyme activity are expressed as percent activity observed in the absence of endogenous ScHal2 (hal2 strain harboring empty vector) and are the means (± standard deviation) of four independent experiments. Error bars correspond to the standard deviation.

Figure 3. Three-dimensional model of H. werneckii Hal2B. (A) Superposition of HwHal2B and ScHal2 in the view of secondary structure elements conservation. The positions of the ANA and META sequences are represented. Note that the META sequence folds into the short α-helix, intruding the β5 sheet of conserved α+β domain within the metal-dependent/Li- inhibited phosphomonoesterase protein family. The conserved active site aminoacids are represented with dark sticks surrounding the substrate PAP represented by big stick. The position of the inhibitory sodium ion is represented by the sphere and the displaced β12α9- loop in HwHal2B is labeled.

(B) The surface view of the HwHal2B. The highly exposed META sequence is shown in dark (on the top), on the surface away from the active site cavity represented by a darker area. The solvent exposed portion of the ANA sequence is shown in dark grey (left side). The plot was created with the program PyMOL (version 0.97; DeLano Scientific, USA. [http://www.pymol.org]). Figure 4. The META sequence is structural determinant of HwHa/2β-dependent halotolerance. S.cerevisiae wild-type (WT) and hal2 strain were transformed with pRD53 empty vector (Vector) or vectors carrying indicated inserts. (A) Deletion of ANA or/and META sequences reduce the halotolerant properties of HwHAL2B for high concentration of NaCI and LiCI. Insertion of the META sequence into ScHAL2 improves halotolerance of the strain. 10- fold serially diluted cultures of transformed S. cerevisiae cells were plated on YNB-Ura+Gal plates without salt (No salt) or plates containing indicated concentration of NaCI and LiCI. (B) Deletion of META or ANA sequences in HwHAL2B or insertion of META sequence into ScHAL2 does not affect the Hal2 protein amount of the transformed strains as determined by western blots using anti-Hal2 antibody. PAP phosphatase activities of indicated transformants are presented. The results of the enzyme activity are obtained and expressed as described in Figure 2.

Figure 5. HAL2-\\ke genes in other saltern-enhabited halotolerant fungi. Deduced partial amino acid sequences of Hal2-like phosphatases from halotolerant fungi were aligned with the corresponding regions of the Hal2 from S. cerevisiae (Gen Bank accession no. X72847) and D. hansenii (GenBank accession no. AY340817) by the program ClustalW. The output was graphically presented by the program BOXSHADE (version 3.21 ; [http://www.ch. embnet.org/software/BOX_form. html]). Similar amino acid residues are highlighted in gray and more than 75% identical residues are highlighted in black. The amino acid residues crucial for the enzyme activity are denoted by black arrows and the hypervariable region comprising the META-like motifs is framed. For fungi description see text.

Figure 6. Phylogenetic tree of deduced Hal2-like phosphatases from saltern enhabited fungi. The tree was constructed with the neighbor-joining method (Saitou and Nei, 1987) based on amino acid sequences alignment by the program MEGA 3.1 (Kumar et al., 2004) and arbitrarily rooted with W. ichthyophaga. The numbers at nodes are bootstrap confidence values based on 1000 replicas. The tree was corrected with Poisson correction. H. werneckii Hal2A and Hal2B proteins are exposed with grey box. EXAMPLES

Materials and method to the examples

Strains and growth conditions.

Cultures of black yeasts H. werneckii (MZKI B736), P. triangularis (MZKI B748), E.amstelodami (MZKI A561 ), A. pullulans (EXF 150), W. muriae (MZKI B952), W. ichthyophaga (EXF 994), W. sebi (EXF 757), T. salinum (EXF 295), C. sphaerospermum (EXF 385) and H. acidophila (CBS 1 13389) from culture collections of the Slovenian National Institute of Chemistry (MZKI) or University of Ljubljana Biotechnical Faculty Department of Biology (EXF) and the Centraalbureau voor Schimmelcultures Utrecht (CBS), The Netherlands were used in this study. The reference salt-sensitive strains were S. cerevisiae S288C (BY4741 ; MATa; his3Δ1 ; leu2Δ0; met15Δ0; ura3Δ0) (Brachmann et al., 1998) and the S. cerevisiae HAL2/MET22 deletion strain (BY4741 : Mat a; his3Δ1 ; leu2Δ0; met15Δ0; ura3Δ0; YOL064c::kanMX4) both obtained from the Euroscarf Yeast Deletion Strain Collection, Germany. Cells were mantained and grown in defined medium YNB (0.17% (w/v) Yeast nitrogen base (Qbiogene), 0.08% (w/v) Complete supplement mixture (Qbiogene), 0.5% (w/v) ammonium sulphate, 2% (w/v) glucose in deionized water) adjusted to indicated NaCI concentrations and to pH 7.0 and incubated at 28 ⁰C in a rotary shaker at 180 rpm. Cells were harvested in the mid-exponential phase by centrifugation at 4000^χ g for 10 min. For hypersaline stress, H. werneckii cells were grown in YNB with 1 M NaCI to ODΘOOnm 1.0 and then the concentration of the medium was adjusted to 3 M NaCI. Aliquots of the medium were removed before and 10, 30, 60, 90, and 120 min after the stress. Cells were separated from growth medium by fast filtration through 0.45 μm-pore filter and frozen in liquid nitrogen.

