Kevin Garcia
email: kgarcia2@ncsu.edu

Maria Del

Carmen Guerrero-Galan

Muhammad Hannah E R Frank

Zulqurnain Haider

Amandine Delteil

Geneviève Conejero

Raphaël Lambilliotte

Cécile Fizames

Hervé Sentenac

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

Carmen Guerrero-Gala ´n

Muhammad Zulqurnain

Haider 2¤

Geneviève Cone ´je ´ro

Raphae ¨l Lambilliotte

Ce ´cile Fizames

Herve ´sentenac

Fungal Shaker-like channels beyond cellular K+ homeostasis: a role in ectomycorrhizal symbiosis between Hebeloma cylindrosporum and Pinus pinaster

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

Introduction

Potassium (K + ) is an essential cation involved in many biological processes in plants, such as growth, photosynthesis and stress tolerance [START_REF] Zimmermann | Potassium[END_REF][START_REF] Anschu ¨tz | Going beyond nutrition: Regulation of potassium homoeostasis as a common denominator of plant adaptive responses to environment[END_REF]. However, most soil K + cations are complexed with minerals such as feldspars or micas, resulting in low mobility and availability. As a consequence, productivity of agricultural and agroforestry ecosystems is strongly dependent on large and regular addition of chemical fertilizers [START_REF] Zo ¨rb | Potassium in agriculture-Status and perspectives[END_REF]. Plants developed various strategies to overcome this lack of K + availability and to improve its uptake, such as expression of highaffinity transport systems, exudation of organic acids by the root system, and symbiotic associations with soil-living microbes (reviewed in [START_REF] Garcia | The role of mycorrhizal associations in plant potassium nutrition[END_REF][START_REF] Meena | Does a rhizospheric microorganism enhance K + availability in agricultural soils?[END_REF][START_REF] Shin | Strategies for improving potassium use efficiency in plants[END_REF][START_REF] Haro | The role of soil fungi in K + plant nutrition[END_REF]).

Ectomycorrhizal (ECM) symbiosis is an intimate relationship between the roots of woody plants and the mycelium of soil-borne fungi, and constitutes a major component of boreal and temperate forest ecosystems [START_REF] Smith | Mycorrhizal Symbiosis[END_REF]. The main benefit of this association for the host plants is the improvement of macro-and micronutrient acquisition [START_REF] Becquer | The ectomycorrhizal contribution to tree nutrition[END_REF]. In ECM structures, nutrients are transferred from the soil-exploring extraradical mycelium to the hyphae developing in planta, through a fungal sheath surrounding the roots. The intra-radical colonization by the fungus forms a specific structure called the Hartig net. This net develops between the host root epidermis and cortex, where various transport proteins are expressed to drive nutrient exchanges between the two partners [START_REF] Becquer | The ectomycorrhizal contribution to tree nutrition[END_REF][START_REF] Guerrero-Gala ´n | The role of plant transporters in mycorrhizal symbioses[END_REF]. While most reports have focused on nitrogen and phosphorus nutrition in ECM plants, some studies have reported a significant transfer of fungal K + towards the host, particularly in K + -limited and stressful conditions [START_REF] Jentschke | Interdependence of phosphorus, nitrogen, potassium and magnesium translocation by the ectomycorrhizal fungus Paxillus involutus[END_REF][START_REF] Garcia | Potassium nutrition of ectomycorrhizal Pinus pinaster: Overexpression of the Hebeloma cylindrosporum HcTrk1 transporter affects the translocation of both K + and phosphorus in the host plant[END_REF]. One hypothesis is that the ectomycorrhiza-mediated K + nutrition could also be a strategy for the host plant to alleviate salt stress responses [START_REF] Guerrero-Gala ´n | Ectomycorrhizal symbiosis helps plants to challenge salt stress conditions[END_REF]. The transfer of K + through the mycorrhizal pathway requires the expression and organization of fungal transport proteins specific to the soil-fungus or plantfungus interfaces, allowing K + movements from the soil into colonized roots. The fine tuning of this putative fungal hyphae polarization seems to be required for the distribution of K + from the soil to plant cortical apoplasm [START_REF] Garcia | The role of mycorrhizal associations in plant potassium nutrition[END_REF].

Several proteins from ECM fungi involved in plant nutrient allocation have been identified and characterized [START_REF] Becquer | The ectomycorrhizal contribution to tree nutrition[END_REF][START_REF] Casieri | Biotrophic transportome in mutualistic plant-fungal interactions[END_REF][START_REF] Courty | The transportome of mycorrhizal systems[END_REF][START_REF] Garcia | Take a trip through the plant and fungal transportome of mycorrhiza[END_REF], and some of these might be responsible for the maintenance of ECM symbiosis, in addition to exclusive involvement in trophic exchanges [START_REF] Garcia | Molecular signals required for the establishment and maintenance of ectomycorrhizal symbioses[END_REF]. To our knowledge, only a handful of transport proteins involved in K + transport has been reported in ECM fungi thus far. Among them, HcTrk1 from Hebeloma cylindrosporum [START_REF] Corratge | Molecular and functional characterization of a Na + -K + transporter from the Trk family in the ectomycorrhizal fungus Hebeloma cylindrosporum[END_REF] is specifically localized in extra-radical hyphae of H. cylindrosporum-Pinus pinaster ectomycorrhizas, suggesting a role in K + uptake from the soil [START_REF] Garcia | Potassium nutrition of ectomycorrhizal Pinus pinaster: Overexpression of the Hebeloma cylindrosporum HcTrk1 transporter affects the translocation of both K + and phosphorus in the host plant[END_REF]. Genome sequencing of H. cylindrosporum [START_REF] Kohler | Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists[END_REF] has allowed the identification of other putative transporters possibly involved in K + acquisition from the soil, HcTrk2 and HcHAK [START_REF] Garcia | The role of mycorrhizal associations in plant potassium nutrition[END_REF], but their roles remain to be elucidated. Recently, we also described for the first time three fungus-specific tandem-pore outwardly rectifying K + (TOK) channels from H. cylindrosporum [START_REF] Guerrero-Gala ´n | Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum[END_REF][START_REF] Guerrero-Gala ´n | HcTOK1 participates in the maintenance of K + homeostasis in the ectomycorrhizal fungus Hebeloma cylindrosporum, which is essential for the symbiotic K + nutrition of Pinus pinaster[END_REF]. Excitingly, we were able to use an overexpression approach to demonstrate that one of them, HcTOK2.2, participates in the release of K + towards colonized P. pinaster roots.

In plants and animals, the efflux of K + from cells is a crucial biological process involved in the electrical polarization and energization of the plasma membrane, as well as in adaptation to biotic and abiotic stresses. Potassium channels and non-selective cation channels are involved in such mechanisms [START_REF] Gambale | Properties of shaker-type potassium channels in higher plants[END_REF]. The large family of tetrameric voltage-dependent K + channels (K V ), often called Shaker-like channels, constitutes a key component of the plasma membrane conductance. Many members are major contributors to the voltage-dependent influx or efflux of K + in plants and animals [START_REF] Zimmermann | Plant ion channels: from molecular structures to physiological functions[END_REF][START_REF] Hedrich | Ion channels in plants[END_REF][START_REF] Jegla | Evolution and structural characteristics of plant voltage-gated K + channels[END_REF]. To our knowledge, although some Shaker-like channels were identified in silico in ECM fungi, none has been studied so far [START_REF] Garcia | The role of mycorrhizal associations in plant potassium nutrition[END_REF]. Previous evidence in plants and animals establishes the relevance of this type of channels in the regulation of cellular K + homeostasis, and suggests fungal Shaker-like channels are likely to have a similar function. Moreover, they are possible candidates for K + allocation from ECM fungi towards the host plant at the symbiotic interface and/or K + storage into the vacuole.

In this work, we provide the first description of the fungal Shaker-like K V channel family by in silico and phylogenetic analyses, and investigate the physiological role of one of them, HcSKC from H. cylindrosporum. To assess the role of this channel in the allocation of fungal K + to the host plant P. pinaster, we generated transgenic fungal lines downregulated in HcSKC expression. We demonstrate that HcSKC silencing affects the fungal K + homeostasis, alters the expression of other fungal K + transport systems, and attenuates the K + acquisition of ECM P. pinaster.

Materials and methods

Wild-type and transgenic fungal strains

The homokaryotic strain h7 of the ECM Basidiomycota H. cylindrosporum Romagnesi [START_REF] Debaud | In vitro fruiting under controlled conditions of the ectomycorrhizal fungus Hebeloma cylindrosporum associated with Pinus pinaster[END_REF] was grown in the dark at 26˚C in YMG medium (Yeast extract, Malt extract, Glucose) [START_REF] Rao | Carbohydrate metabolism during morphogenesis of Coprinus lagopus (sensu Buller)[END_REF] either on agar-solidified Petri dishes or in liquid cultures.

The Agrobacterium tumefaciens-mediated genetic transformation process and production of the empty vector fungal strain (pPZP-133 control) have been detailed previously [START_REF] Garcia | Potassium nutrition of ectomycorrhizal Pinus pinaster: Overexpression of the Hebeloma cylindrosporum HcTrk1 transporter affects the translocation of both K + and phosphorus in the host plant[END_REF]. Using the same protocol and the bacterial strain LBA1126, carboxin-resistant RNAi-SKC fungal lines were produced.

Construction of RNAi-SKC plasmids

A 320 bp HcSKC-specific region was amplified on cDNA using RNAi-specific primers (S1 Table ) for the construction of the silencing vector. The sense (S) and antisense (AS) fragments obtained were inserted into the two MCS of pSILBAγ vector used for gene silencing in Laccaria bicolor [START_REF] Kemppainen | pHg/pSILBAγ vector system for efficient gene silencing in homobasidiomycetes: optimization of ihpRNA-triggering in the mycorrhizal fungus Laccaria bicolor[END_REF]. The silencing cassette was digested using XbaI enzyme and inserted in pPZP-133 plasmid in order to get the pPZP-RNAi-SKC vector.

Ectomycorrhiza production

Maritime pine seeds (P. pinaster Soland in Ait. from Medoc, Landes-Sore-VG source, France) were sterilized with 37% H 2 O 2 [START_REF] Ali | Pinus pinaster seedlings and their fungal symbionts show high plasticity in phosphorus acquisition in acidic soils[END_REF] and sown on Petri dishes containing agarose (Eurobio Molecular Biology Grade) and 0.2% glucose. The co-culture method in glass tubes as well as the composition of standard K + (SK, 1 mM K + ) and low K + (LK, 0.05 mM K + ) liquid N1 media were described previously [START_REF] Garcia | Potassium nutrition of ectomycorrhizal Pinus pinaster: Overexpression of the Hebeloma cylindrosporum HcTrk1 transporter affects the translocation of both K + and phosphorus in the host plant[END_REF]. Culture conditions were 16 h-photoperiod (210 μmol. m -2 .s -1 ), 20˚C and 60% relative humidity. SK medium was used for in situ hybridization experiments, and both SK and LK media for assays analyzing the ECM phenotype of RNAi-SKC fungal lines. Six 2-month-old plants per condition were collected for each experiment.

