# Evidence of nickel (Ni) efflux in Ni-tolerant ectomycorrhizal P isolithus albus isolated from ultramafic soil

Clarisse Majorel, Laure Hannibal, Marc Ducousso, Michel Lebrun, Philippe Jourand

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Clarisse Majorel, Laure Hannibal, Marc Ducousso, Michel Lebrun, Philippe Jourand. Evidence of nickel (Ni) efflux in Ni-tolerant ectomycorrhizal P isolithus albus isolated from ultramafic soil. Environmental Microbiology Reports, 2014, 6 (5), pp.510-518. 10.1111/1758-2229.12176 . ird-04927890

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# Evidence of nickel (Ni) efflux in Ni-tolerant ectomycorrhizal *Pisolithus albus* isolated from ultramafic soil

Clarisse Majorel,¹ Laure Hannibal,¹ Marc Ducoussou,² Michel Lebrun³ and Philippe Jourand¹*

¹IRD, Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM UR040), ²CIRAD, Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM UR82) and ³Université Montpellier 2, Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM UMR28), TA A-82/J Campus International de Baillarguet, 34398 Montpellier cedex 5, France.

## Summary

Nickel (Ni)-tolerant ectomycorrhizal *Pisolithus albus* was isolated from extreme ultramafic soils that are naturally rich in heavy metals. This study aimed to identify the specific molecular mechanisms associated with the response of *P. albus* to nickel. In presence of high concentration of nickel, *P. albus* Ni-tolerant isolate showed a low basal accumulation of nickel in its fungal tissues and was able to perform a metal efflux mechanism. Three genes putatively involved in metal efflux were identified from the *P. albus* transcriptome, and their over-expression was confirmed in the mycelium that was cultivated *in vitro* in the presence of nickel and in fungal tissues that were sampled *in situ*. Cloning these genes in yeast provided significant advantages in terms of nickel tolerance (+31% Ni EC₅₀) and growth (+83% µ) compared with controls. Furthermore, nickel efflux was also detected in the transformed yeast cells. Protein sequence analysis indicated that the genes encoded a P-type-ATPase, an ABC transporter and a major facilitator super-family permease (MFS). This study sheds light on a global mechanism of metal efflux by *P. albus* cells that supports nickel tolerance. These specific responses to nickel might contribute to the fungal adaptation in ultramafic soil.

## Introduction

Various heavy metals are essential at low concentrations in plant and microorganisms nutrition but become toxic at increasing concentrations (Tyler et al., 1989; Marschner, 2012). As a consequence, soils that contain high concentrations of heavy metals, whether from natural origins or anthropogenic activity, may be toxic for plants and soil microorganisms (Giller et al., 2009; Marschner, 2012). To avoid heavy metal toxicity in soils, plants and microorganisms have developed many strategies based on molecular and physiological responses (Haferburg and Kothe, 2007; Manara, 2012). Ectomycorrhizal fungi (ECM) are also known to tolerate heavy metals (Hartley et al., 1997; Colpaert et al., 2011). These ECM participate in crucial symbiotic relationships with plants that grow in soils with high concentrations of heavy metals and alleviate metal toxicity for their host plants (Gadd, 1993; Leyval et al., 1997; Jentschke and Godbold, 2000; Jourand et al., 2014). The ECM fungal metal tolerance is supported by various mechanisms that include (i) reduction of uptake of metals into the cytosol by extracellular chelation through extruded ligands and binding onto cell-wall components, (ii) intracellular chelation of metals in the cytosol by a range of ligands (glutathione and metallothioneins) and (iii) increase of efflux from the cytosol out of the cell or into sequestering compartments (Bellion et al., 2006). In addition, metal tolerance may involve supplementary mechanisms, such as modification of a magnesium transport system, as reported for nickel tolerance in yeast and fungi (Joho et al., 1995).