Amplification of S. cerevisiae HAL2-like genes from genome of halotolerant fungi. Highly purified fungal genomic DNA was isolated from mid-exponential phase cells grown in YNB media without salt by phenol/chloroform/isoamyl alcohol method modified for the DNA isolation from filamentous fungi as described (Rozman et al., 1994). Partial sequences of HAL2 orthologs from halotolerant fungi were amplified with touch-down PCR using the degenerate primers δ'-TTCYTIMGIGGIGGICARTAYGC-S' and 5'-

RTGRTCCCAIATYTTYTCCTGGTA-3', corresponding to conserved amino acid regions in the alignment of the few known Hal2 orthologs. 50 ng of genomic DNA was used as a template in 50 μl_ PCR reaction with Gotaq DNA polymerase (Promega). PCR was performed by 12 touch- down cycles with an annealing temperature from 61⁰C to 55°C followed by 23 cycles with annealing at 55°C. Expected 500 bp PCR products were cloned into pGEM-T Easy vector (Promega) and sequenced. Cloning of the HwHAL2 cDNA and southern blot analysis.

The partial sequence of HwHAL2 was amplified by PCR using the primers 5'- TTCCTGGTAGTCCTTGCGCA-3' and δ'-AATTCGGGGTCCTGTTCTCC-S' and using H. werneckii genomic DNA as a template. A 302 bp PCR product was labeled with [32P]dCTP using the Prime-It random primer labeling kit (Stratagene) according to the manual and then used as a probe. To clone the HwHAL2 cDNA, we constructed a H. werneckii cDNA library in pBK-CMV phagemid vector using a Zap express predigested gigapack cloning kit (Stratagen) according to the manufacturer's protocol. Total RNA was isolated from mid-exponential cells of H. werneckii grown in YNB medium with 3 M NaCI by the classic acid guanidinium thiocyanate/phenol/chloroform method modified for RNA isolation from fungi as described (Breskvar et al., 1991 ). Isolation of mRNA was performed with PoIy(A) Quick mRNA isolation kit (Stratagene). Reverse transcription of mRNA and adaptor ligation was performed using Zap express predigested cDNA synthesis kit (Stratagen) according to the manual. Screening of the cDNA and genomic DNA library were performed by procedure described previously (Turk et al., 2002). Briefly, plaque lifts with 6x105 pfu of recombinant phage were made on the nitrocellulose membranes (Amersham Bioscience), DNA was fixed by baking at 80⁰C for 2 h and the membranes were then hybridized with the [32P]-labeled HwHAL2 probe in a hybridization solution (6^χSSC, 0.5% SDS, 5^χDenhardt reagent, 150μg/ml_ salmon sperm DNA) at 65°C overnight. The blots were then washed three times in 2xSSC, 0.1 % SDS for 15 min at 65°C. Positive clones were visualized by autoradiography, plaques were excised from plates and phages were then eluted in 200 μl_ of SM buffer. E. coli XL1 Blue cells were infected with cDNA phages and the helper phage, then grown for 3h at 37°C, lysed at 70⁰C and centrifuged 15 min at 1000^χg. Supernatant was used to transfect the E. coli XLOLR cells and then selected on LB Kanamycin plates. The pBK-CMV plasmids with cDNA fragment were isolated from resulting colonies using Wizard Plus SV Minipreps Purification System (Promega) and then sequenced. For Southern blot analysis of H. werneckii genomic DNA, 15 μg of high purity genomic DNA was digested with the restriction enzymes Not\, BamYW, Hind\\\ and BambW + Hind\\\ (Roche) overnight on 37°C, resolved by electrophoresis in 1.5% agarose gel, transferred to the nitrocellulose membrane by capillary transfer in 20* SSC buffer overnight and processed as described above for the hybridization with [32P]-labeled HwHAL2 probe.

Reverse transcription PCR. The total RNA contents from H. werneckii cells were isolated using TRI Reagent (Sigma- Aldrich) according to manufacturer's instructions, using 200-300 mg of mid-exponential phase cells grown in YNB media containing 1 , 3 or 4.5 M NaCI, or hypersaline shocked cells. RNA was treated with DNase I (Fermentas). 1 μg of DNA-free RNA was used for 20 μl_ of reverse transcription reaction using Superscript III reverse transcriptase (Invitrogen) and random primers (Promega) according to the manufacturer's protocols. PCR with Gotaq DNA polymerase was performed using 1 μl of cDNA for 25 μl PCR reaction with primers specific for HwHAL2A (5'-TCTCGGACTCCGAGCCCT-S' and 5'- AAGTCCGTCCAAGCAGCATGAACT- 3'), HwHAL2B (5'- GTTTCCGATTCGGAACCGC-3' and δ'-CTCCATCCCGTTTCCCCAT-S') or H. werneckii 26S rRNA (δ'-CATCACTGTACTTGTTCGCTATCGGTC-S' and 5'- GTAACGGCGAGTGAAGCGGC-3') as an internal control. Thermal cycling was programmed for 30 cycles each consisting of 30 sec at 94°C, 30 sec at 60⁰C and 60 sec at 72°C.

Expression constructs and deletion mutagenesis.

