Maria Del

Carmen Guerrero

Amandine Delteil

Kevin Garcia

Gabriella Houdinet

Geneviève Conejero

Isabelle Gaillard

Herve Sentenac

Sabine D Zimmermann
email: sabine.zimmermann@cnrs.fr

Carmen Guerrero-Galán

Genevieve Conéjéro

Hervé Sentenac

Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122

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Originality -Significance Statement

Nutrient transfer in ectomycorrhizal symbiosis requires specialized and bidirectional transport, ensuring uptake of mineral nutrients from the soil by fungal hyphae and secretion towards the plant root. Potassium nutrition has been shown to be improved in ectomycorrhizal plants but the molecular players involved are not yet completely uncovered. Tandem-pore outward-rectifying potassium (TOK) channels that are described in yeast to mediate K + efflux have been found specifically in fungi. Here, TOK channels are reported for the first time in an ectomycorrhizal fungus, and represent good candidates to be involved in symbiotic potassium transfer. Hebeloma cylindrosporum harbors three TOK channels belonging to two subfamilies with distinct properties and roles.

Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122

Introduction

Potassium (K + ) is the most abundant inorganic cation in plant cells, and participates in many processes, including regulation of osmotic pressure, energization of the plasma membrane and resistance to abiotic and biotic stresses [START_REF] Wang | The critical role of potassium in plant stress response[END_REF][START_REF] Benito | The twins K + and Na + in plants[END_REF]. Although it is one of the most important elements in the Earth crust, its availability for plant roots is limited due to its strong adsorption to soil particles [START_REF] Schroeder | Potassium in the soil/plant root system[END_REF]. Plants overcome the difficulty to acquire K + through various strategies, such as expression of high-affinity transport systems

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Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 4 and efficient storage capabilities [START_REF] Kochian | Potassium transport in roots[END_REF][START_REF] Anschütz | Common denominator of plant adaptive responses to environment[END_REF] as well as interaction with soil microorganisms (Garcia and Zimmermann, 2014).

The mycorrhizal symbiosis is a mutualistic interaction occurring between the roots of most land plants and hyphae of soil-borne fungi. This intimate relationship results in the formation of new structures in plant roots, the mycorrhizae, responsible for nutrient exchanges between both partners [START_REF] Smith | Mycorrhizal Symbiosis. 3rd Edn[END_REF]. Among the different types of mycorrhizal associations, the ectomycorrhizal symbiosis is a dominant component of temperate and boreal forests (Fransson et al., 2000). Ectomycorrhizal fungi represent up to 34% of all fungal biodiversity in the soil, where they play major roles in the functioning of these ecosystems [START_REF] Courty | Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum[END_REF][START_REF] Tedersoo | Ectomycorrhizal lifestyle in fungi: Global diversity, distribution, and evolution of phylogenetic lineages[END_REF]. Ectomycorrhizal fungi improve plant water and mineral nutrition, whereas the host provides the fungus with photosynthates.

Upon colonization, fungal hyphae penetrate the roots and form a specific structure between plant cortical cells, called the Hartig net. The Hartig net is responsible for nutrient and water exchanges between both partners through the specific expression of a wide range of transport proteins [START_REF] Casieri | Biotrophic transportome in mutualistic plant-fungal interactions[END_REF][START_REF] Garcia | Take a trip through the plant and fungal transportome of mycorrhiza[END_REF]. Characterization of fungal transport systems active at uptake or release sites in soil-exploring hyphae or at the interface between plant and fungus is a crucial prerequisite for understanding of these biological fluxes.

Although phosphorus and nitrogen are the main macronutrients shuttled by ectomycorrhizal fungi from the soil toward plant roots in many ecosystems [START_REF] Smith | Mycorrhizal Symbiosis. 3rd Edn[END_REF], recent studies have also highlighted a significant improvement of K + acquisition in colonized plants [START_REF] Jentschke | Interdependence of phosphorus, nitrogen, potassium and magnesium translocation by the ectomycorrhizal fungus Paxillus involutus[END_REF]Garcia et al., 2014;Garcia and Zimmermann, 2014).

Maritime pine (Pinus pinaster Ait.) is a forest-forming tree in Southern Europe typically found in coastal regions where it can tolerate unfavourable environments.

Furthermore, P. pinaster has a high economic value due to its exploitation as a softwood and resin source [START_REF]Indicateurs de gestion durable des forêts françaises métropolitaines[END_REF]. The ectomycorrhizal fungus Hebeloma cylindrosporum

Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122

(Basidiomycota) interacts with a broad range of woody plant species, including its natural host P. pinaster [START_REF] Marmeisse | Hebeloma cylindrosporum -A model species to study ectomycorrhizal symbiosis from gene to ecosystem[END_REF]. Both the plant and the fungus are well adapted to acidic sandy soils, like those present along the European Atlantic coast, an environment often suffering from salinity and nutrient deprivation (Lousteau et al., 1995;[START_REF] Gryta | Fine-scale structure of populations of the ectomycorrhizal fungus Hebeloma cylindrosporum in coastal sand dune forest ecosystems[END_REF][START_REF] Barbéro | Pines of the Mediterranean Basin[END_REF]. This tolerance is probably due to efficient nutrient absorption, transfer and storage abilities provided by the mutualistic association.

Molecular insights into the ectomycorrhizal fungus H. cylindrosporum became facilitated by the generation of an expressed sequence tag library [START_REF] Lambilliotte | Large-scale identification of genes in the fungus Hebeloma cylindrosporum paves the way to molecular analyses of ectomycorrhizal symbiosis[END_REF], and more recently the sequencing of its whole genome [START_REF] Kohler | Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists[END_REF] and transcriptomic analyses (Doré et al., 2015(Doré et al., , 2017)). This collection of data enabled the identification of putative channels and transporters that might be involved in K + transfer from the soil to the plant through ectomycorrhizal structures (Garcia and Zimmermann, 2014). We recently described one of these K + transporters, HcTrk1, which contributes to the uptake of K + from the soil (Corratgé et al., 2007;Garcia et al., 2014). In this study, we aim at functionally characterizing three tandem-pore outward-rectifying K + (TOK) channels from H. cylindrosporum, and investigating their role in ectomycorrhizal symbiosis.