In situ hybridization

Two successive PCR amplifications of HcSKC cDNA led to the synthesis of sense and antisense probes (300 bp). The first amplification was performed with ISHSKC-T7-S-F/ISHSKC-S-R or ISHSKC-AS-F/ISHSKC-T7-AS-R primers (S1 Table ). A 1:100 dilution of the first PCR product was used for the second amplification with ISHT7-Prom and specific SKC-F or -R primers. Ectomycorrhizas were embedded in paraffin and 8 μm sections were obtained using a microtome. Sample preparation and hybridization were performed as previously described [START_REF] Guerrero-Gala ´n | Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum[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]. Slides were observed with a Leica DM6000 wide-field microscope (Montpelier RIO Imaging platform, www.mri.cnrs.fr) and pictures were analyzed by Volocity Acquisition 5.1.0 software (Perkin Elmer, www.perkinelmer.com).

Potassium shortage in Hebeloma cylindrosporum pure culture

Empty vector and RNAi-SKC fungal strains were cultivated in N6 liquid medium [START_REF] Louche | Efficiency of acid phosphatases secreted from the ectomycorrhizal fungus Hebeloma cylindrosporum to hydrolyse organic phosphorus in podzols[END_REF] containing 10 mM K + (6 mM KNO 3 , 4 mM KCl). Fresh N6 was supplied on days 7 and 12. On day 14, thalli were washed five times with N6 medium without added K + and cultivated in these media up to 48 h. In medium without K + , NO 3 was added in the form of Ca(NO 3 ) 2 at the same concentration as in the standard K + medium. Fungi were collected 0, 12, 24 and 48 h after K + deprivation and washed twice in CaSO 4 (0.2 mM)-glucose (5 gl -1 ) solution. Half of each sample was flash-frozen in liquid nitrogen for qRT-PCR analyses and the rest was dried for one week at 60˚C, weighed and used for intracellular K + content determination.

Additionally, four media with high or low concentrations of K + (1 or 0.05 mM, respectively) and sodium (Na + ; 1 or 0.2 mM, respectively) were used to assess fungal biomass of transgenic strains (S2 Table ). These media were named: +K/-Na, +K/+Na, -K/-Na, and -K/+Na. Transgenic fungal strains were cultured on a solid version of these media for 1 week at 26˚C, subcultured into 50 ml of their corresponding liquid media and placed at 26˚C for 28 days. Thalli were collected, dried at 70˚C, and dry weights were determined.

Quantification of potassium contents

Plant tissue and mycelia were collected and weighed to determine the dry biomass (DW). Acid extraction of tissue ion contents and assays of K + content by flame atomic absorption spectrophotometry were performed as previously described [START_REF] Garcia | Potassium nutrition of ectomycorrhizal Pinus pinaster: Overexpression of the Hebeloma cylindrosporum HcTrk1 transporter affects the translocation of both K + and phosphorus in the host plant[END_REF].

qRT-PCR analyses

H. cylindrosporum RNA extraction, cDNA synthesis and qRT-PCR protocol were described [START_REF] Garcia | Promoter-dependent expression of the fungal transporter HcPT1.1 under Pi shortage and its spatial localization in ectomycorrhiza[END_REF]. Expression levels of the transport systems HcSKC (protein ID 79961; https://mycocosm. jgi.doe.gov/Hebcy2/Hebcy2.home.html), HcTOK1 (31571), HcTOK2.1 (129509), HcTOK2.2 (127201), HcTrk1 (445173), HcTrk2 (176376), and HcHAK (435192) were determined relatively to the internal control α-tubulin (24108) on mycelium samples (for primers see S1 Table ).

In silico analysis of HcSKC

Hydrophobicity profiles of HcSKC (H. cylindrosporum), LbSKC (L. bicolor), and XlK V 2.1 (Xenopus laevis) subunits were obtained using Kyte-Doolittle algorithm with a window size of 11 amino acids [START_REF] Kyte | A simple method for displaying the hydropathic character of a protein[END_REF] (http://gcat.davidson.edu/DGPB/kd/kyte-doolittle.htm).

Amino acid alignment of HcSKC, LbSKC, and XlK V 2.1 subunits was performed using Clustal Omega program (http://www.ebi.ac.uk/Tools/msa/) and formatted with BoxShade program (http://www.ch.embnet.org/software/BOX_form.html).

Homology structure of the HcSKC subunit was modeled by Swiss-Model server using the template structure of Rattus norvegicus RnK V 2.1 subunit [START_REF] Biasini | SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information[END_REF] (http://swissmodel.expasy.org/). Amino acid alignment between these two subunits was obtained using the same program.

Phylogenetic tree construction

The protein sequences of SKC subunits of 185 fungi, 4 animals and 2 plants were collected from JGI [START_REF] Grigoriev | Fueling the future with fungal genomics[END_REF], NCBI (NCBI Resource Coordinator 2015), UniProt (The UniProt Consortium 2015) or TAIR (www.arabidopsis.org) databases by BLASTP analysis based on HcSKC of H. cylindrosporum (S2 Table ). All fungal databases used here are publicly available. All sequences without a predicted G[YF]G[DE] pore motif were removed manually. Protein sequences were aligned with MUSCLE algorithm v3.8.31 [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF] and the alignment was cured by Gblocks program [START_REF] Castresana | Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis[END_REF]. Phylogenetic analysis was performed by PhyML [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF] using the maximum likelihood method (1000 bootstraps) and the tree was visualized by Dendroscope software v3.2.10 [START_REF] Huson | Dendroscope: An interactive viewer for large phylogenetic trees[END_REF].

Construction of the HcSKC-EGFP fusion and expression in Saccharomyces cerevisiae

The HcSKC-EGFP C-terminal cassette was created with a PCR fusion approach, amplifying separately HcSKC from H. cylindrosporum cDNA and EGFP from a preexisting construct, with primers overlapping the fusion region containing both HcSKC and EGFP sequences (S1 Table ). A third PCR with the flanking primers was required to amplify the whole construct, which was later digested with restriction enzymes and inserted in the corresponding vectors. Subcellular localization of the HcSKC-EGFP construct in the yeast strain ply232 was attempted with two approaches, first a galactose-induced expression in the vector pYES2 (ThermoFisher Scientific) and later under the constitutive promoter PGK in the pFL61 vector [START_REF] Minet | Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs[END_REF].

Statistical analyses

Data normality was checked using the Wilk-Shapiro test. Differences between averages were analyzed by one-way ANOVA followed by Tukey HSD post-hoc tests, or by Student's test, depending on the experiment. Mean comparisons were carried out using Dunnett's test. Dunnett's test was also used to search for significant differences in pairwise comparisons. All statistical analyses were performed with R software at the 5% level of statistical significance.

Results

Identification of a Shaker-like K V channel in Hebeloma cylindrosporum

We previously identified a putative K + channel (STC no.: 009A07R1.0A1.1) in EST resources obtained from H. cylindrosporum pure cultures [START_REF] Lambilliotte | Large-scale identification of genes in the fungus Hebeloma cylindrosporum paves the way to molecular analyses of ectomycorrhizal symbiosis[END_REF]. BLASTP analysis of 009A07R1.0A1.1 protein sequence against the L. bicolor S238N genomic database allowed the identification of a unique orthologue (protein ID 297800; https://mycocosm.jgi.doe.gov/Lacbi2/Lacbi2.home. html). The hydrophobicity profile of these two proteins generated by the Kyte-Doolittle algorithm revealed the presence of 7 hydrophobic domains, similar to the XlK V 2.1 Shaker channel from X. laevis (Fig 1A). The amino acid alignment of these fungal Shaker-like proteins with XlK V 2.1 confirmed the presence of 6 conserved transmembrane domains and one pore domain characterized by the GYGE motif ensuring K + selectivity (Fig 1B). Consequently, we named these proteins SKC (Shaker-like K + -Channel). The S4 domain (constituting the voltage sensor in Shaker channels) of HcSKC and LbSKC harbored several arginine residues (R). Such positively charged residues have been shown in other K V channels to contribute to the channel voltage sensitivity and gating charge [START_REF] Seoh | Voltage-sensing residues in the S2 and S4 segments of the Shaker K + channel[END_REF]. When compared with XlK V 2.1, HcSKC and LbSKC have a shorter C-terminal region, suggesting reduced "built-in" regulation mechanisms (Fig 1A). Based on these in silico analyses, we used the R. norvegicus RnK V 2.1 channel (NP_037318.1; S3

Distribution of Shaker-like channels in the fungal kingdom

Using HcSKC as a reference, we performed BLASTP analysis on publicly available sequenced fungi found on NCBI and JGI fungal portals (http://genome.jgi-psf.org/programs/fungi/index. jsf). In addition, a cure based on the presence of a putative G[YF]G[DE] pore motif was achieved. In the Basidiomycota phylum, the presence of putative SKC proteins was detected only in the Agaricomycotina subphylum, as is defined in the JGI phylogenetic tree (Fig 2). In addition, putative SKC proteins were found in Wallemia melicola, which belongs to Wallemiomycetes, a sister group to Agaricomycotina [START_REF] Matheny | Resolving the phylogenetic position of the Wallemiomycetes: An enigmatic major lineage of Basidiomycota[END_REF]. Two putative Shaker-like channels were also detected in the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis (Glomeromycotina subphylum) and other basal fungi belonging to Cryptomycota, Chytridiomycota, Blastocladiomycota, Zoopagomycota and Mucoromycota phyla (Fig 2). Surprisingly, no SKC was found in the Pucciniomycotina and Ustilaginomycotina subphyla of Basidiomycota, and in the Ascomycota phylum. Moreover, proteins similar to K V channels, but without the G[YF]G[DE] motif, were not found in these fungi, indicating the complete loss of Shakerlike genes in Pucciniomycotina, Ustilaginomycotina and Ascomycota species, with the exception in the Ascomycota phylum of Saitoella complicata, which presents one putative SKC channel (Fig 2).

The functional properties of HcSKC could not be revealed in various heterologous systems

Functional characterization is an important step for the analysis of K + channel activity to predict transport features, such as selectivity and direction of rectification. Many strategies were applied to characterize HcSKC, using multiple heterologous systems (X. laevis oocytes and yeast) and co-injection of putative regulatory proteins (protein kinase K and putative channel β-subunits from H. cylindrosporum: HcKCNAB1 and HcKCNAB2) (S4 Table ). No significant result was obtained from any of these experiments. Moreover, the expression of HcSKC triggers endogenous currents in X. laevis oocytes, making the observation of specific exogenous currents tricky (S4 Table ). In addition, the previously identified HcSKC channel in the H. cylindrosporum h1 strain [START_REF] Lambilliotte | Large-scale identification of genes in the fungus Hebeloma cylindrosporum paves the way to molecular analyses of ectomycorrhizal symbiosis[END_REF] has been extensively tested by voltage-clamp experiments in X. laevis oocytes, without any conclusive result (S4 Table ). As an alternative, a functional characterization of the homologous channel in L. bicolor LbSKC was also attempted in X. laevis oocytes, but unsuccessfully (S4 Table ). Finally, attempts to perform subcellular localizations in yeast using HcSKC-EGFP fused constructs driven by a constitutive or a galactose-inducible promoter failed too (S2 Fig) . None of the experiments yielded a clear localization pattern of HcSKC, probably due to misprocessing of the protein product, reinforcing the issues in expressing HcSKC in the tested heterologous systems.