For expression of HwHAL2A and HwHAL2B in S. cerevisiae, 1.3-kb cDNA fragments containing full-length HwHAL2A or HwHAL2B ORFs from pBK-CMV vectors were subcloned into BamY\\IXho\ sites of the low copy number plasmid pRD53 [CEN, ARS, URA3, GAL1/10 promoter, AmpR), resulting in the plasmids pRD53-HwHAL2A and pRD53-HwHAL2B respectively. Deletions in the HwHAL2B coding region were performed by PCR-based splicing (Horton et al., 1993) using the primers 5'- GCTGGCGTCCGCCCCGGAAACCGGCAGGTT-3' and 5'- AACCTGCCGGTTTCCGGGGCGGACGCCAGC-3' for the deletion ΔM or 5'- CAACCCGTAGACTAGCTCCCGCAAGTCCTT-3 and 5'-AAGGACTTGCGGGAGC TAGTCTACGGGTTG-S' for the deletion ΔA, both primer pairs in combination either with T3- or T7-promoter universal primers in separate reactions and the 100 ng of plasmid pBK-CMV- HwHAL2B as a template in 50 μl_ of primary PCR reaction. PCR products of both reactions for each deletion were gel purified, mixed together in ratio 1 :1 and used as a template for secondary PCR reaction with T3 and T7 primers. Both primary and secondary PCR reactions were performed with 30 cycles each consisting of 30 s at 94°C, 60 s at 55°C and 60 s at 72°C with GoTaq DNA polymerase. Secondary PCR products were resolved on the agarose gel, eluted and cloned into BamH\/Xho\ sites of plasmid pRD53 resulting in plasmids pRD53- HwHAL2BΔM and pRD53-HwHAL2BΔA. An additional round of mutagenesis was performed on plasmid pRD53-HwHAL2BΔM using primers for the deletion ΔA as described to create a double deletion (ΔAΔM) in HwHAL2B coding region resulted in plasmid pRD53- HwHAL2BΔAΔM.

RNAPoI-ChIP lmmunoprecipitation of cross-linked chromatin was performed as described by Sandoval (Sandoval et al., 2004) with some modifications. Briefly, cells of H. werneckii growing in YNB media with 1 M, 3 M or 4.5 M NaCI were cross-linked at OD600 0.8 by formaldehyde treatment in a final concentration of 1 % for 15 min at room temperature with gentle shaking. Cross- linking was stopped with solution of glycin at a final concentration of 0.125 M with gentle shaking for 5 min at room temperature. Cells were harvested, washed twice with ice-cold PBS, pelleted, frozen in liquid nitrogen, and broken with a dismembrator. 2.5 g of cells were resuspended in 10 ml. of ChIP-L buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCI, pH 8.1 ) containing a cocktail of fungal protease inhibitors (Sigma) and mixed by inversion for 30 min on ice. The samples were sonicated on ice with 100-watt sonicator (MSE) at medium power setting for five 15-s pulses, resulting in DNA fragments with average length of 300 bp, and then centrifuged for 15 min at 10.000^χg to remove insoluble debris. For each RNAPoI-ChIP experiment, 100 μl_ of supernatant was frozen as an input and 1 ml. of supernatant was diluted in 9 ml. of ChIP-D buffer (0.01 % SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCI, pH 8.1 , 167 mM NaCI) with inhibitors. Chromatin solutions were pre-cleared with the salmon sperm DNA-precoated protein G-sepharose (Amersham) and then incubated with 5 μg of mouse monoclonal anti-RNAPolll antibody 4H8 (Abeam) overnight at 8°C. Immunocomplexes were precipitated using precoated protein G-sepharose for 2 h at 8°C with shaking, pelleted 1 min at 1000^χg and pellets were washed with 2 ml. of the following washing buffers: twice with ChIP-WI (150 mM NaCI, 0.1 % SDS, 1 % Triton X-100, 2mM EDTA, 20 mM Tris-HCI, pH 8.1 ), once with ChlP-W2 (500 mM NaCI, 0.1 % SDS, 1 % Triton X-100, 2mM EDTA, 20 mM Tris-HCI, pH 8.1 ), once with CMP-W3 (250 mM LiCI, 1 % Na-deoxycholate, 1 mM EDTA, pH 8.0) and twice with ChlP-W4 (10 mM Tris-HCI, 1 mM EDTA, pH 8.0). Immunocomplexes were eluted twice with 100 μL of ChIP-E buffer (1 % SDS, 100 mM NaHCO3) for 10 min at 65°C and eluates were collected. ChIP eluates and inputs were reverse cross-linked in 0.2 M NaCI for 5 h at 65°C, incubated with 20 μg of RNase for 30 min at 37°C, followed by treatment with 10 μg of proteinase K. DNA was purified using the Wizard PCR cleanup purification system (Promega) and eluted with 150 μL of water. PCR with Gotaq DNA polymerase was performed using 1 μl of eluted DNA from immunoprecipitated samples or 1 μl of 100-fold diluted input in 20 μl PCR reaction with Gotaq DNA polymerase and 15 nmol of specific primers (HwHAL2A promoter: δ'-AATGGATGACGTTGTCGCGT-S' and δ'-TTACGTGAGCAGGACAGTAG-S'; HwHAL2B promoter: δ'-AATTCACTAGTGATTAATGG-S' and δ'-AAGCAGCATCCCGGGCTT- 3'; the primer pairs used for RT-PCR analyses were used for the coding region amplification). Thermal cycling was programmed for 30 cycles each consisting of 30 sec at 94°C, 30 sec at 55°C and 30 sec at 72°C.