The TOK channel family is specific to fungi. The first TOK channel was originally identified in yeast, ScTOK1, and functionally characterized by heterologous expression in Xenopus laevis oocytes, which revealed outward-rectifying K + -selective transport properties [START_REF] Ketchum | A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem[END_REF][START_REF] Lesage | A pH-sensitive yeast outward rectifier K + channel with two pore domains and novel gating properties[END_REF][START_REF] Reid | The S. cerevisiae outwardly-rectifying potassium channel (DUK1) identifies a new family of channels with duplicated pore domains[END_REF]. Previous studies in yeast had reported a depolarization-dependent outward current that was strongly selective for K + [START_REF] Gustin | Ion channels in yeast[END_REF][START_REF] Bertl | Gating and conductance in an outwardrectifying K + channel from the plasma membrane of Saccharomyces cerevisiae[END_REF]. Later on, TOK channels were found in filamentous fungi, including Neurospora crassa [START_REF] Roberts | TOK homologue in Neurospora crassa: First cloning and functional characterization of an ion channel in a filamentous fungus[END_REF], and in several other yeasts [START_REF] Ramos | Alkali-metal-cation influx and efflux systems in nonconventional yeast species[END_REF]. TOK polypeptides typically harbor eight transmembrane domains and two pore regions. The functional channel is expected to display a dimeric structure, where the four pore

This article is protected by copyright. All rights reserved.

Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 6 regions assemble in the centre of the protein to give rise to the channel pore representing the ion permeation pathway [START_REF] Baev | Killing of Candida albicans by human salivary histatin 5 is modulated, but not determined, by the potassium channel TOK1[END_REF][START_REF] Roberts | TOK homologue in Neurospora crassa: First cloning and functional characterization of an ion channel in a filamentous fungus[END_REF][START_REF] Prole | Identification and analysis of cation channel homologues in human pathogenic fungi[END_REF]. This structure suggests that TOK channels probably evolved from the fusion of two K + channels, a Shaker-like voltage-dependent subunit harboring six transmembrane segments and a subunit with two transmembrane segments like the inward rectifiers [START_REF] Lesage | A pH-sensitive yeast outward rectifier K + channel with two pore domains and novel gating properties[END_REF][START_REF] Bertl | Physiological characterization of the yeast plasma membrane outward rectifying K + channel, DUK1 (TOK1), in situ[END_REF][START_REF] Saldaña | Splitting the two pore domains from TOK1 results in two cationic channels with novel functional properties[END_REF] or the bacterial KcsA channel (Doyle et al., 1998). However, in contrast to Shaker-like channels, no voltage-sensor could be identified in TOK channels [START_REF] Ketchum | A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem[END_REF][START_REF] Lesage | A pH-sensitive yeast outward rectifier K + channel with two pore domains and novel gating properties[END_REF].

Two subfamilies of TOK channels have been found in the genome of many ectomycorrhizal fungi, like in H. cylindrosporum, but not in the arbuscular mycorrhizal fungus Rhizophagus irregularis (Garcia and Zimmermann, 2014). Given the fact that their homologues in yeast and N. crassa behave as outward-rectifying K + channels allowing K + efflux [START_REF] Ketchum | A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem[END_REF][START_REF] Lesage | A pH-sensitive yeast outward rectifier K + channel with two pore domains and novel gating properties[END_REF][START_REF] Roberts | TOK homologue in Neurospora crassa: First cloning and functional characterization of an ion channel in a filamentous fungus[END_REF][START_REF] Ariño | Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum[END_REF], it was tempting to assume that members from these subfamilies in ectomycorrhizal fungi could contribute to K + secretion from the fungal Hartig net into the plant apoplasm. Here, we describe three TOK channels from the ectomycorrhizal fungus H. cylindrosporum. Their sequences have been analyzed in silico and they have been functionally characterized by heterologous expression and voltage-clamp experiments in oocytes, and complementation of a yeast mutant strain defective in K + transport systems. We also analyzed their expression patterns in ectomycorrhizae by in situ hybridization. Their role in axenic and symbiotic conditions was studied by the phenotyping of transgenic fungal lines overexpressing these proteins.

Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122

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Results

In silico analysis of TOK channels from the ectomycorrhizal fungus H. cylindrosporum

Putative TOK channel genes belonging to two subfamilies have been identified in the genome of ectomycorrhizal fungi (Garcia and Zimmermann, 2014). In H. cylindrosporum, the TOK family comprises three members, named HcTOK1, HcTOK2.1 and HcTOK2.2. Their in silico predicted secondary structure highlighted a major difference between HcTOK1 and the two HcTOK2-type channels in the intracellular domain that connects the two parts of the protein harboring each one pore domain, the Shaker-like moiety (six transmembrane segments) and the inward rectifier-like moiety (two transmembrane segments) (Fig. 1A). This cytosolic loop is about 150 amino acids longer in HcTOK2.1 and HcTOK2.2 compared to HcTOK1. In contrast, HcTOK2.1 and HcTOK2.2 harbored a shorter C-terminal domain than HcTOK1 resulting in a similar total length for all three proteins (around 780 amino acids). Comparison of protein sequences between H. cylindrosporum, S. cerevisiae and N. crassa revealed that

HcTOK2.1 and HcTOK2.2 share about 30 % of similarity with ScTOK1 and NcTOKA (Fig. 1B). HcTOK1 shares the same level of similarity, ca. 30%, with ScTOK1, as well as with

HcTOK2.1 and HcTOK2.2, but a higher level of similarity, ca. 42%, with NcTOKA. Also, the two pore sequences display differences between species, particularly in the hallmark motif of K + -selective channels, TxGYGD, which is not strictly conserved (Fig. 1C). The pore P1 is the most variable because the position corresponding to the core tyrosine residue is occupied by a leucine in HcTOK1, like in ScTOK1, or by a phenylalanine in HcTOK2.1 and HcTOK2.2, like in NcTOKA. Concerning the second pore, the P2 sequence of HcTOK1 is more similar to that of ScTOK1or NcTOKA than to that of HcTOK2.1 or HcTOK2.2.

Putative TOK channels were found in basal fungi belonging to the Neocallimastigomycota, Blastocladiomycota, Chytridiomycota, Zoopagomycota and Mucoromycota phyla, as well as in Basidiomycota and Ascomycota, but not in the This article is protected by copyright. All rights reserved.

Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122

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Glomeromycotina Rhizophagus irregularis (Fig. 2; Garcia and Zimmermann, 2014). The phylogenetic analysis of the TOK sequences divides them into three main clades: an ancestral clade containing basal fungi, a TOK1 subfamily present only in Ascomycota and

Basidiomycota species, and a Basidiomycota-specific clade to which the newly-described TOK2 subfamily belongs (Fig. 2). In summary, this analysis enlarges our previous phylogenetic studies (Garcia and Zimmermann, 2014) and indicates that HcTOK2.1 and HcTOK2.2 are members of a subfamily of TOK proteins that had not been described in fungi thus far.