HcSKC transcripts are localized in all types of ectomycorrhizal hyphae

To elucidate the role of HcSKC channels in the symbiotic association with P. pinaster, the localization of corresponding transcripts was analyzed in 2-month-old ectomycorrhizas through in situ hybridization experiments. Cross-section and probe hybridization processes resulted in the detection of HcSKC transcripts in extra-radical hyphae, the hyphal mantle, and

Silencing of HcSKC affects Hebeloma cylindrosporum potassium homeostasis

Decoding the role of HcSKC in axenic conditions was attempted by RNAi downregulation of the corresponding gene. HcSKC-silencing lines of H. cylindrosporum were produced by Agrobacterium tumefaciens-mediated transformation. The expression level of HcSKC was determined by qRT-PCR in fungal mycelia growing on selection medium. Two isolates displaying a five-fold decrease in HcSKC expression, named RNAi-SKC-5 and RNAi-SKC-7, were selected for ECM assays (S4 Fig) . We then investigated the K + accumulation and expression of K + transport-related genes in RNAi-SKC-5 and RNAi-SKC-7 lines. In fungi grown in standard medium (i.e., in presence of a relatively high external K + , 1 mM), internal K + content was significantly higher in RNAi-SKC transformants than in the control line, indicating an increase in K + acquisition and/or a decrease in K + efflux (Fig 4). Once these fungal isolates were transferred into the K + -free medium, no difference in K + content between RNAi-SKC and control lines could be detected at 12, 24 and 48 h after the transfer (Fig 4). In addition, we assessed the effect of high and low K + (1 and 0.05 mM, respectively) and Na + (1 or 0.2 mM, respectively) detected in extra-radical hyphae, the fungal sheath and the Hartig net of ectomycorrhizas. Bars, 50 μm. c.c., cortical cell; exh, extraradical hyphae; Hn, Hartig net; sh, fungal sheath. https://doi.org/10.1371/journal.pone.0242739.g003 availability on fungal growth in all transgenic strains (S5 Fig) . All media were prepared on the basis of the media used for the subsequent ECM assay (S4 Table). After 28 days of culture in axenic condition, dry weights were recorded. Although the biomass of both RNAi-SKC transgenic lines differed in all conditions, they did not significantly differ from the control line, except for that showed a slightly reduced biomass in +K/-Na condition compared to the control line (S5 Fig) .  Besides HcSKC, six other putative K + transport-related genes can be identified in the H. cylindrosporum genomic database [START_REF] Garcia | The role of mycorrhizal associations in plant potassium nutrition[END_REF][START_REF] Kohler | Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists[END_REF]. To figure out whether their expression levels were affected by the downregulation of HcSKC, the same fungal samples as those used for K + content measurements were analyzed by qRT-PCR. When grown in K + standard pure culture conditions (SK medium), none of the six targeted K + transport system genes was significantly differentially expressed in the RNAi-SKC lines compared to the control (Fig 5). In contrast, 12, 24 and 48 h after the transfer into the K + -free medium, HcTrk2 and HcTOK2.1 were upregulated and HcTOK1 was downregulated in the two RNAi-SKC lines (Fig 5B , 5D and5E). Interestingly, HcHAK was upregulated in both RNAi lines only 48 h after the transfer into the K +free medium (Fig 5C). The expression in the transgenic lines of the other two transport systems, HcTrk1 and HcTOK2.2, was not significantly affected by the transfer into the K + -free medium (Fig 5A -5F). Taken together, these data indicate that the downregulation of HcSKC is sufficient to modify the K + homeostasis in H. cylindrosporum at standard K + . However, in conditions of K + shortage, the modification of the expression of three K + transport-related genes compensated for the effect of HcSKC downregulation and restored a wild-type K + content in the fungus.

Downregulation of HcSKC affects K + transfer to the host plant at standard K + conditions

To investigate the role of HcSKC in symbiotic conditions, P. pinaster seedlings were inoculated with control, RNAi-SKC-5, or RNAi-SKC-7 lines, or kept non-inoculated, and grown in SK and LK conditions (Fig 6). Mycorrhization with the control fungal strain (transformed with the empty plasmid) resulted in statistically significant higher K + contents in the plant roots and shoots when grown in LK, but not in SK, conditions. In comparison with these control plants, mycorrhizal association with the SKC-silencing fungal lines resulted in a significant decrease of K + contents in shoots and roots in SK conditions, indicating a less cooperative behavior of H. cylindrosporum that affected K + transfer to P. pinaster (Fig 6A). Surprisingly, this negative effect of HcSKC silencing on K + contents in the host plant was not observed in LK conditions (Fig 6B). Hence, in low K + conditions, the mycorrhizal pathway for plant K + nutrition was not affected by the reduced expression of HcSKC. This result suggests that, in this LK condition, other underlying mechanisms are at work to achieve an efficient mycorrhizal K + transport towards the host plant. In other words, HcSKC seems to play a significant role in K + delivery toward the plant in SK conditions (1 mM), and not in conditions of K + shortage (0.05 mM).

Discussion

As has recently been revealed, K + acquisition in higher fungi seems to be mediated by membrane transport proteins belonging to the Trk/Ktr/HKT and KT/KUP/HAK families [START_REF] Garcia | The role of mycorrhizal associations in plant potassium nutrition[END_REF][START_REF] Corratge | Potassium and sodium transport in non-animal cells: The Trk/Ktr/HKT transporter family[END_REF]. In addition, ACU ATPases have been reported as being involved in K + and Na + uptake in Ustilago maydis, and more generally envisioned as key players of K + transport in fungi [START_REF] Haro | The role of soil fungi in K + plant nutrition[END_REF][START_REF] Benito | Novel P-type ATPases mediate high-affinity potassium or sodium uptake in fungi[END_REF]. PAT ATPases could emerge as mechanisms for absorption of these cations as well, but they have not been studied thoroughly [START_REF] Fietto | Characterization and expression of two genes encoding isoforms of a putative Na, K-ATPase in the Chytridiomycete Blastocladiella emersonii[END_REF][START_REF] Benito | Potassium and sodium uptake systems in fungi. The transporter diversity of Magnaporthe oryzae[END_REF]. Regarding outward K + transport systems, yeast and filamentous fungi developed TOK and Shaker-like K + channels, from which only some members have been investigated thus far [START_REF] Guerrero-Gala ´n | Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum[END_REF][START_REF] Guerrero-Gala ´n | HcTOK1 participates in the maintenance of K + homeostasis in the ectomycorrhizal fungus Hebeloma cylindrosporum, which is essential for the symbiotic K + nutrition of Pinus pinaster[END_REF]. To our knowledge, this work reports the first description of a Shaker-like K V channel in higher fungi and highlights its role in both K + homeostasis and mycorrhizal symbiosis.

In silico analysis of HcSKC revealed features from animal Shaker K + channels

The initial identification of the HcSKC channel was performed from an EST library of H. cylindrosporum produced in free-living conditions [START_REF] Lambilliotte | Large-scale identification of genes in the fungus Hebeloma cylindrosporum paves the way to molecular analyses of ectomycorrhizal symbiosis[END_REF]. In this study, one putative Shaker-like K + channel was found to be highly expressed (STC no.: 009A07R1.0A1.1), HcTOK2.1 (e) were altered in RNAi-SKC-5 and RNAi-SKC-7 lines in comparison to the transgenic control line (Ctrl) under K + limiting conditions. The relative expressions of HcTrk1 (a) and HcHAK (c) K + transporters, and of the K + channel HcTOK2.2 (f) were not affected by K + shortage in RNAi-SKC-5 and RNAi-SKC-7 lines in comparison to the transgenic control line (Ctrl). Mean values are provided with the standard error (n = 6). Statistical differences were determined using a one-way ANOVA followed by Dunnett's test relative to Ctrl strain for each data point P < 0.05 ( � ), P < 0.01 ( �� ) and P < 0.001 ( ��� ). https://doi.org/10.1371/journal.pone.0242739.g005

suggesting an important role of this transport system in the fungus. Thanks to the release of H. cylindrosporum h7 genomic data [START_REF] Kohler | Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists[END_REF][START_REF] Dore | Comparative genomics, proteomics and transcriptomics give new insight into the exoproteome of the basidiomycete Hebeloma cylindrosporum and its involvement in ectomycorrhizal symbiosis[END_REF][START_REF] Dore | The ectomycorrhizal basidiomycete Hebeloma cylindrosporum undergoes early waves of transcriptional reprogramming prior to symbiotic structures differentiation[END_REF], in silico analyses confirmed the homology of this fungal candidate to animal and plant K V channels (Fig 1). Moreover, phylogenetic analyses provided additional evidence on the proximity of fungal Shaker channels to animal ones (Fig 2). The major difference between HcSKC/LbSKC and animal channels is the length of their Cterminal regions. Several studies have evidenced a crucial role of this C-terminal region in the regulation of plant and animal Shaker channel activity [START_REF] ´rel | Regulation of K + channel activities in plants: from physiological to molecular aspects[END_REF][START_REF] Li | Voltage-gated potassium channels: regulation by accessory subunits[END_REF]. In plants, it was reported that the C-terminal regions are involved in intracellular K + sensing, heteromerization or voltagedependent gating mechanisms [START_REF] Dreyer | Assembly of plant Shaker-like K out channels requires two distinct sites of the channel α-subunit[END_REF][START_REF] Liu | Intracellular K + sensing of SKOR, a Shaker-type K + channel from Arabidopsis[END_REF][START_REF] Naso | The role of the C-terminus for functional heteromerization of the plant channel KDC1[END_REF]. In animal Shaker-type channels, the C-terminus can play multiple roles e.g. in regulation by β-subunits [START_REF] Sokolova | Conformational changes in the C terminus of Shaker K + channel bound to the rat Kvβ2-subunit[END_REF], by voltage [START_REF] Zandany | Alternative splicing modulates Kv channel clustering through a molecular ball and chain mechanism[END_REF], or in localization [START_REF] Ruiz-Cañada | DLG differentially localizes Shaker K + -channels in the central nervous system and retina of Drosophila[END_REF]. The fact that the C-terminal domains of H. cylindrosporum and L. bicolor are significantly shorter than those of the animal Shaker channels (Fig 1) suggests differences in channel activity regulation. Moreover, LbSKC seems to diverge by an additional sequence in the region of the S4 domain (Fig 1B) with unknown function.