Expression constructs and mutagenesis.

For expression of HwHAL2A and HwHAL2B in S. cerevisiae, 1.3-kb cDNA fragments containing full-length HwHAL2A or HwHAL2B ORFs from pBK-CMV vectors were subcloned into BamH\/Xho\ sites of the low copy number plasmid pRD53 (C£Λ/, ARS, URA3, GAL1/10 promoter, AmpR), resulting in the plasmids pRD53-HwHAL2A and pRD53-HwHAL2B respectively. ScHAL2 ORF was amplified from S. cerevisiae genomic DNA using flanking primers δ'-CCCGGGATGGCATTGGAAAGAG-S' and 5'-

CTCGAGATAGGCGTTTCTTGACTGAATG-3', containing Cfrd\ and Xho\ restriction sites (underlined), respectively and cloned into pGEM-T Easy vector or pRD53 vector resulting in the plasmid pRD53-ScHAL2. Deletions in the HwHAL2B coding region were performed by PCR-based splicing (Horton et al., 1993) using the primers 5'- GCTGGCGTCCGCCCCGGAAACCGGCAGGTT-3' and 5'-

AACCTGCCGGTTTCCGGGGCGGACGCCAGC-3' for the deletion ΔM or 5'-10 CAACCCGTAGACTAGCTCCCGCAAGTCCTT-3 and 5'- AAGGACTTGCGGGAGCTAGTCTACGGGTTG-S' for the deletion ΔA, both primer pairs in combination either with T3- or T7-promoter universal primers in separate reactions and the 100 ng of plasmid pBK-CMV-HwHAL2B as a template in 50 μl_ of primary PCR reaction. PCR products of both reactions for each deletion were gel purified, mixed together in ratio 1 :1 and used as a template for secondary PCR reaction with T3 and T7 primers. Both primary and secondary PCR reactions were performed with 30 cycles each consisting of 30 s at 94°C, 60 s at 55°C and 60 s at 72°C with GoTaq DNA polymerase. Secondary PCR products were resolved on the agarose gel, eluted and cloned into BamY\\IXho\ sites of plasmid pRD53 resulting in plasmids pRD53-HwHAL2BΔM and pRD53-HwHAL2BΔA. An additional round of mutagenesis was performed on plasmid pRD53-HwHAL2BΔM using primers for the deletion ΔA as described to create a double deletion (ΔAΔM) in HwHAL2B coding region resulted in plasmid pRD53-HwHAL2BΔAΔM. Insertion of META sequence into ScHAL2 was performed as described for deletion mutagenesis using insertion (underlined) primers 5'- GATGCCTTCGCGTAGCGGTTCCGAATCACTTAAAACTAAGTTGGG-S' and 5'-

GATTCGGAACCGCTACGCGAAGGCATCGGGGCCCAAGATTTGAAA-S' together with flanking primers δ'-CCCGGGATGGCATTGGAAAGAG-S' and 5'-

CTCGAGATAGGCGTTTCTTGACTGAATG-3'. Plasmid pGEM-T Easy containing ScHAL2 ORF was used as a template. Resulted PCR product was cloned into Cfr9\/Xho\ sites of plasmid pRD53 resulting in plasmid pRD53-ScHAL2iM.

Functional expression of HwHAL2 in S. cerevisiae and salt tolerance assays.

Yeast cells were grown overnight in YPD media (1 % yeast extract, 2% peptone, 2% glucose; pH 7.0) at 30⁰C and 180 rpm to mid-exponential phase and then transformed with 1 μg of pRD53 constructs containing HwHAL2A ORF or various HwHAL2B forms, using Alkali-cation yeast transformation kit (Qbiogene) according to the manufacturer's protocol. Transformants were selected on YNB plates without uracil (YNB-Ura). For functional complementation of the HAL2/MET22 gene, positive colonies were grown overnight in YNB-Ura medium to mid- exponential phase, adjusted to OD600 nm 0.5, 10- fold serially diluted (1-10⁴ dilutions) with fresh medium and spotted in 3 μl_ onto plates without uracil, methionine and with 2% (w/v) galactose instead of glucose (YNB-Ura- Met+Gal) to induce transcription from GAL1/10 promoter. For salt tolerance assays, the same dilutions were spotted on YNB-Ura+Gal plates with indicated concentrations of NaCI or LiCI. The nontransformed hal2 mutant strain was grown in YNB+Gal medium prior to plating. Plates were incubated at 30⁰C for 3-5 days and then scanned. For the growth assay in the liquid media, transformants were incubated overnight in YNB- Ura+Gal media, reinoculated into YNB-Ura+Gal media containing 1.6 M NaCI at starting ODΘOOnm 0.05, and incubated at 30⁰C at 180 rpm for 4 days. At various time intervals, aliquots of the cultures were measured for optical density at 600 nm. Western blot analysis of cell lysates.