HcTOK1 and HcTOK2.1 exhibit an outward-rectifying channel activity in Xenopus laevis oocytes

In order to figure out whether the HcTOK1, -2.1 and -2.2 channels are able to transport K + , their activity was investigated by two-electrode voltage-clamp experiments in X. laevis oocytes. Different membrane potentials were imposed to the oocytes (Fig. 3A) and the corresponding currents were recorded. First, oocytes were tested in 1 mM external K + at pH 6.5. Ion channel activity could be detected in oocytes expressing HcTOK1 (Fig. 3C) or

HcTOK2.1 (Fig. 3D), where it gave rise to outwardly directed currents upon membrane depolarization, but not in oocytes injected with HcTOK2.2 cRNA (Fig. 3E), which displayed no difference in comparison with the control oocytes (Fig. 3B). Both channels displayed an instantaneous and a time-dependent activation as observed for the yeast channel [START_REF] Lesage | A pH-sensitive yeast outward rectifier K + channel with two pore domains and novel gating properties[END_REF]. However, the time dependent component of activation differed remarkably, the current activation being slower for HcTOK2.1 than for HcTOK1. Steady-state current-voltage curves (I-V) indicated much larger current amplitudes in HcTOK1-than in HcTOK2.1expressing oocytes in these conditions (Fig. 3F, Fig. S1).

Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122

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To further characterize the activity of HcTOK1 and HcTOK2.1, bath solutions containing either 1, 10 or 100 mM K + at pH 6.5, or three pH conditions (pH 5.5, 6.5 or 7.5) in 1 mM K + were used. These analyses were also performed with HcTOK2.2, but currents different from control oocytes were never recorded (Fig. S1). To summarize, HcTOK1 and HcTOK2.1 behaved as outward-rectifying channels whose currents were enhanced by low external K + concentrations shifting activation to more negative potentials (Fig. 3G andH). A slight inward current was observed for HcTOK1 at hyperpolarizing potentials (Fig. 3G) as previously observed for NcTOKA and ScTOK1 [START_REF] Roberts | TOK homologue in Neurospora crassa: First cloning and functional characterization of an ion channel in a filamentous fungus[END_REF][START_REF] Roller | Functional consequences of leucine and tyrosine mutations in the dual pore motifs of the yeast K + channel, Tok1p[END_REF]. The reversal potential shifted with the decrease of external K + concentrations towards more negative potentials as detectable in I-V curves for HcTOK1 (Fig. 3G) indicating selectivity for K + . However, tail current analyses revealed that this shift did not follow completely the K + Nernst potential, especially at 1 mM K + (Fig. S2 A and B), suggesting either permeability to other ions such as Na + which replaced external K + in this experiment or a reduced sensitivity of K + dependent gating at low K + as observed for NcTOK [START_REF] Roberts | TOK homologue in Neurospora crassa: First cloning and functional characterization of an ion channel in a filamentous fungus[END_REF]. Nevertheless, replacing 100 mM KCl in the bath solution by 100 mM NaCl allowed the observation of large K + outward currents as predicted for a reduced external K + concentration (Fig. S2 C andD).

Concerning HcTOK1, no effect of external pH was observed (Fig. 3I), similarly to ScTOK1 [START_REF] Lesage | A pH-sensitive yeast outward rectifier K + channel with two pore domains and novel gating properties[END_REF]. In contrast, HcTOK2.1 was regulated by the external pH, the outward currents being increased by acidification of the external solution from 7.5 to 6.5 and 5.5 (Fig. 3J, Fig. S1). Altogether, these results revealed that HcTOK1 displays functional properties reminiscent of the yeast outward-rectifying K + channel ScTOK1, and that HcTOK2 behaves also as an outward-rectifying channel but with distinct functional features, at least in terms of pH sensitivity, kinetics and rectification capacity. or PLY246-ScTOK1). These yeast strains were tested for functional growth complementation by serial drop assays (Fig. 4). The PLY246 strain transformed with the control vector (PLY246-pYES2) was able to grow at high (50 mM) but not at low (10 mM) K + , confirming its defective phenotype due to the lack of K + transport systems [START_REF] Bertl | Characterization of potassium transport in wild-type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations[END_REF]. Cells transformed with the positive control vector containing ScTOK1 (PLY246-ScTOK1) were able to grow on both media. Yeast cells transformed with the HcTOK1 channel (PLY246-HcTOK1) were also able to grow at 10 mM K + , although at a rate lower than that of the PLY246-ScTOK1 cells. Such a capacity of HcTOK1 to partially complement the defect in K + uptake of the PLY246 strain, indicating actually K + influx into the cells, is consistent with the fact that this transporter can mediate small inward currents observed at hyperpolarizing voltages in oocytes (Fig. 3G). On the contrary, HcTOK2.1 did not complement the PLY246 mutant strain, indicating a lack of absorption activity. This result was also consistent with the voltage-clamp recordings highlighting a strict outward rectification capacity of HcTOK2.1 (Fig. 3H). Interestingly, yeast cells transformed with the vector containing HcTOK2.2 were able to grow at 10 mM K + , and displayed a growth rate on this medium similar to that of the positive control PLY246-ScTOK1 (Fig. 4). This result indicates that HcTOK2.2 encodes a functional polypeptide endowed with K + transport activity allowing K + influx across the yeast cell membrane.

Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122

11

The three TOK genes from H. cylindrosporum have different expression patterns in pine ectomycorrhizae

Recently, transcriptional studies in axenic and symbiotic conditions revealed changes in the expression of H. cylindrosporum genes (Doré et al., 2015(Doré et al., , 2017)). Based on these results, the expression levels of the three HcTOK channel genes were compared between both conditions (Fig. S3). HcTOK1 and HcTOK2.1 expression levels did not undergo major changes.

Strikingly, HcTOK2.2 was more than 30 times induced upon establishment of the symbiotic interaction, suggesting a specific role in ectomycorrhizae.

The spatial expression patterns of the three HcTOK genes were investigated in ectomycorrhizae. In situ hybridization experiments were performed on colonized P. pinaster roots using either sense or antisense ribosomal probes as negative and positive controls, respectively, or probes for the three TOK channels (Fig. 5). As expected, concerning the control probes, signals (blue coloration) were observed in samples treated with the antisense ribosomal probe, but not with the sense one (Fig. 5A andB). Similarly, concerning the three TOK channels, hybridization with the antisense probes gave rise to signals (Fig. 5C, E and G), while using the sense probes did not (Fig.