Recently, nickel-tolerant ECM *Pisolithus albus* (Cooke, 1891) has been isolated from ultramafic soils in New Caledonia (Jourand et al., 2010a). Ultramafic soils are a weathered product from ultramafic bedrock that covers less than 1% of the earth's surface (Coleman and Jove, 1992). These soils are characterized by high concentrations of iron oxides (up to 85% w/w), unbalanced calcium (Ca)-to-magnesium (Mg) ratios (up to 1:30 that consequently may influence Ca and Mg plant nutrition) and the presence of various heavy metals at high concentrations, such as chromium, cobalt, manganese and nickel, all of which are toxic for many plants (Brooks, 1987; Harrison and Rajakaruna, 2011). These soils are also extremely deficient in elements that are essential for plant nutrition,

<footnote>Received 14 November, 2013; accepted 1 May, 2014. *For correspondence. E-mail philippe.jourand@ird.fr; Tel. +33467593955; Fax +33467593802.</footnote>
including nitrogen, phosphorus and potassium (Brooks, 1987; Chiarucci and Baker, 2007). In New Caledonia, the ultramafic soils cover one third of the main island because of natural geological evolution. As a result of the presence of such ultramafic outcrops, specific biological endemic ecosystems have developed (Jaffré, 1992), making the main island a biodiversity hot spot (Myers et al., 2000). In addition, in neo-Caledonian ultramafic ecosystems, ectotrophic mycorrhizal symbioses are dominant and contribute to plant adaptation and fitness to the extreme soil conditions (Jentschke and Godbold, 2000; Prin et al., 2012). Thus, the dominant ECM *P. albus* was isolated from ultramafic soils in New Caledonia, and Ni-tolerant and Ni-sensitive isolates were reported in one ecotype (Jourand et al., 2010a). To track biomarkers of nickel tolerance in *P. albus*, the Ni differential transcriptomes with or without nickel were determined. The expression of genes involved in the specific molecular response to nickel was monitored in a nickel-tolerant *P. albus* isolate (Majorel et al., 2012). Comparison of expression levels revealed that 30% of the identified genes were modulated by nickel treatment.

The present study aimed to investigate the molecular mechanisms supporting nickel tolerance in this *P. albus* transcriptome, particularly the metal efflux mechanisms recently reported by Ruytinx and colleagues (2013) in *Suillus bovinus* in response to zinc. To test this hypothesis, (i) nickel efflux and accumulation were analysed in *P. albus*, (ii) genes potentially involved in the metal efflux were investigated, (iii) and cloning of these genes in *levuro* was achieved to assess their expression in the presence of nickel and their role in metal efflux.

## Results and discussion

### Nickel efflux and accumulation in *P. albus*

Ectomycorrhizal fungi *P. albus* fungal isolates used in the study are described in Table 1. These fungal isolates were sampled from ultramafic soils in New Caledonia: the Ni-tolerant isolate (MD06-337) and the Ni-sensitive isolate (MD09-078) as reported in Jourand and colleagues

(2010b) and Majorel and colleagues (2012) respectively. To test the nickel accumulation in *P. albus*, the fungal isolates were cultivated in liquid in presence of nickel at different concentrations, and then nickel was analysed in the mycelial tissue. As shown in Fig. 1 (A and A'), the mycelium of *P. albus* presented a linear relationship between (i) the nickel concentrations tested in the liquid medium and (ii) the residual nickel content in its tissues. For the *P. albus* Ni-tolerant isolate (Fig. 1A), the slope of the linear regression was 0.0173 (R² = 0.98734), and the slope for the Ni-sensitive isolate (Fig. 1A'), was 0.025 (R² = 0.99881). At nickel concentrations of 50 µM and 100 µM, the residual nickel content in the mycelia of Ni-sensitive *P. albus* was 34% and 25% higher than the nickel content in Ni-tolerant *P. albus* respectively. To test the existence of a nickel efflux, the fungal isolates were cultivated for 1 week to generate a suitable quantity of biomass. Then, the kinetics of the metal efflux were tested by adding nickel at different concentrations, and residual nickel in mycelial tissue was measured at different times. Figures 1B and 1B present a comparison of the time-courses of residual nickel in mycelium between *P. albus* Ni-tolerant and Ni-sensitive isolates when exposed in liquid medium to 50 µM Ni (B) or 250 µM Ni (B'). After 48 h of nickel exposure and compared with nickel content at time 2 h, the residual nickel content in the mycelium of Ni-tolerant *P. albus* was increased by 25.7% at Ni = 50 µM and by 6.9% at Ni = 250 µM. In contrast, at the same time exposure (48 h) and compared with the nickel content at time 2 h, the residual nickel content in the mycelium of Ni-sensitive *P. albus* increased by 46.3% at Ni = 50 µM and 27.4% at Ni = 250 µM.