Cell lysates were prepared from exponentially growing cells by disruption with glass beads (for S. cerevisiae cells) or with microdismembrator (for H. werneckii cells) in hyperosmotic buffer (10 mM Tris-HCI pH 7.4, 10 mM KCI, 1.5 mM MgCI2, 10 mM DTT, 500 mM sucrose) containing a cocktail of fungal protease inhibitors (Sigma). They were fractionated into soluble and membrane fraction by centrifugation for 30 min at 27.000^χg and the supernatants were further centrifuged for 1 h at 100.000^χg to obtain cytosolic fraction. Protein concentration was measured by spectrophotometry at 590 nm by the Bradford method with Nanoquant reagent (Roth) and an equal amount of total cytosolic protein (40 μg) was boiled for 10 min in 5^χ Protein loading buffer (Fermentas) before loading. Proteins were separated by SDS-PAGE in 10% polyacrylamide gel and transferred to PVDF membrane (Roth). Immunodetection with rabbit polyclonal anti-Hal2 antibodies (Glaser et. al., 1993) or rabbit polyclonal anti-β-actin antibodies (Santa Cruz Biotechnology) and secondary goat polyclonal anti-rabbit antibodies conjugated with HRP (Santa Cruz Biotechnology) was performed using the ECL detection system (Amersham Bioscience).

PAP phosphatase acitivity assay

PAPase activity assays were carried out in a total volume of 100 μL containing 50 mM Tris (pH 7.5), 0.5 mM magnesium acetate, 1 mM PAP (Sigma) and 10 μg of cytosolic proteins, as described by Murguia (Murguia et al., 1995). After 30 min at 30°C, the released inorganic phosphate was quantified by the colorimetric malachite green method (Baykov et al., 1988). The values were corrected first by subtracting the blank readings obtained by hydrolysis of PAP in the absence of cytosolic proteins and additionally by subtracting the level of input phosphate from cytosolic protein fraction. The amount of phosphate released in nmol in each sample was calculated from standard curve made by serial dilution of standard monobasic sodium phosphate solution. Enzyme activity was expressed as nmol phosphate released per min per mg of proteins. Homology based 3D molecular modeling.

The three-dimensional (3D) model of HwHal2B was built by homology based protein structure modeling with the program MODELER 8v2 (Sanchez et al., 1997), which implements comparative modeling by satisfaction of spatial restraints (SaIi et al., 1993). The input consisted of the crystallographic template structure of S. cerevisiae Hal2 (Protein Data Bank (PDB), 1QGX) and the alignment of the HwHal2B sequence with this structure. The output obtained were ten slightly different 3D models of the HwHal2B with all non-hydrogen atoms. The 3D model of HwHal2 with lowest energy function was chosen for the interpretation. This model was derived by minimizing violations of many distance and dihedral angle restraints extracted from the template structure. The constructed 3D model passed the tests in the PROSAII (Sippl, 1993) and PROCHECK (Laskowski et al., 1993) programs.

Example 1 : H. werneckii contains two HAL2-\\ke genes: Isolation and characterization of HwHAL2A and HwHAL2B.

To identify a HAL2 ortholog in the unsequenced genome of H. werneckii, a conserved partial sequence of H. werneckii HAL2-\\ke gene (HwHAL2) was first obtained and used as a hybridization probe for screening the H. werneckii cDNA library. Analysis of the expressed sequence tags of positive clones revealed two highly similar coding regions with distinct 5'- and 3'-untranslated regions: we assigned them the names HwHAL2A and HwHAL2B. cDNA clones contained noninterrupted 1074 bp ORF encoding proteins of 357 amino acids, that differ only in 6 amino acid residues. The deduced amino acid sequences showed significant similarity with known fungal orthologs as retrieved by a BLASTX algorithm. HwHal2A and HwHal2B protein sequences show 73% identity with a Neurospora crassa ortholog and 42% or 43% identity with S. cerevisiae Hal2 (ScHal2), respectively. Predicted molecular masses of proteins were 37.8 kDa and 37.9 kDa, respectively. Their predicted isoelectric point values were 5.22 and 5.42 for HwHal2A and HwHal2B, respectively, which ware much lower when compared with 6.14 of ScHal2. The ClustalW (Thompson et al., 1994) alignment of HwHal2A and HwHal2B with

ScHal2 (Fig. 1A) highlighted that the sequence motifs, which are fully conserved among Hal2- like phosphatases, were present in HwHal2A and HwHal2B (Fig. 1A, dashed areas). HwHal2A and HwHal2B also contain two extra amino acid regions NSQLRD and DSEPLREGI, named ANA and META respectively, which are not found in ScHal2 (Fig. 1A, framed). One of the striking features of HwHal2B is the presence of the ATP binding motif or P-loop which can be found neither in HwHal2A nor in ScHal2. Two different, but closely related HwHAL2 genes and their sequences were further confirmed by screening the H. werneckii genomic DNA library with the same probe as used for cDNA library screening, confirming the absence of introns within coding regions of both genes. The number of HwHAL2 genes present in the genome of H. werneckii was further assessed by a restriction fragment Southern blot analysis with high stringency hybridization conditions. As shown in Figure 1 B, two bands were detected with HwHAL2 probe in the BamYW, Hind\\\ and BamH\+Hind\\\ digests of H. werneckii genomic DNA, indicating the existence of two different HAL2 genes in the genome of H. werneckii.

Example 2: Expression of HwHAL2A and HwHAL2B genes in H. werneckii is different in adapted and salt stress cells.