Shaker-like channels were lost in Pucciniomycotina, Ustilaginomycotina and Ascomycota phyla

Although well-described in plants and animals, the presence of K V channels in the fungal kingdom has been hypothetical until now. Indeed, no Shaker channel gene could be detected in the Saccharomyces cerevisiae genome sequence. Our phylogenetic analysis based on many fungal genomes belonging to Basidiomycota and Ascomycota phyla, as well as basal fungi (Cryptomycota, Chytridiomycota, Blastocladiomycota, Zoopagomycota and Mucoromycota phyla) revealed the absence of putative SKC proteins in Pucciniomycotina and Ustilaginomycotina, which are sub-phyla of Basidiomycota, and the entire Ascomycota phylum. Interestingly, one SKC protein was found in S. complicata, which is related to the Ascomycota phylum. It is, however, worth noting that the affiliation of S. complicata to Ascomycota remains unclear [START_REF] Goto | Saitoella, a new anamorph genus in the Cryptococcaceae to accommodate two himalayan yeast isolates formerly identified as Rhodotorula glutinis[END_REF][START_REF] Nishida | Draft genome sequencing of the enigmatic yeast Saitoella complicata[END_REF], and its SKC protein sequence clusters with those of basal fungi, in agreement with the idea that this species might belong to a more basal phylum than previously thought. Given that Shaker-like channels were found in basal fungi and in the most recent subphylum of Badisiomycota (Agaricomycotina), their absence in Pucciniomycotina, Ustilaginomycotina, and Ascomycota strongly suggests that they have been lost in the ancestors of these phyla. This indicates that other transport systems can fill the roles of Shaker channels and play a central role in the maintenance of K + homeostasis in these organisms. The survey and analysis of other K + channels found in fungi, such as the TOK channels [START_REF] Garcia | The role of mycorrhizal associations in plant potassium nutrition[END_REF], have been integral in describing other key regulators of K + transport and homeostasis in fungi (e.g. [START_REF] Guerrero-Gala ´n | Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum[END_REF][START_REF] Guerrero-Gala ´n | HcTOK1 participates in the maintenance of K + homeostasis in the ectomycorrhizal fungus Hebeloma cylindrosporum, which is essential for the symbiotic K + nutrition of Pinus pinaster[END_REF]).

HcSKC cannot be functionally characterized in heterologous systems

Although several attempts using different setups, strategies and heterologous systems were performed, the functional characterization of H. cylindrosporum and L. bicolor Shaker-like channels is still missing (S4 Table ). Similarly, no conclusive data were obtained after expressing a HcSKC-EGFP construct driven by a constitutive or a galactose-inducible promoter in S. cerevisiae (S2 Fig). This might result from the fact that conditions or proteins required for the expression, function, localization, and regulation of fungal SKC channels were missing in the tested heterologous systems. Recently, the similar failed expression of a zinc transporter from the ECM fungus Suillus luteus was reported, showing the risky nature of these experiments [START_REF] Ruytinx | Identification, evolution and functional characterization of two Zn CDF-family transporters of the ectomycorrhizal fungus Suillus luteus[END_REF]. It is well-known that animal and plant Shaker channels can physically interact with modulatory β-subunits [START_REF] Torres | A marriage of convenience: β-subunits and voltagedependent K + channels[END_REF]. For example, rat K V 1 Shaker channels are able to form stable complexes with cytosolic β-subunits (K V β), which modify the channel activity [START_REF] Rettig | Inactivation properties of voltage-gated K + channels altered by presence of β-subunit[END_REF][START_REF] Pan | Functional coupling between the Kv1.1 channel and aldoketoreductase Kvbeta1[END_REF]. In the model plant Arabidopsis thaliana, KAB1 is a protein related to animal β-subunits that interacts with the K + channel KAT1 [START_REF] Tang | Physical association of KAB1 with plant K + channel alpha subunits[END_REF]. We identified ten putative β-subunits in the genomic database of H. cylindrosporum and two of them, HcKCNAB1 and HcKCNAB2, were co-expressed with HcSKC in X. laevis oocytes, resulting in no exogenous current detection. At this stage we cannot conclude whether fungal Shaker channels require β-subunits or other types of peptides for their activation. It is known that some compounds can alter the activity of voltage-dependent K + channels, such as human hormones that inhibit K V 1.3 and K V 1.5 [START_REF] Yu | Inhibitory effects of cortisone and hydrocortisone on human Kv1.5 channel currents[END_REF]. Thus, the presence in X. laevis oocytes of undefined compounds that would inhibit fungal SKC currents might be possible. Another possibility would also be a regulation by other membrane proteins. For instance, in the nematode Caenorhabditis elegans, the two-pore K + channel SUP-9 forms a complex with two proteins, UNC-93 (Major Facilitator Superfamily) and SUP-10 (The Potassium Channel Regulatory Protein Sup-10 Family), which coordinate muscle contraction [START_REF] De La | sup-9, sup-10, and unc-93 may encode components of a two-pore K + channel that coordinates muscle contraction in Caenorhabditis elegans[END_REF]. In A. thaliana, the homolog of UNC-93 regulates K + translocation from the root to the aerial parts of the plant. Disruption of this gene leads to a phenotype similar to that of knockout mutants of the AtSKOR Shaker-like channel, indicating that they may function together, although no physical interactions have yet been observed [START_REF] Xiang | The Arabidopsis AtUNC-93 acts as a positive regulator of abiotic stress tolerance and plant growth via modulation of ABA signaling and K + homeostasis[END_REF][START_REF] Gaymard | Identification and disruption of a plant Shaker-like outward channel involved in K + release into the xylem sap[END_REF]. There is one UNC-93 gene in the genome of H. cylindrosporum which is induced in ectomycorrhizas, compared to freeliving mycelium, and could be a promising candidate to test as a regulator of the activity of HcSKC.

Calcium signaling pathways are well-known regulators of K + transport systems in plants and animals. In plants, calcium mediates signals during biotic interactions and environmental stress, and regulates a series of proteins that can regulate the activity of K + channels through phosphorylation, dephosphorylation, structural modification, and physical interaction [START_REF] ´rel | The complex fine-tuning of K + fluxes in plants in relation to osmotic and ionic abiotic stresses[END_REF]. In animals these pathways are different, but there is also evidence of the modulation of K + channel transport by calcium-dependent phosphorylation and dephosphorylation [START_REF] Roeper | Frequency-dependent inactivation of mammalian A-type K + channel KV1.4 regulated by Ca 2+ /Calmodulin-dependent protein kinase[END_REF]. In filamentous fungi, there are few links between the regulation of K + transport and calcium signaling, probably because most of the studies have been carried on the model Ascomycota yeast S. cerevisiae [START_REF] Casado | Regulation of Trkdependent potassium transport by the calcineurin pathway involves the Hal5 kinase[END_REF]. However, the first description of Shaker-like K + channels in fungi may lead to the discovery of new pathways. Supporting this hypothesis, a rapid survey of the genome of H. cylindrosporum yields many putative calcium-regulated genes: three calmodulins (Protein ID 385514, 443591 and 443596), one calcineurin B (444455) and a wide range of Ca 2+ /calmodulin-dependent and serine/threonine protein kinases. It is also tempting to imagine that the most promising heterologous system for the characterization of fungal transport systems would be S. cerevisiae, which is phylogenetically closer to higher fungi than animal cells. In general, the use of yeast seems more adapted for the characterization of inward transport systems than of outward systems, but so far it has not provided encouraging results in our uptake experiments (S4 Table ). Direct transport measurements by the electrophysiological patchclamp method might provide missing insights in fungal SKC functional properties.

Potassium homeostasis mediated by HcSKC is a prerequisite for K + allocation to pine

In order to investigate in vivo the role of the K V channel SKC in both free-living and ECM conditions, we decided to generate RNAi fungal lines affected in HcSKC expression. Analysis of the phenotype of these transgenic fungal lines revealed a close relationship between HcSKC expression, K + accumulation in the fungus, and improvement of K + nutrition of the colonized plants. Indeed, HcSKC silencing resulted in contrasting phenotypes of both the fungus and the mycorrhizal plant, depending on external K + availability. At 1 mM K + , the downregulation of HcSKC expression altered K + accumulation in the free-living mycelia and the ECM plants. It should be noted that, in our previous report [START_REF] Garcia | Potassium nutrition of ectomycorrhizal Pinus pinaster: Overexpression of the Hebeloma cylindrosporum HcTrk1 transporter affects the translocation of both K + and phosphorus in the host plant[END_REF], H. cylindrosporum had a significant effect on K + allocation to P. pinaster only in K + limiting conditions and not in sufficient conditions. Such a phenotype is further confirmed in the present report by the comparison between plants inoculated by the control transformed fungus harboring the empty vector and the control non-mycorrhizal plants ( Fig 4). However, this does not mean that K + transfer from the fungus to the plant did not occur in standard (SK) conditions. Indeed, the direct uptake of K + from the medium by the root system may be reduced upon mycorrhizal association, and this reduction could be compensated by fungal K + allocation to the plants. Parts of the ECM root system are somehow isolated from the external medium due to a loss of root hairs and to the presence of the fungal mantle surrounding the roots, and thus are dependent on the ECM symbiont to acquire nutrients. Given that HcSKC RNAi transformants accumulated more K + than the control strain at standard external K + , it may be assumed that, when such transformants were grown in association with P. pinaster, their reduced expression of HcSKC either modified the pool of fungal K + available for the plant and/or affected its symbiotic transfer to host roots. This would result in impaired K + nutrition of the host. It is worth noting that varying external concentrations of K + and Na + did not drastically impact the growth of the RNAi-SKC transgenic fungal lines (S5 Fig) . This indicates that the alteration of K + nutrition observed in plants colonized by the RNAi-SKC lines at standard K + cannot be explained by a modification of fungal growth and sensitivity to external conditions, but by the actual downregulation of HcSKC. Interestingly, the over-accumulation of K + in the RNAi-SKC transformants was not found to involve the upregulation of putative inward K + transporters, and thus, might result from a decrease in K + efflux from the fungus. In contrast, in low K + conditions, three other putative K + transport systems were up-or downregulated in the RNAi lines. We postulate that the altered expression of HcTrk2, HcHAK, HcTOK1 and HcTOK2.1 restored the overall fungal K + transport activities and homeostasis, as suggested by the recovery of the fungal K + content observed in free-living conditions (Fig 4). This could explain the restoration of the K + contents displayed by the mycorrhizal plants, suggesting that efficient ectomycorrhiza-mediated K + nutrition occurred again. Another hypothesis, that HcSKC is involved in K + movements from/ to the vacuole, cannot be completely excluded, even though only plasma membrane localization of this type of ion channel has been described in any organism so far. Subcellular localization at either the plasma membrane or at the tonoplast should allow us to fully appreciate the role of HcSKC, regarding its manifest importance in H. cylindrosporum biology under axenic and symbiotic conditions.

To summarize, homology of HcSKC with animal outward K V channels and the observed K + nutrition-related mycorrhizal and fungal phenotypes lead to the simplest hypothesis: that the fungal Shaker-like channel HcSKC may be involved in K + efflux from fungal cells, allowing K + translocation towards the host plant. Thus, the role played by HcSKC in K + homeostasis in H. cylindrosporum seems crucial for fungal K + availability and/or release to the host plant.

Fig 1 .