5D, F and H). The signals indicated that

HcTOK1 was highly expressed in extra-radical hyphae, the sheath and the Hartig net (Fig. 5C). In contrast, HcTOK2.1 was barely expressed and only inside the root (Fig. 5E). H. cylindrosporum, we generated transgenic overexpressing fungal lines. Among the lines produced, the two displaying the highest overexpression (OE) levels were selected for each gene after real-time PCR screenings (Fig. S4). These transgenic fungal lines were tested in symbiosis with P. pinaster (Fig. 6 A) along with the corresponding wild-type line (h7) and a transgenic line transformed with the empty pPZP133 vector (CT, control). Prior to the harvest, the remaining liquid medium from each tube was collected. The K + concentration in the culture medium was decreased in six days from 1 mM right after the last watering to approximately 100 to 150 µM in six days (Fig. 6 B).

The total amounts of K + assayed in the plants (roots and shoots) were higher in the plants inoculated with the TOK1-OE2, the line in which the transforming construct resulted in a very high increase in expression level, and in the plants inoculated with the TOK2.1-OE3 and TOK2.2-OE2 and -4 lines, than in the control plants (Fig. 6 C). Expressed on a dry weight basis, the major differences in K + contents were displayed by the shoots rather than by the roots in seedlings colonized by TOK2.1-OE3 and TOK2.2-OE2 and -4 lines (Fig. 6D).

Finally, when grown in axenic fungal cultures, only the TOK1-OE2 line contained significantly more K + than the control lines h7 and pPZP133 (Fig. S5). These results suggest that seedlings inoculated with TOK1-OE2 accumulated more K + because of a higher K + content in the fungus, whereas the effect observed in the seedlings colonized by TOK2.1-OE3 and TOK2.2-OE2 and -4 lines was due to an enhanced efflux of K + towards the plant. The present results indicate that two from the three TOK channels from H. cylindrosporum, HcTOK1 and HcTOK2.1, can be expressed heterologously in a functional state in X. laevis oocytes, allowing electrophysiological analyses. The reasons why the activity of the third one, HcTOK2.2, is undetectable in this expression system might be linked to e.g. inappropriate cytosolic conditions in oocytes or missing regulatory proteins. For instance, several plant K + channels from the Shaker family remain electrically silent when expressed in oocytes except when they are co-expressed with CBL-CIPK phosphorylating partners [START_REF] Xu | A protein kinase, interacting withtwo calcineurin B-like proteins, regulates K + transporter AKT1 in Arabidopsis[END_REF]Cuéllar et al., 2013). Nonetheless, expression in mutant yeast cells defective for K + transport provides evidence that HcTOK2.2 is permeable to K + (Fig. 5).

No member from the TOK2-type subfamily had been characterized so far. The analysis of HcTOK2.1 in oocytes provides the first information on such channels. Overall, the functional properties of this channel, as outward rectification and selectivity for K + , are reminiscent of those of its H. cylindrosporum HcTOK1 relative, as well as of those of ScTOK1 and NcTOKA from S. cerevisiae and N. crassa [START_REF] Ketchum | A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem[END_REF][START_REF] Bertl | Characterization of potassium transport in wild-type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations[END_REF][START_REF] Roberts | TOK homologue in Neurospora crassa: First cloning and functional characterization of an ion channel in a filamentous fungus[END_REF]. Even that all three HcTOK channels did not display pH-dependent expression levels (Fig. S6), the unique sensitivity of HcTOK2.1 to external pH at the functional level could allow the fungal cells to adapt to pH changes in the soil, the fungal sheath and/or the Hartig net. Indeed, the currents induced by HcTOK2.1 were stronger at pH 5.5 than 6.5 or 7.5, meaning that acidification of the external medium in a physiological range of pH values can enhance the activity of this channel. Moderately acid pH can be expected in forest soils and has been reported for the Landes forest [START_REF] Jolivet | Les sols du massif forestier des Landes de Gascogne: formation, histoire, propriétés et variabilité spatiale[END_REF]. Interestingly, acidification and pH signaling has been described for ectomycorrhizal roots in Eucalytus globulus-Pisolithus microcarpus interaction [START_REF] Ramos | A pH signaling mechanism involved in the spatial distribution of calcium and anion fluxes in ectomycorrhizal roots[END_REF]. Regulation of the TOK Outward rectification was analyzed and controversially discussed for the S. cerevisiae channel TOK1. Two models for the possible gating mechanism have been proposed

suggesting either an external (presence of other cations) or a protein-intrinsic voltagedependent block [START_REF] Ketchum | A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem[END_REF][START_REF] Lesage | A pH-sensitive yeast outward rectifier K + channel with two pore domains and novel gating properties[END_REF]. Further analyses of this ion channel rectification would be interesting for the new fungal channels from H.

cylindrosporum. Furthermore, distinct behavior found here for HcTOK1 and HcTOK2.1 with respect to the channel activation kinetics, pH dependence and outward rectification could allow addressing questions concerning the corresponding molecular bases and physiological meaning of these differences.

Towards the physiological role of TOK channels in ectomycorrhizal symbiosis

Different types of K + and/or Na + transport systems have been identified in yeasts, amongst which the TOK channels are considered as the sole systems dedicated to K + -selective secretion [START_REF] Ariño | Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum[END_REF][START_REF] Ramos | Alkali-metal-cation influx and efflux systems in nonconventional yeast species[END_REF]. In yeasts, TOK channels have been shown

to be involved in control of the cell membrane potentials and in apoptosis mechanisms by allowing K + efflux [START_REF] Ramos | Alkali-metal-cation influx and efflux systems in nonconventional yeast species[END_REF]. Although no more precise function has been described thus far, the conservation of the TOK family across the fungal kingdom provides evidence of its importance in fungal physiology [START_REF] Bertl | Characterization of potassium transport in wild-type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations[END_REF][START_REF] Maresova | Measurements of plasma membrane potential changes in Saccharomyces cerevisiae cells reveal the importance of the Tok1 channel in membrane potential maintenance[END_REF][START_REF] Roller | Functional consequences of leucine and tyrosine mutations in the dual pore motifs of the yeast K + channel, Tok1p[END_REF][START_REF] Zahrádka | Plasma-membrane hyperpolarization diminishes the cation efflux via Nha1 antiporter and Ena ATPase under potassium-limiting conditions[END_REF].