It has recently been demonstrated that cells growing with elevated NaCI concentration counteract the sodium inhibition of Hal2 by increased production of Hal2 protein (Todeschini et al., 2006). Authors showed that transcriptional activation of HAL2 in S. cerevisiae by translational derepression of transcriptional activator Gcn4 is induced by sodium chloride. Significant differences were observed in the promoter sequences of the genomic DNA clones of both HAL2 genes in H. werneckii, suggesting a differential transcriptional regulation of these genes. We examined how different NaCI concentrations in growth media affect the expression of HwHAL2 genes and their protein products. The level of HwHal2 protein was examined by immunodetection of western blots with anti-Hal2 antibodies (Glaser et al., 1993). As shown in Figure 1 C the accumulation of HwHal2 protein in H. werneckii was approximately 3.5-fold higher at very high salinity (4.5 M NaCI) than at the low and moderate salinity (1 M and 3 M NaCI, respectively). The transcript levels of the genes were examined by RT-PCR. As shown in Figure 1 D (middle panel), different profiles of HwHAL2 genes was observed in H. werneckii adapted to various salinity levels. Whereas the expression of HwHAL2A gene was strongly induced only at very high salinity (4.8-fold induction in 4.5 M NaCI), the transcript level of HwHAL2B shows a U-shaped profile with high level of gene expression at both the low and very high NaCI concentrations (6.1-fold induction in 1 M and 4.5 M NaCI). A low level of gene expression was observed at the moderate salinity of 3 M NaCI, which has been previously assigned as the optimal metabolic condition for H. werneckii (Petrovic et al., 2002). The transcriptional response of both genes to acute salt-stress was relatively slow (Fig. 1 D, right panel). The first significant increase in gene expression was observed only after 60 min. The transcription of HwHAL2A gene responded more intensively to hypersaline shock (7.2-fold induction after 120 min) than in the case of HwHAL2B gene (1.8-fold induction after 120 min). To confirm the actual transcription rate of both HwHAL2 isoforms in adapted cells we performed a chromatin immunoprecipitation of RNA polymerase Il (RNAPoI-ChIP). As shown in Figure 1 E, the RNA polymerase Il was detected well in HwHAL2A coding region predominantly at very high salinity, whereas paused at the promoter at the low and moderate salinity. In the case of HwHAL2B, the RNA polymerase Il was detected in coding region both at low and very high salinity, whereas paused at the promoter predominantly at the moderate salinity. The observed RNAPoI-ChIP data strongly correlate with the actual level of each HwHAL2 isoform transcripts as shown by RT-PCR.

Example 3: HwHal2A and HwHal2B complement the ScHal2 function in methionine biosynthesis and increase salt-tolerance in yeast.

Since more than 20 genes seem to be involved in methionine biosynthesis in yeast, the only apparent phenotype of /-//A/.2-disrupted cells is an auxotrophy for methionine (Glaser et al., 1993). To determine whether the HwHAL2A and HwHAL2B genes are true functional homologues of the yeast HAL2 gene, we carried out a complementation assay. The S. cerevisiae hal2 mutant cells were transformed either with pRD53-HwHAL2A or pRD53- HwHAL2B plasmids and selected on methionine and uracil minimal medium. Both HwHAL2A and HwHAL2B were seen to complement the S. cerevisiae hal2 mutant (Fig. 2A, lower left panel). The HAL2 gene was originally identified by functional cloning as supporting the growth of yeast under high salinity stress and improving halotolerance upon overexpression (Glaser et al., 1993). Therefore we tested whether HwHAL2A and HwHAL2B genes can improve halotolerance of S. cerevisiae to NaCI and LiCI when expressed from low copy number vector. The growth patterns of the wild-type and hal2 transformants were monitored on plates containing increased amount of salts, up to 1.8 M NaCI or 0.8 M LiCI. Our results clearly indicate the substantial increase in halotolerance to both NaCI and LiCI in HwHAL2 transformants as compared to the strain overexpressing ScHAL2 and parent strain used as controls. For both wild-type and hal2 transformants, the tolerance to NaCI was achieved even at the concentration of 1.8 M (Fig. 2A, upper panel). Especially noteworthy is the tolerance to LiCI where the growth of S. cerevisiae transformants was achieved even on a medium of 0.8 M LiCI (Fig. 2A, lower panel). Results in Figure 2A also show the difference between the two HwHAL2 genes on improving halotolerance, with HwHAL2B being more potent than HwHAL2A. In all cases, transformants with vector containing ScHAL2 were less halotolerant than transformants containing HwHAL2 isoforms. The influence of HwHAL2A, HwHAL2B and ScHAL2 expression on the halotolerance was also followed by growth measurements in liquid cultures containing 1.6 M NaCI. The observed growth kinetics of individual hal2 transformants were in accordance with previously observed growth properties on the plates (Fig. 2A, upper panel). Again, the ScHAL2 transformant showed very poor growth in the presence of 1.6 M NaCI, whereas hal2 strains harbouring HwHAL2A and HwHAL2B grew remarkably well under this condition (Fig. 2B). The linear portion of the growth curves of the HwHAL2A transformant exhibits the same slope as HwHAL2B transformant, although a small delay was observed in the lag time of the HwHAL2A transformant. Considering the lower slopes of the linear portion of the growth kinetics of ScHAL2 transformants and control strain containing only the empty vector, HwHal2 proteins conferred halotolerance evidently better than ScHal2. The PAP phosphatase (PAPase) activity was further assessed, to see whether different halotolerant properties of ScHal2 and HwHal2 proteins arise from differences in the enzyme activity. As showed in Figure 2C, the PAPase activity of cell lysates of HwHAL2A, HwHAL2B and ScHAL2 transformants were approximately the same, with HwHal2B showing only slightly higher PAPase activity as HwHal2A and ScHal2. To check if the increased halotolerance of HwHAL2 strains is the consequence of higher expression level of the HwHal2A and HwHal2B proteins when compared with the level of expression of the ScHal2, the immunoblot analysis of Hal2 proteins in cell lysates used for PAPase activity assay was performed. Results in Figure 2C revealed that the Hal2 protein level was the same in all samples tested, confirming that the improved halotolerance is related to better halotolerant properties of the HwHal2 proteins, which are independent of the enzyme amount and activity.