These data showed that the Ni-tolerant *P. albus* isolate accumulates a low basal level of nickel in its mycelial tissue, notwithstanding the nickel concentration applied in the medium. In liquid medium, a linear relationship exists between the nickel concentrations applied in the medium and the residual nickel content in the fungal tissue for Ni-tolerant and Ni-sensitive isolates *P. albus*. A comparison of the slopes (Fig. 1A and 1A') highlighted that the Ni-sensitive isolate presents faster nickel uptake than the Ni-tolerant isolate. Correlated with

<table><caption>**Table 1.** Isolates of *Pisolithus albus* used in this study. A description of the sites where isolates were sampled (map location, global positioning system (GPS) coordinates); isolate codes, DNA internal transcribed spacer (ITS) sequences accession number in the EMBL database and *in vitro* tolerance to nickel (phenotype and Ni EC₅₀) are provided for each fungal isolate.</caption><thead><tr><th rowspan="2">Site description (ultramafic soils)<br>Site location and GPS coordinates</th><th colspan="5">Pisolithus albus isolates</th></tr><tr><th>Isolate code</th><th>DNA-ITS EMBL<br>accession code</th><th>Nickel<br>phenotype</th><th>Ni EC₅₀ µM in<br>MMN medium</th><th>References</th></tr></thead><tbody><tr><td>Trazy-Guerioum. Koniambo Massif<br>(21°00'28" S; 164°49'50" E)</td><td>MD06-337</td><td>AM947121</td><td>Tolerant</td><td>569 ± 3</td><td>Jourand et al., 2010a</td></tr><tr><td>Pindjen Waterfall Road Koné<br>(21°02'19" S; 164°46'26" E)</td><td>MD09-078</td><td>FR852890</td><td>Sensitive</td><td>42 ± 4</td><td>Majorel et al., 2012</td></tr></tbody></table>
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Nickel content in *Pisolithus albus* mycelial tissue, expressed in µmol of Ni per g of DW mycelium. A and A'. Residual nickel in mycelium of the *P. albus* Ni-tolerant (A) or Ni-sensitive (A') isolates after 15 days of culture in liquid medium containing increasing nickel concentrations. B and B'. Time-course comparison between *P. albus* Ni-tolerant and Ni-sensitive isolates when exposed in liquid medium to 50 µM Ni (B) or 250 µM Ni (B'). Bars represent the means, and error bars represent the standard deviation of the mean (n = 3) of three independent experiments. The different letters above the columns indicate significant differences as determined by Tukey's honest significant difference test (P ≤ 0.05). Abbreviation: DW: dry weight.</figcaption></figure>

these data, the residual nickel content in the Ni-sensitive isolate was higher (+ 29%) than in the Ni-tolerant isolate. The profiles of the time-courses of residual nickel in the mycelia (Fig. 1B and 1B') were significantly different for both isolates. Indeed, the Ni-tolerant isolate was able to manage the nickel flux according to time, but the Ni-sensitive isolate could not. Altogether, these data clearly demonstrate that metal does not accumulate in the fungal tissue of Ni-tolerant *P. albus*, which suggests a metal efflux. Similar fungal behaviour in the presence of heavy metal has been recently reported in other ectomycorrhizal fungi, such as in *Suillus* and its tolerance to cadmium (Cd) and zinc (Zn) (Colpaert *et al*.,

2011; Ruytinx *et al.*, 2013). To argue this efflux hypothesis, we investigated the genes in the transcriptome of the Ni-tolerant *P. albus* isolate (i) that showed Ni-induced expression (Majorel *et al.*, 2012) and (ii) that were potentially involved in the metal efflux, such as the P-type ATPase (Portillo, 2000; Coleman and Mylonakis, 2009).

*Expression and selection of putative genes involved in nickel efflux in P. albus*