Overexpression of HcTOK channel genes in H. cylindrosporum was poorly informative on the roles of the corresponding proteins in the free-living fungal mycelia grown in axenic cultures, except for the TOK1-OE2 line, regarding fungal K + content (Fig. S5). 16 increase of fungal internal K + contents. Such an effect is consistent with the fact that the rectification provided by HcTOK1 is not absolute, allowing this channel to mediate K + influx in oocytes (Fig. 3) as well as in yeast (Fig. 4). Another hypothesis would be that the strong overexpression of HcTOK1 affects the cell membrane potential, shifting the potential towards more negative values and thereby increasing the driving force for K + uptake. It is worth to note that, in S. cerevisiae, tok1 KO mutant yeast cells do not display significant changes in K + contents, K + (Rb + ) uptake and K + efflux [START_REF] Bertl | Characterization of potassium transport in wild-type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations[END_REF].

Intriguingly, the very strong overexpression of

Here we have investigated the roles of HcTOK channels in ectomycorrhizal symbiosis.

The colonization of P. pinaster roots by the fungal lines overexpressing the HcTOK channel genes resulted in higher K + amounts in the plant, in one line out of two in the cases of HcTOK1 and HcTOK2.1, and in both lines in the case of HcTOK2.2. Such an effect is consistent with the fact that TOK channels can mediate K + efflux, a property that can enable them, when ectopically overexpressed, to contribute to K + transfer towards the host plant. It is worth to note that the increase in the plant K + amounts appeared to be essentially due to increased K + contents in shoots (Fig. 6C &D). In other words, the K + translocation towards the shoots was stimulated in the plants colonized by the fungal strains overexpressing the TOK channel genes, suggesting an enhanced K + efflux from the fungus towards the plant.

HcTOK2.2 is a promising candidate for K + movements in ectomycorrhizae

With respect to the fungal capacity to transfer K + to the host plant, the whole set of results supports the hypothesis that a single TOK channel is principally dedicated to this function, 17 plant, particularly in the aerial parts, suggesting an increased K + translocation from the mycorrhizal roots to the shoot. [START_REF] Bücking | Subcellular compartmentation of elements in nonmycorrhizal and mycorrhizal roots of Pinus sylvestris: an X-ray microanalytical study. II. The distribution of calcium, potassium and sodium[END_REF] reported that, in some conditions, K + can be retained by the hyphae of the Hartig net, without a transfer to the host cortical cells.

Therefore, the effect we observed here could be due to a decrease of this K + retention in the mycelium due to the overexpression of HcTOK2.2.

In contrast, HcTOK1 expression was detected in all hyphae forming the ectomycorrhizae, without any specific localization, and HcTOK2.1 transcripts were barely labeled in our in situ hybridization experiments probably due to a reduced expression level within ectomycorrhizae (Fig. S3). Furthermore, the transcript levels of these two channels were not enhanced upon mycorrhizal association. Altogether, these results indicate that HcTOK1 and HcTOK2.1 are not essentially dedicated to K + secretion in the Hartig net towards the cortical cells of the host roots, but are involved in fungal K + homeostasis through the control of K + efflux and partly of K + influx, as already proposed for ScTOK1 [START_REF] Maresova | Measurements of plasma membrane potential changes in Saccharomyces cerevisiae cells reveal the importance of the Tok1 channel in membrane potential maintenance[END_REF].

Conclusion

In summary, our results indicate that the three TOK channels from H. cylindrosporum display specific rather than redundant functions. HcTOK1 is closer to the previously characterized ScTOK1 and NcTOKA than the other two channels and could play a role in the basal K + homeostasis of the fungus. Moreover, we described and characterized for the first time another subfamily of TOK channels. Both TOK2 members of H. cylindrosporum display original properties compared to HcTOK1, which could help in investigating the structurefunction relationships of these channels. The first member from the H. cylindrosporum TOK2 subfamily, HcTOK2.1, is not likely to play a major role in K + secretion towards the host plant, but could provide a pH sensing mechanism in the regulation of K + homeostasis. In

This article is protected by copyright. All rights reserved.

Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 18 contrast, the strong induction of the second one, HcTOK2.2, upon mycorrhizal symbiosis, its preferential localization in the Hartig net, and the fact that its overexpression results in higher accumulation of K + in colonized plants provide evidence for a role of this channel in K + secretion towards the host root in mycorrhizal symbiosis.

Experimental Procedures

Fungal materials and cultures

The H. cylindrosporum (Romagnesi) monokaryotic strain h7 used in our studies was isolated from the germination of a basidiospore by Debaud and Gay (1987). Fungal cultures were maintained on solid YMG medium [START_REF] Rao | Carbohydrate metabolism during morphogenesis of Coprinus lagopus (sensu Buller)[END_REF] at 26ºC in the dark.

The mycelia were grown on N6 medium [START_REF] Louche | Efficiency of acid phosphatases secreted from the ectomycorrhizal fungus Hebeloma cylindrosporum to hydrolyse organic phosphorus in podzols[END_REF]6 For pH-assays, culture medium pH was adjusted additionally to pH 4.5, 6.5, and 7.5 by HCl or Ca(OH) 2 . Fungal transgenic lines were obtained by Agrotransformation using Agrobacterium tumefaciens LBA1126 as described by [START_REF] Ngari | The dominant Hc.Sdh R carboxin-resistance gene of the ectomycorrhizal fungus Hebeloma cylindrosporum as a selectable marker for transformation[END_REF] and [START_REF] Garcia | Promoter-dependent expression of the fungal transporter HcPT1.1 under Pi shortage and its spatial localization in ectomycorrhiza[END_REF].

Ectomycorrhization assays

Plant seeds (P. pinaster Soland in Ait. From Medoc, Landes-Sore-VG source, France) were sterilized in 30% H 2 O 2 for 15 min, rinsed with sterile water, hydrated at 4ºC for 48-72 h and germinated on solid medium (2 g l -1 glucose, 10 g l -1 agar) for two weeks. The device used for co-cultures was described by [START_REF] Plassard | Nitrate uptake in maritime pine (Pinus pinaster) and the ectomycorrhizal fungus Hebeloma cylindrosporum: effect of ectomycorrhizal symbiosis[END_REF]. Briefly, four implants of

Cloning and in silico analyses

Total RNAs were extracted from 2-week old H. cylindrosporum axenic cultures grown in N6 medium by tissue grinding in liquid nitrogen, followed by a TRIzol® Reagent purification (Invitrogen, Carlsbad, CA, USA). RNAs were treated with DNAse I (Invitrogen), checked for integrity by electrophoresis in 1 % agarose gel, and retrotranscribed to cDNA using the Superscript III enzyme (Invitrogen). Primers with flanking SpeI restriction sites were designed to amplify the sequences coding for HcTOK1 (Protein ID 31571), HcTOK2.1

(Protein ID 129509) and HcTOK2.2 (Protein ID 127201) (H. cylindrosporum h7 genome database v2.0; http://genome.jgi-psf.org/Hebcy2/Hebcy2.home.html; Table S1). A poly-A tail was added to the product of the previous reaction and the construct was inserted into a TOPO-TA cloning vector (Invitrogen). The HcTOK coding sequences were amplified by PCR with primers with flanking HindIII (for HcTOK1) or EcoRI (for HcTOK2.1 and HcTOK2.2) restriction sites and cloned in the Zero Blunt® vector (Invitrogen) (Table S1).