 1 Fig 1. In silico prediction of a Shaker-like channel in the fungus Hebeloma cylindrosporum. The hydrophobicity profiles of the Shaker subunits from H. cylindrosporum (HcSKC), L. bicolor (LbSKC), and X. laevis (XlK V 2.1) (a) and their partial amino acid sequence alignment (b) predicted six transmembrane domains (S1 to S6). One pore domain in HcSKC and LbSKC subunits containing the conserved G[YF]G[DE] motif of the Shaker channel selectivity filter was also detected. The S4 transmembrane domain corresponds to the predicted voltage-sensor domain. (c) The plausible 3-D model of the fungal HcSKC subunit showing the transmembrane (S1 to S6) and pore (P) domains was generated by homology with the 3-D structure of RnK V 2.1 K + channel from R. norvegicus (NP_037318.1; S2 Table) as a template using Swiss-Model server [33] (http://swissmodel.expasy.org/). The GYGE signature motif of the pore region was highlighted in green. The sequence alignment of HcSKC and RnK V 2.1 polypeptides is displayed in S1 Fig. https://doi.org/10.1371/journal.pone.0242739.g001

Fig 2 .Fig 3 .

 23 Fig 2.Phylogenetic tree of K V channels in fungi, and representative animal and plant species. The Shaker-like K + channel tree of publicly available sequenced fungi from the Basidiomycota phylum and from basal fungi (Cryptomycota, Chytridiomycota, Blastocladiomycota, Zoopagomycota and Mucoromycota) was calculated by the maximum likelihood method (1000 bootstraps). Shaker-like proteins were found only in one putative Ascomycota species (Saitoella complicata) and not in the Pucciniomycotina and Ustilaginomycotina subphyla of Basidiomycota. Ectomycorrhizal (ECM), arbuscular mycorrhizal (AM), and orchid mycorrhizal (OM) fungi are indicated by yellow, green, and blue dots, respectively. K V channels from representative land plant and animal species were included in the tree. All species displayed in this tree are listed in the S2 Table. Shaker-like proteins from H. cylindrosporum (HcSKC) and L. bicolor (LbSKC) used in this study are indicated by black arrows. https://doi.org/10.1371/journal.pone.0242739.g002

Fig 4 .

 4 Fig 4. Potassium content in Hebeloma cylindrosporum HcSKC downregulated lines. K + content was measured in fungal lines transformed with the empty (Ctrl) or HcSKC-silencing vectors (RNAi-SKC-5 and RNAi-SKC-7) using an atomic absorption flame spectrophotometer. Statistical differences were evaluated using the Student's test with respect to the Ctrl strain ( � , P < 0.05; �� , P < 0.01). n = 6. https://doi.org/10.1371/journal.pone.0242739.g004

Fig 5 .

 5 Fig 5. Relative expression of putative K + channels and transporters in Hebeloma cylindrosporum control and HcSKC-silencing lines. Relative expressions of the K + transporters HcTrk1 (a), HcTrk2 (b), HcHAK (c) and the K + channels HcTOK1 (d), HcTOK2.1 (e) and HcTOK2.2 (f) were analyzed in dependence on K + shortage in control and RNAi fungal lines. The relative expressions of the K + transporter HcTrk2 (b) and the K + channels HcTOK1 (d) andHcTOK2.1 (e) were altered in RNAi-SKC-5 and RNAi-SKC-7 lines in comparison to the transgenic control line (Ctrl) under K + limiting conditions. The relative expressions of HcTrk1 (a) and HcHAK (c) K + transporters, and of the K + channel HcTOK2.2 (f) were not affected by K + shortage in RNAi-SKC-5 and RNAi-SKC-7 lines in comparison to the transgenic control line (Ctrl). Mean values are provided with the standard error (n = 6). Statistical differences were determined using a one-way ANOVA followed by Dunnett's test relative to Ctrl strain for each data point P < 0.05 ( � ), P < 0.01 ( �� ) and P < 0.001 ( ��� ).

Fig 6 .

 6 Fig 6. Potassium content in 2-month-old Pinus pinaster plants growing alone or in co-culture with Hebeloma cylindrosporum under K + -sufficient or -deficient conditions. Root and shoot contents were measured in nonmycorrhizal plants (NM) and plants inoculated with fungi transformed with the empty (Ctrl) or HcSKC-silencing vectors (RNAi-SKC-5 and RNAi-SKC-7), and growing at standard K + (SK medium, 1 mM K + , a) or low K + (LK medium, 0.05 mM K + , b). Mean values are provided with the standard error (n = 5-6). Different letters indicate significant differences between treatments according to one-way ANOVA followed by Tukey HSD post-hoc tests (P < 0.05). https://doi.org/10.1371/journal.pone.0242739.g006

  Table) as a template to propose a structural model for the fungal Shaker-like proteins (Fig 1C, S1 Fig).

PLOS ONE | https://doi.org/10.1371/journal.pone.0242739November 20, 2020

Acknowledgments

We would like to thank Dr. A.G. Pardo and Dr. M.J. Kemppainen (Laboratorio de Micologı ´a Molecular; Universidad Nacional de Quilmes, Argentina) for kindly providing the vector pSILBAγ. We also thank Dr. A. Kafle for assistance with some statistical analyses.

Funding: RL and CGG were financially supported by grants from the French Minister of Research and Technology, AD by funding of the ANR project "TRANSMUT" 2010 BLAN 1604 03. KG acknowledges support of the North Carolina Agriculture Research Service (NCARS) and the North Carolina Soybean Producers Association (2019-1656). SDZ is supported by the French ANR project "MYCOTRANS". The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All relevant data are within the manuscript and its Supporting Information files.

Supporting information

potassium and sodium regimes. Dry weights of empty vector (Ctrl), RNAi-SKC-5 and RNAi-SKC-7 fungal lines were determined after 28 days of culture in media containing high (+K and +Na) or low concentration (-K and -Na) of potassium (K + ) and sodium (Na + ), respectively. +K corresponds to 1 mM of K + , -K to 0.05 mM of K + , +Na to 1 mM of Na + , and -Na to 0.2 mM of Na + . Full recipes are provided in S1 Table . Mean values are provided with the standard deviation (n = 4-6). Different letters indicate significant differences between treatments according to one-way ANOVA followed by Tukey HSD post-hoc tests (P < 0.05). (TIF) S1 Table . Primer list. Restriction enzyme sites indicated in primer name, were underlined in the corresponding 5'-3' sequence. (TIF)

S2 Table. Protocol of media used to assess the impact of high and low potassium and sodium conditions on the biomass of transgenic fungal strains


Ambre Salis
email: ambre.salis@univ-lyon1.fr

Jean-Paul Léna

Thierry Lengagne

Response to allopatric combinatorial calls Great tits (Parus major) adequately respond to both allopatric combinatorial mobbing calls and their isolated parts

Keywords: combinatorial call, heterospecific communication, mobbing, Paridae, Parus major, semantic compositionality

HAL is

| INTRODUCTION

The vast majority of research in animal communication focuses on intraspecific interactions, while heterospecific exchanges of information are often interpreted as simple by-products of conspecific communication. For example, birds responding to each other alarm calls are more readily seen as by-product mutualism, where the information produced is mostly directed toward conspecifics, and by chance, is also useful to heterospecifics listeners, Bradbury & Verhencamp, 2011). However, recent studies have begun to unravel complex interactions between different species, especially in bird flocks [START_REF] Goodale | Response to conspecific and heterospecific alarm calls in mixed-species bird flocks of a Sri Lankan rainforest[END_REF][START_REF] Coppinger | Experimental manipulation of mixed-species flocks reveals heterospecific audience effects on calling[END_REF][START_REF] Carlson | What makes a 'community informant'? Reliability and anti-predator signal eavesdropping across mixed-species flocks of tits[END_REF]. The gathering of birds of different species, either all year round in tropical environments [START_REF] Goodale | The structure of mixed-species bird flocks, and their response to anthropogenic disturbance, with special reference to East Asia[END_REF] or during the non-breeding season in temperate environments (Dutour et al., 2019a), is widespread and usually thought to be beneficial for both foraging efficiency and predator defence [START_REF] Berner | An Experimental Analysis of Mixed-Species Flocking in Birds of Deciduous Woodland[END_REF]. These benefits mainly depend on an increased transfer of information; with vocal communication being one efficient strategy [START_REF] Bradbury | Principles of animal communication[END_REF]. Indeed, food calls [START_REF] Newman | Co-operative and non-co-operative bases of foodcalling[END_REF], contact calls [START_REF] Kondo | Contact calls: Information and social function: The information and function of contact calls[END_REF] and alarm calls have been thoroughly described [START_REF] Caro | Antipredator defenses in birds and mammals[END_REF]. In this latter category, two type of alarm calls are classically opposed. Flee calls, difficult to localize due to their pure tones (Marler, 1955), are produced when facing an urgent threat and provoke an escape or a freezing reaction from birds in the vicinity [START_REF] Caro | Antipredator defenses in birds and mammals[END_REF]. In opposition, mobbing calls are used when facing an intermediate threat (e.g. a non-hunting predator, [START_REF] Andrew | The Motivational Organisation Controlling the Mobbing Calls of the Blackbird (Turdus merula) IV. A General Discussion of the Calls of the Blackbird and Certain Other Passerines[END_REF], and often made of notes with broadband frequencies, helping localisation (Marler 1955). Mobbing typically induces the approach of other potential prey (conspecifics and heterospecifics) and active harassment of the predator [START_REF] Carlson | Mobbing[END_REF][START_REF] Curio | The adaptive significance of avian mobbing: I. Teleonomic hypotheses and predictions[END_REF] and is consequently one great opportunity to study heterospecific communication [START_REF] Hurd | Interspecific attraction to the mobbing calls of black-capped chickadees (Parus atricapillus )[END_REF]Dutour et al., 2019a;[START_REF] Yu | Heterospecific alarm-call recognition in two warbler hosts of common cuckoos[END_REF].

Studies about the mechanisms underlying heterospecific communication suggest that both learning and innate processes are in place: associative learning has been evidenced both in the adult response to mobbing calls and in the ontogeny of mobbing behaviour in fledglings [START_REF] Magrath | Eavesdropping on heterospecific alarm calls : From mechanisms to consequences: Interspecific eavesdropping[END_REF][START_REF] Potvin | Birds Learn Socially to Recognize Heterospecific Alarm Calls by Acoustic Association[END_REF][START_REF] Carlson | Wild fledgling tits do not mob in response to conspecific or heterospecific mobbing calls[END_REF][START_REF] Dutour | The role of associative learning process on the response of fledgling Great tits (Parus major) to mobbing calls[END_REF]. This could well explain higher responsiveness toward sympatric species than toward allopatric ones [START_REF] Wheatcroft | Learning and signal copying facilitate communication among bird species[END_REF]. Still, responsiveness towards allopatric species is rather widespread [START_REF] Johnson | Mechanisms of heterospecific recognition in avian mobbing calls[END_REF][START_REF] Fallow | Sound familiar? Acoustic similarity provokes responses to unfamiliar heterospecific alarm calls[END_REF][START_REF] Randler | A possible phylogenetically conserved urgency response of Great tits (Parus major) towards allopatric mobbing calls[END_REF][START_REF] Dutour | Mobbing calls : A signal transcending species boundaries[END_REF][START_REF] Yu | Heterospecific alarm-call recognition in two warbler hosts of common cuckoos[END_REF] and, although not sufficient in itself, phylogenetic conservation or convergence of mobbing call characteristics could also well explain such a phenomenon (Stefanski & Falls, 1972;[START_REF] Fallow | Sound familiar? Acoustic similarity provokes responses to unfamiliar heterospecific alarm calls[END_REF][START_REF] Randler | A possible phylogenetically conserved urgency response of Great tits (Parus major) towards allopatric mobbing calls[END_REF][START_REF] Wheatcroft | Rates of signal evolution are associated with the nature of interspecific communication[END_REF][START_REF] Dutour | Mobbing calls : A signal transcending species boundaries[END_REF].