The contigs of putative genes involved in nickel efflux were selected from functional data obtained using the gene ontology (GO) analysis of the *Pisolithus albus*
<table><caption>**Table 2.** *Pisolithus albus* contigs selected in the transcriptomic database (Majorel *et al.*, 2012) which are functionally identified as involved in the nickel efflux. Data come from pyrosequencing analysis, and they are showing differential expression in *P. albus* nickel-tolerant isolate MD06-337. Data of the Blast X functional identification were collected from the NCBI database (http://blast.ncbi.nlm.nih.gov).</caption><thead><tr><th colspan="4">Pyrosequencing analysis</th><th colspan="2">Blast X functional identification</th></tr><tr><th>N° Contig</th><th>Size (bp)</th><th>RPKM<br>0 µM Ni</th><th>RPKM<br>250 µM Ni</th><th>Functional classification</th><th>Species</th></tr></thead><tbody><tr><td>C2515</td><td>464</td><td>0</td><td>114</td><td>Aminophospholipid-transporting P-type ATPase</td><td>Laccaria bicolor</td></tr><tr><td>C2624</td><td>295</td><td>0</td><td>102</td><td>Protein transporter</td><td>Coprinopsis cinerea</td></tr><tr><td>C3578</td><td>2428</td><td>101</td><td>134</td><td>0H+-transporting ATPase</td><td>Laccaria bicolor</td></tr><tr><td>C5339</td><td>2324</td><td>219</td><td>334</td><td>ABC transporter</td><td>Coprinopsis cinerea</td></tr><tr><td>C6943</td><td>578</td><td>6</td><td>46</td><td>Aminophospholipid-transporting P-type ATPase</td><td>Laccaria bicolor</td></tr><tr><td>C7177</td><td>1284</td><td>6</td><td>72</td><td>Phospholipid-translocating P-type ATPase</td><td>Coniophora puteana</td></tr><tr><td>C9915</td><td>446</td><td>0</td><td>11</td><td>Cu-transporting P-type ATPase</td><td>Laccaria bicolor</td></tr><tr><td>C16623</td><td>419</td><td>9</td><td>58</td><td>Minophospholipid-transporting P-type ATPase</td><td>Laccaria bicolor</td></tr><tr><td>C17235</td><td>2224</td><td>10</td><td>55</td><td>MFS general substrate transporter</td><td>Coniophora puteana</td></tr></tbody></table>

bp, base pair; RPKM, reads per kilobase of exon model per million mapped reads (Mortazavi *et al.*, 2008).

cDNA library (Majorel *et al.*, 2012). The list of the selected Ni-efflux genes (contigs) obtained from the GO analysis of the *P. albus* cDNA library is presented in Table 2 including the DNA sequences deposited into the EMBL database (from HG530057 to HG530065) and their BlastX functional identification. Their expression was tested by quantitative polymerase chain reaction (PCR) on either the fungal isolates *in vitro* cultivated with or without nickel and on mycelial tissue *in situ* sampled. The results of quantitative PCR analysis of the expression of these genes in *P. albus* are presented in Fig. 2. In mycelium cultivated *in vitro*, most of the genes were mainly expressed in the *P. albus* Ni-tolerant isolate (Fig. 2A). In particular, the contigs C3578 and C5339 showed the highest expression levels. Moreover, the contigs expression showed a clear induction at 250 µM of nickel. Furthermore, most of the genes were expressed in the mycelia and ectomycorrhizal tissue of Ni-tolerant *P. albus* sampled *in situ* (Fig. 2B). Contig C17235 showed the highest level of expression in *in situ*-sampled mycelium and the second highest level of expression in ECM. By combining the data from Table 2 and Fig. 2, contigs C3578, C5339 and C17235 were selected as the best candidate genes putatively involved in nickel efflux. The contigs C3578, C5339 and C17235 belong to the P-type ATPase family, the ATP-binding cassette (ABC) transporter family and a major facilitator superfamily (MSF) respectively. As shown in Fig. 2, these genes were expressed and induced by nickel in the Ni-tolerant *P. albus* isolate but not in the Ni-sensitive isolate. The expression of these genes was also detected in mycelia and ECM tissues that were sampled *in situ*. Both *in vitro* and *in situ* gene expression argue in favour of their role in nickel tolerance by metal efflux in *P. albus*. To test this hypothesis, these contigs were further used in heterologous expression experiments in yeast.