For the generation of HcTOK1, HcTOK2.1 and HcTOK2.2 overexpressing (OE) fungal lines, three plasmids named pPZP-OE-TOK1 or -2.1 or -2.2 were constructed by digestion of the TOPO-TA-TOK1, -2.1 or -2.2 vectors using SpeI and ligation into the pPZP-Pgpd vector [START_REF] Ngari | The dominant Hc.Sdh R carboxin-resistance gene of the ectomycorrhizal fungus Hebeloma cylindrosporum as a selectable marker for transformation[END_REF][START_REF] Garcia | Promoter-dependent expression of the fungal transporter HcPT1.1 under Pi shortage and its spatial localization in ectomycorrhiza[END_REF].

This article is protected by copyright. All rights reserved. In silico analysis of the secondary structure was performed with the online TMHMM software [START_REF] Krogh | Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes[END_REF] and the predicted structures were represented with the Protter website tool [START_REF] Omasits | Protter: interactive protein feature visualization and integration with experimental proteomic data[END_REF]. The phylogenetic analysis was done by choosing TOK protein sequences from representative members of the publicly available fungal tree (https://genome.jgi.doe.gov/programs/fungi/index.jsf; [START_REF] Grigoriev | Fueling the future with fungal genomics[END_REF]2014), aligning them using MUSCLE and Gblocks, building the tree with SeaView 4 [START_REF] Gouy | SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF], and calculating bootstrap values using the PhyML method [START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF]. The phylogenetic tree was created using iTOL tool.

In situ hybridization

Two PCR reactions on cDNA were performed to obtain 350 bp sense and antisense probes for the in situ detection of HcTOK1, -2.1 and -2.2 transcripts, as described by Garcia et al.

(2014), using specific sets of primers (Table S2). PCR products were purified by ethanol precipitation and in vitro transcription was made using the MAXIscript® T7 Transcription Kit (Invitrogen) to synthetize digoxigenin-UTP-labelled RNA probes. The hybridization and detection of the probes were performed on 8 µm-thick sections of mycorrhizal roots fixed in paraffin as described by Garcia et al. (2014). Images were taken on a wide-field microscope Leica DM6000 (Leica Microsystems, Wetzler GmbH, Germany) and analyzed with the Volocity Acquisition 5.1.0 software (Perkin Elmer, Boston, MA, USA) and FiJi [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF].

Electrophysiological analysis

In order to investigate the functional properties of the HcTOK1, -2.1 and -2.2 channels, the two-electrode voltage-clamp (TEVC) experiments were performed using X. laevis oocytes.

The TOPO-TA plasmids containing the HcTOK1, -2.1 or -2.2 coding sequences were

This article is protected by copyright. All rights reserved. MgCl 2 , 5 HEPES, 2.5 sodium pyruvate; supplemented with 50 μg ml -1 gentamycin; pH 7,4) at 18ºC for 3 days before TEVC recordings.

The bath solutions that were percolated contained 2 mM MgCl 2 , 1 mM CaCl 2 and 10 mM HEPES-Tris, pH 6.5. They were supplemented with K + and Na + provided as gluconate salts, the concentration of K + being 1, 10 or 100 mM, and the sum of the concentrations of the two cations K + and Na + being 100 mM. The effect of the external pH on TOK channel activity was investigated with bath solutions at pH 5.5 (the HEPES buffer present in the other bath solutions being then substituted by MES buffer), 6.5 and 7.5. The TEVC protocol started at a holding potential of -60 mV and then imposed voltage pulses to the oocyte membrane from hyperpolarizing potentials (-100 mV or -60 mV) to depolarizing potentials (+80 mV) in steps of +10 mV. Voltage-clamp protocols, measurements and data analyses were performed using the pClamp10 software (Axon Instruments, Foster City, CA, USA).

Functional complementation in yeast

Saccharomyces cerevisiae strains PLY232 (WT) and PLY246 (trk1∆, trk2∆, tok1∆) were used for yeast complementation assays [START_REF] Bertl | Characterization of potassium transport in wild-type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations[END_REF] PLY232 (WT) strain was transformed with the empty pYES2 for use as control. Functional complementation for K + uptake was tested by serial drop assays. Overnight cultures of yeast were rinsed with sterile water and diluted to an OD 600 of 1. Three tenfold serial dilutions were prepared and 10 µl-drops were plated onto selective agar medium containing either 10 or 50 mM KCl, added in the arginine-phosphate medium described by Rodríguez-Navarro and [START_REF] Rodriguez-Navarro | Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum[END_REF].

Real-time quantitative PCR analyses

Total RNAs were extracted from 2-week old cultures of wild-type and transgenic lines of H. cylindrosporum and transcribed to cDNA. Real-time PCRs were performed in the LightCycler® 480 Real-Time system (Roche, Mannheim, Germany) using a reaction mix containing 10 ng cDNA, 5 µl SYBR® Premix Ex Taq ™ II Tli RNaseH Plus (TaKaRa Bio Inc., Dalian, China) and 0.5 µM of each forward and reverse primer (Table S3). The amplification protocol had the following steps: 95ºC for 30 s, 45 cycles of [95ºC for 10 s, 60ºC for 10 s, 72ºC for 15 s] and cooling to 40ºC for 40 s. Two housekeeping genes were used for data normalization, α-tubulin and elongation factor 1α (Protein IDs 24108 and 439881; http://genome.jgi-psf.org/Hebcy2/Hebcy2.home.html) (Table S3). Data analyses were performed as described by Cuéllar et al. (2010).

Phenotyping of transgenic fungal lines in axenic and symbiotic conditions

Fungi were cultured in N6 liquid medium in axenic conditions for two weeks. Mycelia were then sampled, washed twice in water for 1 min, weighed, dried at 60ºC for 7 days and weighed again. K + ions were extracted from the mycelia by 0.1 N HCl (5 ml per sample) for

Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 23 seedlings were weighed and dried at 60ºC for 14 days. Acid extraction was performed with 5 ml of HCl 6 N at 110ºC for 16 h. K + contents in fungal and plant tissues were determined by atomic absorption spectrometry (SpectrAA 220 FS, Varian).