The acoustic structure of mobbing calls has been described in many passerine species [START_REF] Ficken | A Comparative Analysis of Passerine Mobbing Calls[END_REF] and recent studies suggest that some Parids use a combinatorial call sequence to encode information in mobbing calls: the association of introductory notes with strong frequency modulation (here referred as the I notes, Figure 1) and several broadband elements (referred as D notes, following [START_REF] Hailman | The 'chick-a-dee' calls of Parus atricapillus : A recombinant system of animal communication compared with written English[END_REF]. Alone, introductory notes are used to warn neighbours about a threat and induce vigilance behaviour; while D calls have a recruitment function (in various contexts such as mating or foraging) and thus trigger an approach of conspecifics [START_REF] Suzuki | Wild Birds Use an Ordering Rule to Decode Novel Call Sequences[END_REF]. Although the use of a combinatory call sequence in eliciting mobbing behaviour of a conspecific receiver has been evidenced in several passerine birds [START_REF] Suzuki | Wild Birds Use an Ordering Rule to Decode Novel Call Sequences[END_REF][START_REF] Engesser | Meaningful call combinations and compositional processing in the southern pied babbler[END_REF][START_REF] Dutour | Syntax manipulation changes perception of mobbing call sequences across passerine species[END_REF], until now, little has been done to understand to which extent this process also applies to heterospecific mobbing sequences.

Yet, such test may bring information on how birds perceive and deal with combinations, and shed light on whether it could be considered semantic compositionality in the human sense [START_REF] Dutour | The role of associative learning process on the response of fledgling Great tits (Parus major) to mobbing calls[END_REF]. Semantic compositionality (also termed compositional syntax by some authors) occurs when a combination of words has a meaning emerging from its individual parts (each part having its own meaning), and when the order of such parts is important [START_REF] Engesser | Combinatoriality in the vocal systems of nonhuman animals[END_REF][START_REF] Suzuki | Syntactic rules in avian vocal sequences as a window into the evolution of compositionality[END_REF][START_REF] Zuberbühler | Evolutionary roads to syntax[END_REF]. Debates on whether the "I notes followed by D notes" mobbing sequence can approach human compositional syntax have been profuse (Bolhuis et al., 2018a;2018b;Suzuki et al., 2018). Determining how Parids respond to unknown calls but with similar syntax would improve the current knowledge of birds' ability to process syntactic rules. Only one study [START_REF] Suzuki | Wild Birds Use an Ordering Rule to Decode Novel Call Sequences[END_REF] demonstrated that Japanese tits respond to artificial mobbing sequences made of the concatenation of introductory notes of their own species and D notes of another sympatric Parid species, the willow tit (Poecile montanus).

By testing birds with allopatric calls, we here have the advantage of ruling out any learning effect, and focus on birds' ability to generalize from similar acoustic properties.

Several studies have already showed the European great tit's (Parus major) responsiveness to the allopatric black-capped chickadee's (Poecile atricapillus) mobbing call sequence, although the intensity of the responsiveness varied to some extent between studies [START_REF] Randler | A possible phylogenetically conserved urgency response of Great tits (Parus major) towards allopatric mobbing calls[END_REF][START_REF] Dutour | Mobbing calls : A signal transcending species boundaries[END_REF]. Acoustic properties of their mobbing calls have been extensively studied (black-capped chickadee: [START_REF] Landsborough | Variation in chick-a-dee call sequences, not in the fine structure of chick-a-dee calls, influences mobbing behaviour in mixed-species flocks[END_REF], great tit: Kalb et al., 2019b), and both species display a sequence that involves a combination of introductory and D notes in a fixed order [START_REF] Templeton | Allometry of Alarm Calls : Black-Capped Chickadees Encode Information About Predator Size[END_REF][START_REF] Dutour | Syntax manipulation changes perception of mobbing call sequences across passerine species[END_REF], see Figure 1). Therefore, these two species are particularly suitable for testing whether combinatorial heterospecific mobbing calls are decoded in a similar way than intraspecific ones. Here, we first investigated how great tits alter their behavioural responses when exposed to a conspecific mobbing call sequence or its isolated decomposition into introductory or D notes, in order to verify that the combinatory sequence is an indispensable prerequisite to elicit mobbing in the receiver. We measured vigilance effort, willingness to approach [START_REF] Suzuki | Wild Birds Use an Ordering Rule to Decode Novel Call Sequences[END_REF][START_REF] Dutour | Syntax manipulation changes perception of mobbing call sequences across passerine species[END_REF], propensity to mob as well as level of excitement. We then examined whether great tits adopt similar behavioural responses when they are exposed to a black-capped chickadee mobbing call sequences or their isolated decomposition into introductory or D notes. According to previous studies, we should expect that great tits would be sensitive to mobbing call sequences of the black-capped chickadee, but less responsive than to those of conspecifics [START_REF] Randler | A possible phylogenetically conserved urgency response of Great tits (Parus major) towards allopatric mobbing calls[END_REF][START_REF] Dutour | Mobbing calls : A signal transcending species boundaries[END_REF]2019a). We hypothesized that the similarity in call organisation (i.e. introductory notes followed by D notes) between great tit and black-capped chickadee calls would engender similar behaviours (scanning to introductory notes, approaching to D notes, and displaying a complete mobbing behaviour to the complete sequence).

| METHODS

| General organisation

Our aim was to test whether great tits are able to adequately respond to isolated parts of never heard before heterospecific mobbing sequences. We performed a call-to-behaviour experiment, a classical method used to study acoustic communication [START_REF] Schlenker | Formal monkey linguistics[END_REF]. More specifically, we set up 8 different treatments: first, great tits were subjected to stimuli with the complete, natural (I-D) mobbing call sequence of black-capped chickadees (BCCH), then only with the introductory notes or only with the D notes. Second, great tits were subjected to the three same types of sequence (I-D, I alone and D alone) but from other great tits (GRTI).

Finally, two different types of controls were broadcast: background noise or the song of the black-capped chickadee, to distinguish the response due to novelty from the actual mobbing response. For each of the eight treatments, we created 20 different stimuli with different callers, and then performed 20 independent tests (i.e. different receiver and emitter for each of the 160 tests performed).

| Preparation of stimuli

Our stimuli of great tits and black-capped chickadees were designed using recordings obtained from the Xeno-canto online database (www.xeno-canto.org) and the Macaulay Library (www.macaulaylibrary.org). For both species, we only used good quality recording files (A or B grades) under the denomination "Alarm call"/"Call".

Both species produce a mobbing call with an association of introductory and D notes (Fig. 1).

However, as these notes can also be produced in other context [START_REF] Freeberg | Receivers respond differently to chick-a-dee calls varying in note composition in Carolina chickadees, Poecile carolinensis[END_REF][START_REF] Kalb | Subtle variations in mobbing calls are predator-specific in great tits (Parus major)[END_REF], we controlled several features to ensure that the selected recordings represented mobbing call sequences. To begin, selected recordings were all made of the same introductory and D notes reported in [START_REF] Baker | Mobbing calls of Black-Capped chickadees : Effects of urgency on call production[END_REF] and [START_REF] Templeton | Allometry of Alarm Calls : Black-Capped Chickadees Encode Information About Predator Size[END_REF] for the black-capped chickadee and in [START_REF] Randler | A possible phylogenetically conserved urgency response of Great tits (Parus major) towards allopatric mobbing calls[END_REF] and Kalb et al. (2019a) for the great tit.

In addition, for both species, D notes length is known to vary with the context [START_REF] Templeton | Allometry of Alarm Calls : Black-Capped Chickadees Encode Information About Predator Size[END_REF][START_REF] Kalb | Subtle variations in mobbing calls are predator-specific in great tits (Parus major)[END_REF]; we therefore checked that the D notes in our stimuli had the same length (X = 0.05 s ± 0.01 for the great tit, X = 0,18 s ± 0,02 for the black-capped chickadee).

Finally, we verified that no introductory notes used in food or flight related contexts were the most frequent in any of our recordings (i.e. the C notes for the black-capped chickadee associated with flight; and the G, H,I and M notes associated with food for the great tit, [START_REF] Freeberg | Receivers respond differently to chick-a-dee calls varying in note composition in Carolina chickadees, Poecile carolinensis[END_REF][START_REF] Kalb | Subtle variations in mobbing calls are predator-specific in great tits (Parus major)[END_REF].

From these recording files, we designed 40 1 min mobbing sequences of great tits and blackcapped chickadees (20 for each species, each obtained from a different sender) using Avisoft-SASLab software. To allow comparison between the responses of black-capped chickadees and great tits, we constructed every mobbing sequence with a similar duty cycle (i.e. the amount of time a signal is present over a specified time, [START_REF] Landsborough | Variation in chick-a-dee call sequences, not in the fine structure of chick-a-dee calls, influences mobbing behaviour in mixed-species flocks[END_REF] and mobbing call repetition (30 calls per minute, natural range of repetition rate, [START_REF] Suzuki | Wild Birds Use an Ordering Rule to Decode Novel Call Sequences[END_REF].

Consequently, each mobbing call emitted by both species had a similar total introductory note duration (0.31 s ± 0.06, mean ± SD) and D duration (0.50 s ± 0.07), but not the same number of notes, since black-capped chickadee's D notes are longer (Figure 1). Isolated introductory notes and isolated D notes stimuli for each species (20 stimuli for each treatment, for a total of 80 stimuli) were constructed by replacing the unwanted notes (D notes for the I notes treatment, and I notes for the D notes treatment) by background noise from the original recording, therefore keeping the same duty cycle of each call type as in the original soundtrack, since this acoustic feature seems more important than the number of notes/elements in eliciting mobbing behaviour [START_REF] Landsborough | Variation in chick-a-dee call sequences, not in the fine structure of chick-a-dee calls, influences mobbing behaviour in mixed-species flocks[END_REF]. We also constructed 20 background noise stimuli extracted from the original recordings (control treatment hereafter referred as C1) and 20 control stimuli using recordings of songs of black-capped chickadees (the two-note "feebee" song, control treatment hereafter referred as C2) from Xeno-canto database, with a rate of 18 song/min (natural rate, calculated from the original recordings). Each of these 160 stimuli were cleared of any other bird calls, background noise was reduced, and amplitude homogenized.

| Field tests

Tests were performed in the east of France at the beginning of the breeding season, in spring (April 2019) in a radius of 25 km around Lyon (45°53'39.3"N 5°03'05.7"E).