## RACE and gene expression in yeast

Contigs 17235 already showed a full-length sequence, while full sequences contigs of C3578 and C5339 were investigated and submitted into the EMBL database with accession numbers HG530066 and HG530067 respectively. Transformed yeast containing one of the three full-length contigs included transformant A: Pa-MFS (contig 17235), transformant B: Pa-ATPase (contig 3578) and transformant C: Pa-ABCtr (contig 5339). They were tested for their tolerance to nickel (i) using a drop test on solid medium and (ii) by following their kinetics in liquid medium. Figure 3 presents their responses to nickel on yeast nitrogen base (YNB) selective medium with and without nickel (1 mM). The data showed that transformants A, B, and C presented a gain of growth with nickel at 1 mM up to dilution 10E⁻³ compared with controls. The kinetics of growth in liquid medium at different nickel concentrations are presented in Fig. 4. Growth data analyses are summarized in Table 3 as growth rates (µ), the relationship between µ with nickel concentrations, and the determination of the nickel concentration that inhibited growth by 50% (Ni EC₅₀). All of these data showed that the expression of each contig provided a significant advantage in terms of growth rates (µ) in the presence of nickel; at 1 mM Ni, the mean of µ was enhanced significantly by 83% compared with controls (mean of µ of transformants A, B and C as reported in Table 3 compared with the mean of µ of the controls). In addition, the tolerance of the yeast transformants to nickel was improved; the mean of the Ni EC₅₀ was enhanced by 30.7% compared with controls. The nickel efflux of each transformant was also investigated, as shown in Fig. 5. Compared with controls, transformants A, B and C did not accumulate nickel in cells; at t = 90 min, a comparison of the means of nickel content in transformants A, B and C (3.05 ± 0.15 µmol Ni per
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** mRNA accumulation patterns of the nine genes putatively involved in metal efflux that were upregulated by nickel and selected using the 454 data (Table 2). Q-PCR mRNA patterns were tested in the Ni-tolerant *P. albus* isolate MD06-337; A: in mycelium cultivated *in vitro* without nickel or with nickel (+ 50 or + 250 µM) and compared Ni-sensitive *P. albus* isolate (MD09-078); B: in mycelium and ECM sampled *in situ*. Transcript accumulation was quantified by RT-qPCR using the 2^ΔΔCt^ method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and EF4α as reference genes for normalization and expressed as arbitrary units. The mean values were calculated from three technical replicates for each triplicate biological sample ± stdv and are indicated by bars.</figcaption></figure>

OD₆₀₀nm unit) with the means of the nickel content of the controls (7.20 ± 0.36 µmol Ni per OD₆₀₀nm unit) showed that the residual nickel content in yeast cells of the transformants (A, B and C) was significantly reduced by 57.6%. In addition, a comparison of the means of the residual nickel contents between time *t* = 0 min (3.89 ± 0.19 µmol Ni per OD₆₀₀nm unit) and *t* = 90 min (3.05 ± 0.15 µmol Ni per OD₆₀₀nm unit) in the transformants (A, B and C) were significantly reduced by 21.6%. In contrast, the mean of the residual nickel content for the controls was enhanced significantly by 92%.

The results of phenotypic tests and kinetics of the transformed yeast cells showed significant advantages in

terms of tolerance to nickel and growth compared with controls (Figs 3 and 4). In addition, the nickel efflux rates detected in the transformed yeast cells (Fig. 5) were similar to the nickel efflux rates detected in *P. albus*. Together, the transforming results of these genes in yeast support the metal efflux hypothesis. Various mechanisms were expected regarding metal tolerance, such as metal binding on the fungal wall and metal accumulation in the tissue or extra cellular chelation (Bellion *et al.*, 2006). Our data suggest nickel efflux as a main mechanism supporting nickel tolerance in *P. albus*, as recently reported in other ectomycorrhizal fungi, such as *Suillus bovinus* in response to Zn (Ruytinx *et al.*, 2013).
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Sensitivity of transformed *Saccharomyces cerevisiae* strains to nickel after 6 days of exposure. Each yeast strain was transformed with the empty vector (pYES) or the transformant constructions and plated in four serial dilutions onto YNB medium with (right column) or without (control, left column) NiSO₄. The concentration of nickel that was used in the spot assays is indicated above each row (1 mM). Plates were incubated at 30°C for 6 days.</figcaption></figure>

A. The BY4741 wild-type strain and three transformed yeast strains (transformant A: Pa-MFS, transformant B: Pa-ATPase and transformant C: Pa-ABCtr) on control and nickel-amended medium with 2% glucose. Abbreviation: Pa: *Pisolithus albus*.