Example 4: Molecular modelling of HwHa I2 revealed a structural anomaly of the PAP phospatase. Naturally evolved halotolerant properties of HwHal2 proteins could reside in the structural elements of the protein. A three-dimensional model of full-length HwHal2B protein was calculated by program MODELLER (Sanchez and SaIi, 1997) based on the crystallographic structure of S. cerevisiae Hal2 (Albert et al., 2000) as a template to further examine the possible structural rearrangements due to the presence of the two uncommon sequence insertions in HwHal2 proteins, ANA and META, respectively (Fig 1A). Our approach to comparative protein modeling was based on satisfaction of spatial restraints (SaIi and Blundell, 1993) implemented in the program MODELLER. Since the template structure for homology based modeling was more than 40% identical to the sequence of the target, the model created is suppose to have about 90% of the mainchain atoms comparable with the X-ray structure (Sanchez and SaIi, 1997). The calculated 3D model of HwHal2B structure proved a well defined overall fold of the Hal2-like phosphatase superfamily. The superposition of HwHal2B model on the ScHal2 template revealed fine overlapping especially within the conserved regions and the active site amino acid residues (Fig. 3A). Compared to the template structure, the only poorly defined region is the META sequence due to the lack of sequence homology. An additional loop displacement appeared between β12 sheet and C-terminal α9 helix most probably due to Pro338 in the HwHal2B which precedes the corresponding Pro in the ScHal2 for two amino acid residues. The ANA sequence seems to contributes an additional turn at N- terminus of the α4 helix within the α subdomain of N-terminal α+β domain. The META sequence, however, folds into a much more intervening α-helix, interrupting the β5 sheet in the α+β domain core structure of the protein, an area otherwise structurally very conserved among metal-dependent/Li-inhibited phosphomonoesterase protein family (York et al., 1995). This feature has not been reported before in other Hal2-like proteins of this superfamily. Both insertions are exposed on the protein surface far apart from the conserved active site (Fig. 3A and 3B).

Example 5: Genetic validation of the structural requirements for HwHAL2B dependent halotolerance.

Identification of two extra amino acid sequences (ANA and META) in HwHal2 proteins when compared to ScHal2 primary (Fig. 1A) and tertiary (Fig. 3A) structures, led us to examine whether their structural role is related with the halotolerant nature of the protein. We focused particularly on HwHal2B, which increased the halotolerance most effectively. Three mutant forms of the protein HwHal2B were designed, with deleted either 6 amino acid residues of the ANA sequence (deletion ΔA) or 9 amino acid residues of META sequence (deletion ΔM), or both of them (deletion ΔAΔM). Mutated forms of the proteins were expressed in S. cerevisiae hal2 cells and grown on plates with increased NaCI and LiCI concentrations. Indeed, the deletions affected the halotolerant properties of HwHal2B. A shown in Figure 4A, deletion of the META region has a more evident effect on the halotolerance than the deletion of the ANA region, indicating a smaller contribution of the ANA sequence on the halotolerant properties of the HwHal2B protein. The double deletion further reduces the growth at high NaCI concentration. The growth effects are even more pronounced in the presence of lithium ions in the media. To dissect the role of the ANA and META sequences from halotolerant properties and the enzyme activity, we assessed whether deletions made in the coding region of HwHAL2B influenced the enzyme activity. The PAPase activity of HwHal2B and its deletion mutants in the transformed hal2 yeast was determined. As shown in Figure 4B, the PAPase activity of HwHal2BdA isoform was approximately half of the wild-type HwHal2B activity, indicating an important role of ANA sequence for the enzyme activity. The PAPase activity of the HwHal2BdM mutant remained virtually unaffected and similar to those of HwHal2B and the enzyme activity of the double deletion isoform HwHal2BdAdM remained similar to those of HwHal2BdA enzyme activity, excluding the role of META sequence in PAPase activity. Again, the level of all HwHal2 protein forms was similar in all transformants tested, as determined by immunoblot analysis of cell lysates (Figure 4B, lower panel). Results in Figure 4 therefore revealed that the META sequence is involved in the halotolerant nature of the HwHal2B protein which does not depend on enzyme activity or protein level. Example 6: Insertion the META sequence into the S. cerevisiae HAL2 framework is sufficient to confer salt tolerance to S. cerevisiae

The role of the META sequence in the halotolerant nature of the HwHal2 proteins was further validated by constructing the chimeric protein, by the in-frame insertion of the META sequence into the coding region of ScHAL2 at position homologous to HwHAL2 genes, connecting the

ScHal2 amino acid chain at the place of Ser181 and Gly184. The resulted construct ScHAL2iM was expressed and tested in hal2 strain. The ScHAL2iM transformant grew considerably better on high salt plates than ScHAL2 transformant. However, the growth advatage of the ScHAL2iM strain was observed only on the plates containing up to 1.4M NaCI and 0.4 M LiCI.