Each test was performed by two field assistants. One of them was assigned to the playback operation, while the other was kept unaware of the selected stimulus (using headphones with music) and assigned to the behavioural recording of the focal bird. For each test, after detecting an individual using binoculars, the focal bird was observed for at least 1 min, and the pre-test behaviour (usually singing or foraging) was noted. If the animal displayed an alarm behaviour, no test was performed. A loudspeaker was placed 16 meters from the bird (16.79 ± 6.27 m), and at less than 3 meters of a potential roost (bushes/ trees) to allow the approach of the focal bird. The two field assistants were then placed away (minimum of 15 meters to both the bird and the loudspeaker) before playing the stimulus with a remote control. All stimuli were broadcast using a Shopinnov 20 W loudspeaker with an intensity of 79.8 +/-1.9 dB(C) (measured at 1 m from the loudspeaker using Lutron SL-4001, C weighting, slow settings, re: 20 µPa).

Tests were carried out between 06:00 and 13:00 h during calm and dry days. To avoid pseudoreplication, all selected focal birds were distant from one another by at least 100 meters [START_REF] Dutour | Seasonal variation in mobbing behaviour of passerine birds[END_REF]Dutour et al., 2020). Two or three different calling males were usually detected within 100 m, we never went on the same path twice, and we made sure that no bird was following us. We are thus confident that each focal bird participated in only one test.

Moreover, each of the 8 treatments were tested each day in a different order to avoid autocorrelation among the responses of focal birds.

| Behavioural observations

During 1 min of playback, four types of behavioural states were assessed, (1) Vigilance effort as indicated by the number of horizontal scans displayed (the number of movements that birds made with their heads from left to right or right to left, approximately a 180° turn; [START_REF] Suzuki | Wild Birds Use an Ordering Rule to Decode Novel Call Sequences[END_REF]; (2) Approach inferred using a dichotomic variable (approaching at least halfway from their starting point) measured with a Leica DISTO D210 telemeter, (3) Mobbing behaviour, recorded when birds approached, scanned and produced mobbing calls (assessed by the person without headphones). This variable was also a dichotomic variable (1 = mobbing, 0 = not mobbing); we therefore had to transform the continuous scanning variable into a dichotomic one. We considered that a bird had scanned if it produced more than the 99% CI of the BCCH-Song Control, hence 5 scans (99% CI: [1.75; 4.2]). ( 4) The presence of either wing flicking behaviour or tail wagging (both described in [START_REF] Hinde | The behaviour of the Great tit (Parus major) and some other related species[END_REF][START_REF] Carlson | Sparrowhawk movement, calling, and presence of dead conspecifics differentially impact blue tit (Cyanistes caeruleus) vocal and behavioral mobbing responses[END_REF][START_REF] Zuberbühler | Evolutionary roads to syntax[END_REF] was used as a proxy for a higher excitement from the birds, since these behaviours were previously associated with intense mobbing [START_REF] Hinde | The behaviour of the Great tit (Parus major) and some other related species[END_REF][START_REF] Andrew | The Motivational Organisation Controlling the Mobbing Calls of the Blackbird (Turdus merula) IV. A General Discussion of the Calls of the Blackbird and Certain Other Passerines[END_REF][START_REF] Carlson | Sparrowhawk movement, calling, and presence of dead conspecifics differentially impact blue tit (Cyanistes caeruleus) vocal and behavioral mobbing responses[END_REF]. All observations were made using binoculars and recorded on a voice recorder (Sony ICD-PX370) by the same two trained persons. We limited disturbance for birds with short duration tests, and testing birds only once. Moreover, after our tests, we checked that all birds returned to their pre-test behaviour in less than 5min.

| Statistical Analysis

All analyses were done in R Studio 1.2.5033 (R core development team 2018). The propensity to mob and the propensity to approach the loudspeaker (both dichotomic variables) were analysed using a logistic regression (binomial distribution and logit link function) with the treatment (the two type of controls, and each type of sound sequences for each species) as an explanatory term. We first performed a contrast analysis to verify that the responsiveness of great tits did not significantly differ between the two control tests (C1 versus C2). We then pooled the two controls and tested whether the responsiveness to each type of sound sequence (I alone, D alone, or I-D) of both species differed from this pooled control tests using a Dunnet-Hsu correction. A partial analysis (discarding both control tests) was performed to compare the response of tits to the three types of sound sequences (I alone, D alone, or I-D) according to the emitter species. More specifically we examined the interaction between both factors to test whether the responsiveness variation among the sound sequences differed according to the issuing species. We used odds ratio (OR) for post-hoc comparisons (odds.ratio, package questionr). For the approach variable, we first made sure that the complete (I-D) and D alone sequences engendered a similar response, then pooled these two treatments before comparing them to responses elicited by sequences with only with introductory notes.

The number of scans displayed by focal birds during tests was analysed using a generalized linear model (negative binomial error distribution and log-link function, glm.nb, package MASS, since an overdispersion of 2.09 was detected with a Poisson error distribution) and the actual time seeing the bird in the one minute observation as offset. We followed the same framework than for mobbing and approach behaviour; except that further comparisons between the types of sound sequences and emitter species were performed using contrasts analyses (functions emmeans and multcomp::cld, packages emmeans and multcompView).

When reporting the comparisons of interest in the results section, we relate to the different treatments with first the emitter species in the stimuli (BCCH for black capped chickadees stimuli, GRTI for those with great tits' calls), then with the parts of the call sequence present ('Complete' if both Introductory notes and D notes were kept, 'I' for stimuli for which the D notes were discarded, and 'D' for stimuli in which the introductory notes were removed).

Finally, since the occurrence of signs of higher excitement was low (19 out of 160 tests), we used a Fisher exact test to compare the proportion of individuals that displayed signs of higher excitement through the different sound treatments and the pooled controls; then calculated the odds ratio for the highest responses.

| How do great tits respond to the complete conspecific mobbing sequence and its isolated parts?

If a complete mobbing sequence is a prerequisite for eliciting mobbing in receiver, great tits should display a mobbing response (approach, scan and call) towards stimuli with complete mobbing call sequence (Introductory notes + D notes), but should only scan to introductory notes and approach to D notes.

Our results show that birds mobbed more often toward the conspecific complete mobbing sequences than toward the controls (pooled controls vs. GRTI-Complete: OR = 0.01, 95 % CI [0.0006; 0.19], p = .002; Figure 2). For treatments with only introductory notes or D notes, mobbing response was infrequent and did not differ from controls (all p > .05). In addition, only the complete mobbing sequences triggered excitement in birds (Fisher exact test, p < .001, Controls vs GRTI-Complete: OR = 0.03, 95%CI [0.004, 0.27], p = .002).

Great tits scanned more toward any call than the controls (all p < .001; Figure 3), and scanned more toward Control 2 (black-capped chickadee song) than Control 1 (background noise; C1 vs C2: z = 2.73, p = .006). As expected, introductory notes stimuli triggered as much scanning behaviour as complete call sequences (GRTI-I vs GRTI-Complete: z = 1.27, p = .20).

Unexpectedly however, birds also scanned as much toward isolated D notes stimuli as toward stimuli with introductory notes (GRTI-D vs pooled GRTI-I /GRTI-Complete: z = 0.76, p = .45).

Finally, for the approach behaviour, birds responded more often to both the complete and isolated D notes stimuli than to the controls (all p < .05; Figure 4). They also approached complete mobbing sequences and isolated D notes sequences more often (GRTI-D vs GRTI-Complete: OR = 0.50, 95% CI: [0.12; 1.89], p = .31) and approached isolated introductory notes stimuli less often than stimuli with D notes (GRTI-I vs pooled GRTI-D/ GRTI-Complete: OR = 0.20, 95% CI: [0.06; 0.63], p = .008).

| Does responsiveness to different types of call sequences vary according to sender species?

If great tits decrypt allopatric mobbing calls in the same way as conspecific calls, we should not detect interaction between treatment and emitter species whatever the behavioural response.

As for conspecific calls, introductory notes should trigger vigilance, D notes trigger approach, and the complete sequence (I-D) trigger mobbing and excitement signs.

For mobbing behaviour, no interaction between the emitter species and the type of call was detected (GLM, Species*Test: χ² = 1.07, df = 2, p = .58; Figure 2), but great tits mobbed conspecific more than allopatric calls (GLM, Species effect: χ² = 4.68, df = 1, p = .03). Great tits mobbed only towards the complete allopatric mobbing call sequences (Controls vs BCCH-Complete: OR = 0.04, 95 % CI [0.002; 0.89], p = .04), and only this sequence triggered excitement in birds (Controls vs BCCH-Complete: OR = 0.10, 95%CI [0.01, 0.99], p = .049).

For the scanning behaviour, no difference between allopatric and conspecific calls was detected (GLM, Test*Species effect: χ² = 0.23, df = 2, p = .89, Species effect: χ² = 2.11, df = 1, p = .14, n = 120; Figure 3). Specifically, great tits scanned as much black-capped chickadee's complete mobbing sequences than black-capped chickadee's isolated introductory notes stimuli (BCCH-Complete vs BCCH-I: z = 1.27, p = .20), but also scanned as much black-capped chickadee's isolated D notes stimuli as toward calls containing introductory notes (BCCH-D vs pooled BCCH-I / BCCH-Complete: z = 0.76, p = .45).

As was the case for mobbing behaviour, great tits approached black-capped chickadees calls less than great tits calls (GLM, Test*Species effect: χ² = 0.33, df = 2, p = .85, Species effect: χ² = 15.82, df = 1, p < .001, n = 120; Figure 4). Great tits approached black-capped chickadee's complete call sequences and black-capped chickadee's isolated D notes stimuli more than controls (both p < .05). They also approached complete mobbing sequences as much as isolated D notes sequences (BCCH-D vs BCCH-Complete: OR = 0.78, 95% CI: [0.19; 3.15], p = .72).

The slight increase of approach toward sequences with D notes compared to isolated introductory notes sequences was not statistically significant (BCCH-I vs pooled BCCH-D / BCCH-Complete: OR = 0.29, 95% CI: [0.04; 1.25], p = .13). Still, the effects sizes are similar between species' calls (0.20 vs 0.29, confidence intervals largely overlapping, [START_REF] Cumming | Error bars in experimental biology[END_REF], indicating that great tits altered their behaviour the same way (i.e. approached) to both species' calls.

| DISCUSSION

Overall, less birds responded to the allopatric calls than to the conspecific calls. Yet, we found that whatever the emitter species (conspecific or allopatric), Great tits mobbed (scanned, approached and called but also displayed signs of excitement) when the sequence was complete (introductory notes + D notes). Moreover, isolated introductory notes triggered vigilance while isolated D notes triggered approach. Unexpectedly, Great tits scanned also when hearing isolated D notes of both great tits and black capped chickadees.

| Response to conspecific calls: a new argument towards semantic compositionality?