Contig translation into protein

Table 4 summarizes the data on cDNA translation of the contigs into protein. The contig C3578 encodes for a protein belonging to the P-type ATPase family. As most of typical members of this protein family, this *P. albus* P-type ATPase contains 10 predicted trans-membrane segments and conserved sequence motifs such as TGES, CSDKTGT, MLTGD and GDGVN, within the catalytic region (see ESM Table 1). In plant and fungi, such protein, which contains a potential metal-sensitive enzyme system such as ATPase, is the first living functional structure that comes in contact with toxic heavy metals (Hall, 2002). The P-type ATPase proteins are described to form phosphorylated intermediate substrates and to pump a range of cations across cellular membrane including heavy metals (Hall and Williams, 2003). The contig C5339 encodes for an ABC transporter family member. In plants, such transporters are membrane-intrinsic primary-active pumps and are known to play a key role in many biological functions, such as heavy metal cell detoxification by sequestration in the

vacuole or exportation out of the cell (Wanke and Kolukisaoglu, 2010; Kretzschmar *et al.*, 2011; Lee *et al.*, 2014). In fungi, we can assume that the ABC protein acts as trans-membrane shuttle exporting heavy metals while transporting substrates into the cell. Contig 17235 encodes for a major facilitator protein permease (MFS). Major facilitator superfamily is one of the largest groups of secondary active transporters that are conserved from bacteria to humans. They transport a wide spectrum of substrates across bio-membranes and play a pivotal role in multiple physiological processes (Yan, 2013). In plants, MFS transporters respond to plant stresses, such as defending against infection by pathogens and exporting toxic compounds and heavy metals (Peng *et al.*, 2011). In this study, we noticed that the gene expression level of the contig C17235 was higher in mycelium sampled *in situ* than in the mycelium cultivated *in vitro* in presence of nickel. We hypothesize that C17235 gene expression might not specifically be upregulated by nickel but by all metals as found in the ultramafic substrate, i.e. cobalt, chromium, iron, nickel and manganese as reported in Majorel and colleagues (2012).

<table><caption>**Table 3.** Calculated exponential growth rates (10⁻³ µ are expressed in min⁻¹) based on the kinetics of the five *Saccharomyces cerevisiae* strains as shown in Fig. 4: BY4741 WT) strain and four yeast transformants: transformant A: Pa-MFS; transformant B: Pa-ATPase; transformant C: Pa-ABCtr, and the yeast transformed with the empty vector (pYES) and used as a control (Control-pYES).</caption><thead><tr><th rowspan="2">Yeast transformants</th><th colspan="4">Growth rates (10<sup>-3</sup> µ in min<sup>-1</sup>) at<br/>different nickel concentrations tested (µM)</th><th rowspan="2">Exponential regression<br/>parameters µ = f [(Ni)]</th><th rowspan="2">Ni EC<sub>50</sub> (µM)</th></tr><tr><th>Ni = 0</th><th>Ni = 250</th><th>Ni = 500</th><th>Ni = 1000</th></tr></thead><tbody><tr><td>BY4741 WT</td><td>4.8 ± 0.2ab</td><td>3.3 ± 0.2de</td><td>2.6 ± 0.8e</td><td>1.3 ± 0.9gh</td><td>y = 0.0047e<sup>-0.001x</sup>, r<sup>2</sup> = 0.99597</td><td>672 ± 31b</td></tr><tr><td>Transformant A: Pa-MFS</td><td>4.8 ± 0.3ab</td><td>3.9 ± 0.3cd</td><td>2.8 ± 0.6e</td><td>2.1 ± 0.4g</td><td>y = 0.0054e<sup>-0.001x</sup>, r<sup>2</sup> = 0.92556</td><td>811 ± 72ab</td></tr><tr><td>Transformant B: Pa-ATPase</td><td>5.1 ± 0.9a</td><td>4.8 ± 0.4ab</td><td>3.6 ± 0.2cd</td><td>2.4 ± 0.2f</td><td>y = 0.0060e<sup>-0.04x</sup>, r<sup>2</sup> = 0.9946</td><td>950 ± 192a</td></tr><tr><td>Transformant C: Pa-ABCtr</td><td>5.1 ± 0.7a</td><td>4.0 ± 0.2b</td><td>3.3 ± 0.4de</td><td>2.1 ± 0.2g</td><td>y = 0.0051e<sup>-0.04x</sup>, r<sup>2</sup> = 0.99896</td><td>770 ± 161ab</td></tr><tr><td>Control-pYES</td><td>4.9 ± 0.3ab</td><td>3.7 ± 0.2bc</td><td>2.2 ± 0.3fg</td><td>1.1 ± 0.3h</td><td>y = 0.0046e<sup>-0.001x</sup>, r<sup>2</sup> = 0.97492</td><td>630 ± 62b</td></tr></tbody></table>