Statistical analyses

The statistical treatment of the data was performed with R Studio with default parameters (RStudio Team, 2015). Data were analyzed with the Kruskal-Wallis and Wilcoxon-Mann-Whitney tests.

Comment with bootstrap support of over 500 (out of 1000 replicates) are indicated with grey dots. The radial tree was generated using iTOL v4.0.3 (http://itol.embl.de/) [START_REF] Letunic | Interactive tree of life (iTOL) v3: an online tool for the Comment citer ce document[END_REF].

Table and

Basal fungi have an ancestral version of the TOK channels. Channels belonging to the TOK1 subfamily are highlighted with branches in green and channels belonging to the TOK2 subfamily with branches in magenta. Bold characters indicate the Basidiomycota branches.

The TOK1 subfamily is present in Ascomycota and Basidiomycota fungi, whereas the TOK2 subfamily is specific of the latter clade. The list of species used is shown in Table S4. The three TOK channels of H. cylindrosporum are marked in red and other channels described previously are indicated in blue, namely ScTOK1 [START_REF] Ketchum | A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem[END_REF], NcTOKA (Roberts, This article is protected by copyright. All rights reserved.

Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 32 2003), CaTOK1 [START_REF] Baev | Killing of Candida albicans by human salivary histatin 5 is modulated, but not determined, by the potassium channel TOK1[END_REF], as well as HwTOK1A and HwTOK2A [START_REF] Plemenitaš | Adaptation to high salt concentrations in halotolerant/halophilic fungi: a molecular perspective[END_REF]. Units of branch length ("Tree Scale") are in amino acid substitutions per site. Table S1. used for amplification and cloning of the three TOK channels from Hebeloma cylindrosporum.

Table S2. Primers used for in situ hybridization experiments on Hebeloma cylindrosporum -Pinus pinaster ectomycorrhizae.

Table S3. Primers used for Real-time PCR experiments.

Table S4. Sequences used in the phylogenetic tree and abbreviations of their names and species. determined in H. cylindrosporum (h7) mycelia grown for two weeks in liquid cultures at pH 4.5, 5.5, 6.5 or 7.5. Note that the expression level of HcTOK1 was very low in all conditions (8-28 copies per ng of total RNA; cf. also Fig. S4) explaining differences in the relative expression. For each TOK gene, the lowest expression level that was observed was used for normalization and given the value of 1. n=3 pools of 2 fungal cultures.

This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 10 Growth complementation of yeast mutants lacking K + transport systems The S. cerevisiae strain PLY246 defective for K + transport was transformed with the empty pYES2 plasmid as control, or with pYES2 constructs containing either the HcTOK1, HcTOK2.1, HcTOK2.2 or ScTOK1 coding sequences (the corresponding yeast transformants being named PLY246-pYES2, PLY246-HcTOK1, PLY246-HcTOK2.1, PLY246-HcTOK2.2

  Finally, the signals resulting from hybridization with the HcTOK2.2 antisense probe were most apparent in the Hartig net, faint in the fungal sheath surrounding the root, and not detectable in extra-radical hyphae (Fig. 5G). Overall, the expression studies indicate that HcTOK2.2 was strongly induced and displayed the highest expression level, amongst the three HcTOK channel genes, when the fungus engaged in ectomycorrhizal symbiosis. The corresponding HcTOK2.2 transcripts were This article is protected by copyright. All rights reserved. Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 12 specifically present in hyphae internally colonizing the root, within the Hartig net, suggesting a function at this interface between fungus and plant. Analysis of symbiotic in vitro co-cultures of Pinus pinaster and HcTOK overexpression lines Aiming at analyzing the physiological role of the three TOK channels from

  Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 13 Discussion The TOK channel family represented in the ectomycorrhizal fungus H. cylindrosporum by three distinct members Members of the fungal-specific TOK channel family are present in most of the Basidiomycota and Ascomycota species that have been sequenced so far, and also in more basal taxa, including Zygomycota, Neocallimastigomycota and Chytridiomycota (Garcia and Zimmermann, 2014; Fig. 2). Three TOK channel genes were found in the genome of the ectomycorrhizal fungus H. cylindrosporum. The two recently identified TOK-subfamilies, TOK1 and TOK2, are represented in this fungus (Garcia and Zimmermann, 2014; this study), the first one by a single member, HcTOK1, and the second one by two members, HcTOK2.1 and HcTOK2.2. The present in silico analyses reveal different structural features between these two subfamilies. The different position of the largest intracellular loop (Fig. 1), placed at the extreme C-terminus downstream of the P2 in HcTOK1, and between P1 and P2 in HcTOK2 proteins, might modify the functional properties or regulation of the channels. One particularity of TOK channels is the variation of their K + -filter signature in the pore domains compared to the canonical TxGYGD sequence. More specifically, the central tyrosine of the P1 pore, a residue that has been shown to play a major role in the structure and selectivity for K + in K + -selective Shaker and KcsA-type channels (Doyle et al., 1998) is replaced by a leucine or a phenylalanine. In the three members of the two subfamilies in H. cylindrosporum, the leucine is found in HcTOK1 and the phenylalanine in both HcTOK2 channels. Roller et al. (2008) analyzed the significance of such changes by mutating either the leucine of P1 into a tyrosine or the tyrosine of P2 into a leucine, or by introducing these two mutations at the same time. The ion transport activity of the channels was barely altered by any of these mutations, but changes in several parameters of the transport process were This article is protected by copyright. All rights reserved. Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 14 recorded, including opening speed and amplitude of inward currents. Unexpectedly, the effect of the mutation of the pore P2 yielded very slight changes in the properties of the channel.

  Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 15 channel activity by pH as found here for HcTOK2.1 might contribute to the adaptability of the fungus to specific environmental conditions.

  HcTOK1 in the TOK1-OE2 cells resulted in an Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122

HcTOK2. 2 .

 2 Indeed, HcTOK2.2 was strongly and preferentially expressed in the Hartig net, and its expression was remarkably induced, by more than 30 times, in symbiotic condition compared to fungal pure culture. Furthermore, the colonization of pine roots by the fungal strains ectopically overexpressing HcTOK2.2 led to a high accumulation of K + in the host Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122

  This article is protected by copyright. All rights reserved. Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 19 H. cylindrosporum solid culture and a pine seedling were placed in a glass tube between a Whatman paper (cat. No. 1542-240) and the tube wall. Six biological replicates were prepared per condition. Each tube was filled with 20 ml of N1 medium (0.2 mM Ca(NO 3 ) 2 , 0.6 mM KNO 3 , 0.2 mM KH 2 PO 4 , 1 mM MgSO 4 , 0.2 mM KCl, 0.02 mM iron (III) citrate, 0.3 µM thiamine-HCl, complemented with 0.2 ml•l -1 Morizet and Mingeau (1976) microelements solution; pH 5.5). Plants were watered every week with a fresh solution, and harvested after 2 months. The remaining nutritive solution was measured after the harvest.

  Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 20

  Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 21 digested with SpeI and the cDNA inserted in the pGEMXho vector (derived from pGEMDG; D. Becker, Würzburg), which was then linearized and transcribed in vitro with the mMessage mMachine® T7 Ultra kit (Invitrogen). Oocytes were injected with water for negative control or with either 10, 30 or 50 ng of the in vitro described cRNA of HcTOK1, -2.1 or -2.2 respectively, and incubated in ND96 medium (mM: 96 NaCl, 2 KCl, 1.8 CaCl 2 , 1

  using the pYES2 plasmid. Plasmids allowing expression of either HcTOK1, HcTOK2.1 or HcTok2.2 were obtained by the digesting the Zero Blunt®-TOK1, -2.1 or -2.2 vectors and ligation into pYES2. A pYES2 plasmid harboring ScTOK1 cDNA from S. cerevisiae was used as positive control, and the empty pYES2 vector as negative control. Each of these constructs was transferred into Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 22 PLY246 cells using the transformation method as described by Gietz and Schiestl (2007).

  citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 25 The role of ectomycorrhizal communities in forest ecosystem processes: New perspectives and emerging concepts. Soil Biol Biochem 42: 679-698. Cuéllar, T., Pascaud, F., Verdeil, J. L., Torregrosa, L., Adam-Blondon, A. F., Thibaud, J. B., et al. (2010) A grapevine Shaker inward K + channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions. Plant J 61: 58-69. Cuéllar, T., Azeem, F., Andrianteranagna, M., Pascaud, F., Verdeil, J. L., Sentenac, H., et al. (2013) Potassium transport in developing fleshy fruits: The grapevine inward K + channel VvK1.2 is activated by CIPK-CBL complexes and induced in ripening berry flesh cells. Plant J 73: 1006-1018. Debaud, J.C., and Gay, G. (1987) In vitro fruiting under controlled conditions of the ectomycorrhizal fungus Hebeloma cylindrosporum associated with Pinus pinaster. New Phytol 105: 429-435. Doré, J., Perraud, M., Dieryckx, C., Kohler, A., Morin, E., Henrissat, B., et al. (2015) Comparative genomics, proteomics and transcriptomics give new insight into the exoproteome of the basidiomycete Hebeloma cylindrosporum and its involvement in ectomycorrhizal symbiosis. New Phytol 208: 1169-1187. Doré, J., Kohler, A., Dubost, A., Hundley, H., Singan, V., Peng, Y., et al. (2017) The ectomycorrhizal basidiomycete Hebeloma cylindrosporum undergoes early waves of transcriptional reprogramming prior to symbiotic structures differentiation. Environ Microbiol 19: 1338-1354. Doyle, D.A., Morais Cabral, J., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., et al. (1998) The structure of the potassium channel: molecular basis of K + conduction and selectivity. Science 280: 69-77. Fransson, P.M.A., Taylor, A.F.S., and Finlay, R.D. (2001) Elevated atmospheric CO 2 alters This article is protected by copyright. All rights reserved. Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 31 FEMS Yeast Res 12: 439-446.

Figure 1 .

 1 Figure 1. Protein sequence analysis of H. cylindrosporum TOK channels. A. Secondary

Figure 2 .

 2 Figure 2. Maximum Likelihood phylogenetic tree of fungal TOK channels. The branches

Figure 3 .

 3 Figure 3. Analysis of HcTOK1, HcTOK2.1 and HcTOK2.2 K + transport activity by two-

Figure 4 .

 4 Figure 4. Functional complementation of the potassium transport-defective yeast strain

Figure 5 .

 5 Figure 5. In situ hybridization of HcTOK1/2.1/2.2 transcripts in P. pinaster -

Figure 6 .

 6 Figure 6. Determination of the K + content in mycorrhizal pine seedlings. Plants were

Figure S1 .

 S1 Figure S1. Mean currents of Xenopus oocytes expressing HcTOK1, HcTOK2.1 or HcTOK2.2 or injected with water determined by voltage-clamp in dependence on external K + (A) or pH

Figure S2 .

 S2 Figure S2. Determination of the reversal potential of the HcTOK channels by the analysis of

Figure S3 .

 S3 Figure S3. Expression of HcTOK1, HcTOK2.1 and HcTOK2.2 in pure culture and

Figure S4 .

 S4 Figure S4. Determination of transcript levels of HcTOK1, HcTOK2.1. and HcTOK2.2 in

Figure S5 .Figure S6 .

 S5S6 Figure S5. Phenotypical analysis of the transgenic lines overexpressing HcTOK1, HcTOK2.1. and HcTOK2.2. K + contents in the mycelia. The only line showing any difference in

  Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 This article is protected by copyright. All rights reserved. Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 This article is protected by copyright. All rights reserved. Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 This article is protected by copyright. All rights reserved. Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 This article is protected by copyright. All rights reserved. Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122 This article is protected by copyright. All rights reserved. Comment citer ce document : Guerrero, M. D. C., Delteil, A., GARCIA, K., Houdinet, G., Conejero, G., Gaillard, I., Sentenac, H., Zimmermann, S. (Auteur de correspondance) (2018). Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum . Environmental Microbiology, 20 (5), 1873-1887. , DOI : 10.1111/1462-2920.14122

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Acknowledgments

We thank the French Ministry of Higher Education and Research for financing the fellowships of C.G.G., K.G. and G.H., and the ANR Project 'TRANSMUT' 2010 BLAN 1604 03 for the financing of the contract of A.D. K.G. was also supported by the U.S. Department of Agriculture (USDA NIFA 2017-67014-26530). We also thank Baptiste Monterroso for technical support in microscopy experiments, Sylvain Kumansky for participating in this project during an internship and Cécile Fizames for advice in phylogenetic analyses. Kind gift of K + -transport deficient yeast mutants by Adam Bertl is thanked. We are grateful to F. Martin and the Mycorrhizal Genomics Initiative consortium for access to unpublished genome data. The genome sequence data were produced by the US Department of Energy Joint Genome Institute in collaboration with the user community.