Only the combinatorial mobbing sequence (i.e. I-D) triggered mobbing behaviour in Great tits, while isolated introductory notes triggered scanning and D sequences induced an approach to the loudspeaker. This result is in adequacy with the emerging hypothesis of semantic compositionality in Parids [START_REF] Engesser | Combinatoriality in the vocal systems of nonhuman animals[END_REF][START_REF] Suzuki | Syntactic rules in avian vocal sequences as a window into the evolution of compositionality[END_REF][START_REF] Zuberbühler | Evolutionary roads to syntax[END_REF]. To be true, this hypothesis must meet several criteria: (i) A different order should trigger a different response, which has been demonstrated for conspecific calls in the Japanese tit [START_REF] Suzuki | Wild Birds Use an Ordering Rule to Decode Novel Call Sequences[END_REF], but also recently for the great tit toward the black-capped chickadee calls (Dutour et al., 2020). (ii) The whole sequence should not only be the sum of its different parts, but have a new emergent meaning, which is the main result of our study. Here, great tits not only scanned and approached, but also produced mobbing calls and displayed signs of excitement such as wing-flicking and tail wagging. This suggests that they extract a semantic content in I-D call sequences different from the simple sum of introductory and D call sequences: the meaning of the complete call is related to the parts [START_REF] Engesser | Combinatoriality in the vocal systems of nonhuman animals[END_REF], but is not simply the sum of these parts. Finally, (iii) the two parts, when isolated, should still be meaning-bearing units. This latter has been confirmed in [START_REF] Suzuki | Wild Birds Use an Ordering Rule to Decode Novel Call Sequences[END_REF][START_REF] Dutour | Syntax manipulation changes perception of mobbing call sequences across passerine species[END_REF] but is only partly corroborated in the present study. Indeed, for the scanning behaviour, no difference was found between isolated introductory and D calls. Yet, the isolated part meaning criterion is of primary importance to consider the great tits' coding system as semantic and not only phonological compositionality (i.e. when elements are combined but meaningless on their own, like in birdsong, [START_REF] Marler | Animal communication and human language[END_REF]. If great tits do also scan the environment when hearing isolated D calls, the vigilance meaning of introductory notes may not be specific, consequently weakening the compositionality hypothesis.

Several non-mutually exclusive explanations may be proposed: (i) Scanning, interpreted as a vigilance behaviour, may be caused by many environmental and internal parameters. For instance, high stress levels may increase vigilance and scanning behaviour. Nonetheless, individuals are known to deal differently with stress [START_REF] Koolhaas | Coping styles in animals: Current status in behavior and stress-physiology[END_REF] and both a freezing and a highly active individual can have high levels of stress hormones, although behavioural measures such as scanning would be different [START_REF] Careau | Energy metabolism and animal personality[END_REF]. Consequently, we can hypothesize that individuals with different coping styles (i.e. how individuals react to stress) would give opposite results in scanning behaviour, and therefore that scanning behaviour is not a relevant variable in these contexts. (ii) Protocol choices such as the distance to the focal bird may impact bird behaviour. Since mobbing calls are meant to indicate a potential threat in the vicinity, the willingness to scan the environment may be strongly dependent on the proximity to this potential threat [START_REF] Seppänen | Social information use is a process across time, space, and ecology, reaching heterospecifics[END_REF]. While Suzuki and colleagues (2016) were at ~ 5 meters from the birds, the present study tested birds at ~ 16m. We can hypothesize that distance is used by great tits as a proxy for urgency: a more distant sound would engender increased scanning for any sound, while closer sounds would trigger more urgent response such as direct physical movement (i.e. approaching or fleeing away). Nonetheless, the control (black-capped chickadee song) we used provoked less scanning than any of the conspecific calls. Even if distance was probably implicated, this parameter cannot explain the absence of difference between all conspecific treatments. Finally, we cannot rule out the hypothesis that previous work may have been biased by observer's expectations (Rosenthal & Fode, 1963;[START_REF] Holman | Evidence of Experimental Bias in the Life Sciences: Why We Need Blind Data Recording[END_REF], as we are the first study for which the main observer did not hear the broadcast treatment. Nevertheless, our own protocol could also be improved. For example, video recording the great tits to confirm the reliability of our observations was not possible but will be prioritized in future experiments.

| Responding to allopatric calls: less response from great tits, but similar in kind.

A second important result in our study is that great tits always responded more to conspecifics than to heterospecifics. This result is congruent with various studies using heterospecific call sequences, and particularly Randler (2012) that found a lower response of great tits to black-capped chickadees calls, but not with Dutour et al. (2020) who did not detect differences in approach when comparing conspecific and heterospecific calls in the great tit. This low level of response could be explained by the choice we made when preparing our stimuli. Indeed, we chose to control by the duty cycle to compare the two species: whatever the length of introductory and D notes, great tits all heard calls of the same length for both great tits and black-capped chickadees calls. As D notes of black-capped chickadees are longer, the playbacks for this species contained only 2-3 D notes per mobbing call but 6-8 D notes for great tits. Yet, in Parids, D notes are probably used to code for the level of threat [START_REF] Templeton | Allometry of Alarm Calls : Black-Capped Chickadees Encode Information About Predator Size[END_REF][START_REF] Kalb | Subtle variations in mobbing calls are predator-specific in great tits (Parus major)[END_REF], and this information is also considered in heterospecific communication [START_REF] Templeton | Nuthatches eavesdrop on variations in heterospecific chickadee mobbing alarm calls[END_REF][START_REF] Randler | Heterospecifics do not respond to subtle differences in chaffinch mobbing calls : Message is encoded in number of elements[END_REF][START_REF] Randler | A possible phylogenetically conserved urgency response of Great tits (Parus major) towards allopatric mobbing calls[END_REF]. Still, recent work aiming at disentangling the effect of number of notes and duty cycle found that the latter was of greater importance for the birds' response [START_REF] Landsborough | Variation in chick-a-dee call sequences, not in the fine structure of chick-a-dee calls, influences mobbing behaviour in mixed-species flocks[END_REF]; our choice was therefore in adequacy with current knowledge on the subject. Either way, the impact of duty cycle or number of D notes indicates that gradual factors may be as important as semantic compositionality in the decoding processes of the great tits. An additional explanation for this increased response toward conspecific calls may be that great tits vary their investment depending on the reliability of the information [START_REF] Magrath | Eavesdropping on heterospecific alarm calls : From mechanisms to consequences: Interspecific eavesdropping[END_REF]. There is a trade-off between the risk of not responding (potential death) and the energy lost in investing in false mobbing calls. The first option leading to more important negative consequences than the second, responding to not wholly reliable cues could be selected (the "Better safe than sorry" hypothesis, [START_REF] Haftorn | Contexts and Possible Functions of Alarm Calling in the Willow Tit, Parus montanus; The Principle of « Better Safe than Sorry[END_REF]. Still, birds can take into account the quality of the sender [START_REF] Goodale | Response to conspecific and heterospecific alarm calls in mixed-species bird flocks of a Sri Lankan rainforest[END_REF][START_REF] Magrath | An avian eavesdropping network : Alarm signal reliability and heterospecific response[END_REF]. Conspecific calls being more reliable than unknown calls, a lower number of birds responding is therefore expected and is congruent with results in other species [START_REF] Johnson | Mechanisms of heterospecific recognition in avian mobbing calls[END_REF][START_REF] Dutour | Mobbing calls : A signal transcending species boundaries[END_REF].

Even if their response was lower than to conspecifics calls, great tits approached to D notes and scanned to introductory notes of the black-capped chickadee. Moreover, more birds mobbed to complete allopatric mobbing sequence than to either of the two controls. Both the introductory and D notes share some key characteristics (sharp modulation of frequency for the first, broadband frequency resulting in a harsh sound for the latter, Fig 1). Therefore, great tits can respond to allopatric calls that are relatively similar to their own. A misinterpretation due to the notes' similarity may explain these results. Nevertheless, humans easily distinguish these different sounds and it seems thus unlikely that birds, known for their fine acoustic discrimination abilities [START_REF] Bloomfield | All "chick-a-dee" calls are not created equally Part II. Mechanisms for discrimination by sympatric and allopatric chickadees[END_REF][START_REF] Fishbein | Sound sequences in birdsong: How much do birds really care?[END_REF], performed worse than humans. More likely is the generalization of the introductory and D notes, implying that great tits were able to differentiate the two close stimuli, but focused on their similarity when responding [START_REF] Ghirlanda | A century of generalization[END_REF]. Indeed, generalization has recently been proposed as a mechanism explaining heterospecific acoustic communication [START_REF] Fallow | Sound familiar? Acoustic similarity provokes responses to unfamiliar heterospecific alarm calls[END_REF]2013).

The ability to generalize a syntactic rule makes sense since some birds can generalize the underlying structure of a sound (differentiating new XYX sounds from new XXY sounds, [START_REF] Spierings | Budgerigars and zebra finches differ in how they generalize in an artificial grammar learning experiment[END_REF]. We can therefore hypothesize that, when hearing I-D mobbing calls, great tits generalize from their own call structure and expect to find a predator nearby. By responding to allopatric I-D calls by mobbing, isolated introductory notes by scanning, and D notes by approaching, great tits give us another argument toward the compositionality theory.

Consequently, as they respond to heterospecific and conspecific calls in the same manner, we can hypothesize that great tits probably attribute some general meaning of alert to introductory notes and recruitment to D notes, but also a general mobbing meaning to I-D sequences, which states that the concatenation of introductory and D notes may be seen as true semantic compositionality. Nonetheless, although our results seem to corroborate that hypothesis, they do not directly prove the neural processes implied in these responses. Our work is therefore one encouraging first step towards a stronger understanding of syntactic-like processes in the Paridae. We advocate for cautious replicates in other species and trans-disciplinary collaborations such as with neurology to clarify this subject.

FIGURE 1 :

 1 FIGURE 1: Spectrograms of typical mobbing call of (a) great tits (Parus major), and (b) black-

FIGURE 2 :

 2 FIGURE 2: Proportion of individuals that mobbed (approached, scanned the environment, and

FIGURE 3 :

 3 FIGURE 3: Estimates (mean number of scans produced by great tits in 1 min) and 95% CI

FIGURE 4 :

 4 FIGURE 4: Proportion of individuals approaching the speaker given the emitter species and the

Acknowledgments:

We are grateful to Julie Ruffion, Charlotte Bourbon and Jean Capelle for efficient conduct of fieldwork and useful discussions on the protocol. We also thank Bernard Kaufmann for English language checking and for his constructive comments on the manuscript. This work was supported by French Ministry of Research and Higher Education funding (to A.S. PhD grants 2019-2022). All the authors comply with the ASAB/ABS Guidelines for the Use of Animals in Research and with The European Code of Conduct for Research Integrity.

This project was conducted in accordance with the current laws in France and followed rules of procedures of the UMR5023; but did not need any special permit. We have no conflict of interest to declare. Data used for statistical analysis as such as GPS points of all tests can be made available upon request.

ORCID

A.S. https://orcid.org/0000-0003-0753-8981 J-P.L ?? T.L. https://orcid.org/0000-0001-7840-6068 M.D. https://orcid.org/0000-0001-8093-9832