The data are presented as the means ± standard deviation from three independent experiments. The different letters after the data indicate significant differences as determined by a Tukey's HSD test (P ≤ 0.05).
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Growth of *Saccharomyces cerevisiae* cultures exposed to different nickel concentrations. Time-course (in h) of the optical density at 600 nm. The studied *S. cerevisiae* strains included the wild-type (WT) strain BY4741 (♦), four-yeast transformed cells, i.e. transformant A: Pa-MFS (□), transformant B: Pa-ATPase (△), transformant C: Pa-ABCtr (X) and the wild-type strain BY4741 that contained the empty plasmid pYES and was used as control (control-pYES) (●).</figcaption></figure>

Altogether, our data and the protein structure analyses indicated that the three systems (i.e. P-type ATPase, ABC transporter and MFS) (i) are induced in the presence of nickel *in vitro*, (ii) expressed in *in situ* fungal tissues and (iii) are involved in nickel efflux in transformed yeast. They may act simultaneously in the fungi *P. albus* to export the

metal. These distinct and various mechanisms of metal efflux in *P. albus* could be involved in nickel tolerance and may contribute to the global metal homeostasis as an adaptive response to the ultramafic edaphic constraint, as suggested by Majorel and colleagues (2012). Further experiments such as (i) a comparison of metal accumu-

<table><caption>**Table 4.** Deoxyribonucleic acid full-length encoding contig sequences with their accession numbers in the EMBL database, translation into amino acids and prediction of (i) protein family identification, (ii) number of transmembrane domains and (iii) putative transport processes.</caption><thead><tr><th>Contig code</th><th>EMBL accession number</th><th>Size translation in amino acids</th><th>Protein family identification</th><th>Number of trans membrane domains</th><th>Putative transport process</th></tr></thead><tbody><tr><td>C17235</td><td>HG530065</td><td>515 aa</td><td>MFS family</td><td>14</td><td>Proton motive force (H+ anti-porter)</td></tr><tr><td>C3578</td><td>HG530066</td><td>940 aa</td><td>P-type ATPase</td><td>10</td><td>Electrochemical gradient</td></tr><tr><td>C5339</td><td>HG530067</td><td>727 aa</td><td>ABC transporter</td><td>6</td><td>Hydrolysis of ATP</td></tr></tbody></table>
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Nickel efflux in *Saccharomyces cerevisiae* cultures exposed to nickel at 1 mM. The time-course (in min) of residual nickel content is expressed in µmol of Ni per unit of optical density (OD) and was measured at 600 nm. The studied *S. cerevisiae* strains included the wild-type (WT) strain BY4741, four yeast transformed cells, i.e. transformant A: *Pa-MFS*, transformant B: *Pa-ATPase*, transformant C: *Pa-ABCtr* and the WT strain BY4741 that contained the empty plasmid pYES and was used as the control (control-pYES). Bars represent the means, and error bars represent the standard deviation of the mean (n = 3) of three independent experiments.</figcaption></figure>

lation and efflux in fungal tissues among metal tolerant and non-tolerant of other *P. albus* isolates, (ii) the construction of a GFP-fusion protein and its expression in yeast for each of sequences of the gene putatively involved in the metal efflux, and (iii) the use of metal radioelement when available (such as ⁶³Ni), should be carried out to confirm the metal efflux hypothesis as major mechanism in the fungal metal tolerance.

## Acknowledgements

This work was supported by (i) the GIP CNRT ‘Nickel and its Environment’ [grant number GIPCNRT98] and (ii) the National Agency for Research (ANR) Financial Support through the BIOADAPT Program 2012 [grant number ANR-12-ADAP-0017 ADASPIR]. We wish to thank L. Jamet and its colleagues for the chemical analyses (ICP-EOS) conducted at Laboratoire d’Analyses Chimiques (LAMA), Centre IRD de Nouméa, Nouvelle-Calédonie.

**Conflict of interest:** The authors declare having no conflict of interest.

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## Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher's web-site:

**Table S1.** Amino acid (aa) sequence of the translated contigs. In bold red: the domains that are conserved. Underlined in green: the trans-membrane domains. In blue: the putative walker B, ABC signature. Bold underlined: the metal binding site. Bold in black: the histidine (H) and the cysteine (C).

**Table S2A.** Primer sequences (Primer Express software), efficiency of the selected ESTs and the two reference genes (GAPDH and EF4α) that were used for qPCR analysis.

**Table S2B.** RACE primer DNA sequences.

**Table S2C.** Primer cloning DNA sequences.