The PAPase activity of the ScHal2iM chimera was comparable to those of ScHal2 and

HwHal2B and so was the level of the protein expression (Fig 4B).

Example 7: HAL2 genes in other halotolerant fungi.

Since our results suggest that HwHal2 proteins from H. werneckii play an important role in halotolerance, further evidence was therefore sought concerning the validity of the prediction that Hal2 proteins from other halotolerant fungi might possess common structural elements. This was achieved by searching for HAL2 genes in other fungal species isolated from solar salterns (Gunde-Cimerman et al., 2000): Phaeotheca triangularis, Eurotium amstelodami, Aureobasidium pullulans, Wallemia muriae, Wallemia ichthyophaga, Wallemia sebi, Trimatostroma salinum and Cladosporium sphaerospermum. A salt sensitive species Hortaea acidophila was used for comparison as the only other member of the genus Hortaea. The translated partial Hal2 sequences were compared with the two well described Hal2 proteins from the salt sensitive S. cerevisiae and the halotolerant D. hansenii, using multiple sequence alignment with ClustalW (Fig. 5). The flanking regions of the aligned sequences and the amino acidresidues, shown earlier to be implicated in the catalytic mechanism (Atack et al., 1995), are well conserved among all fungi studied here (Fig. 5, black arrows). On the other hand, the META sequence identified by this study in the HwHal2 proteins appears to be the most heterogeneous portion of the Hal2 primary structure (Fig. 5, dashed area). While the sequence is evident in the majority of halotolerant melanized black yeasts from the ordo Dothideales H. werneckii, T. salinum, C. sphaerospermum, A. pullulans, P. triangularis and is also present but different in related E. amstelodami from Eurotiales, it is absent in salt-sensitive S. cerevisiae and halotolerant saccharomycete D. hansenii as well as in the members of the genus Wallemia, W. muriae, W. sebi and W. ichthyophaga. A functional phylogenetic tree based on the amino acid alignment of Hal2 sequences from the above mentioned halotolerant and halophilic fungi was generated using the neighbor-joining method implemented in ClustalW (Fig. 6). According to the amino acid sequence, the studied fungi are grouped into three major clusters. The first group is represented by different halotolerant to extremely halotolerant black yeasts. This group also contains the salt sensitive H.acidophila, the only other known Hortaea species. The two fungi, which are not matched with ecotype of black yeast, represent separate lineages. The second group is represented by the salt-sensitive S. cerevisiae and the halotolerant D. hansenii, both Saccharomycetales. And the third one is composed of the genus Wallemia, with the most halophilic so far known W. ichthyophaga being distinct from the salt sensitive W. muriae and W. sebi.

Example 8: HwHAL2B confers salt resistance to plant cells

The HwHAL2B coding sequence is placed under control of the CMV 35S promoter, and transformed into Arabidopsis using standard plant transformation techniques as described by De Block et al. (1987). Plants are grown in pots on peat-based compost containing fertilizer and were watered with demineralized water to which, after bolting, 1 vol % of a commercial fertilizer (NPK 6-3-6) is added. The plants are grown in a 12 h light/12 h dark cycle, with day and night temperatures of about 22 and 16°C respectively. In salt stress experiments, plants are watered with demineralized water, to which 6OmM NaCI is added from 12 days after sowing. The HwHAL2B transformants grow significantly better under saline conditions than the non transformed control. There is no significant difference in growth rate and yield between the transformants in saline conditions, and both the transformants under normal conditions and the non transformed plants under normal conditions.

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Claims

1. An artificial recombinant gene, encoding a protein comprising SEQ ID N°1.

2. An artificial recombinant gene according to claim 1 , encoding a protein comprising SEQ ID N° 2.

3. An artificial recombinant protein, comprising SEQ ID N° 1.

4. An artificial recombinant protein according to claim 2, comprising SEQ ID N° 2.

5. The use of a recombinant gene encoding a protein comprising SEQ ID N° 1 or SEQ ID N°2 to obtain salt tolerance in eukaryotic cells.

6. The use of a recombinant gene according to claim 5, whereby said gene comprises a coding sequence according to SEQ ID N° 4 (HAL2A DNA) or SEQ ID N° 6 (HAL2B DNA).

7. The use of a recombinant gene according to claim 5 or 6 whereby said eukaryotic cell is a fungal cell.

8. The use according to claim 7, whereby said fungal cell is S. cerevisiae.

9. The use of a recombinant gene according to claims 5 or 6 whereby said cell is a plant cell.

10. A transgenic plant, comprising an artificial recombinant gene according to claim 1 or 2.

1 1. A transgenic plant, comprising an artificial recombinant protein according to claim 3 or 4.