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Zinc (Zn) is a plant essential micronutrient involved in a wide range of cellular processes. Ectomycorrhizal fungi (EMF) are known to play a critical role in regulating plant Zn status. However, how EMF control uptake and translocation of Zn and other nutrients in plant roots under different Zn conditions is not well known. Using X-ray fluorescence imaging, we found the EMF species Suillus luteus increased pine root Zn acquisition under low Zn concentrations and reduced its accumulation under higher Zn levels. By contrast, non-mycorrhizal pine roots exposed to high Zn indiscriminately take up and translocate Zn to root tissues, leading to Zn stress. Regardless of S. luteus inoculation, the absorption pattern of Ca and Cu was similar to Zn. Compared to Ca and Cu, effects of S. luteus on Fe acquisition were more marked, leading to a negative association between Zn addition and Fe concentration within EMF roots. Besides, higher nutrient accumulation in the fungal sheath, compared to hyphae inhabiting between intercellular space of cortex cells, implies the fungal sheath serves as a barrier to regulate nutrient transportation into fungal Hartig net. Our results demonstrate the crucial roles EMF play in plant nutrient uptake and how fungal partners ameliorate soil chemical conditions either by increasing or decreasing element uptake.

Trees are able to colonize, establish and survive in a wide range of soils through associations with ectomycorrhizal (EcM) fungi. Proper functioning of EcM fungi implies the differentiation of structures within the fungal colony. A symbiotic structure is dedicated to nutrient exchange and the extramatricular mycelium explores soil for nutrients. Eventually, basidiocarps develop to assure last stages of sexual reproduction. The aim of this study is to understand how an EcM fungus uses its gene set to support functional differentiation and development of specialized morphological structures. We examined the transcriptomes of Laccaria bicolor under a series of experimental setups, including the growth with Populus tremula x alba at different developmental stages, basidiocarps and free-living mycelium, under various conditions of N, P and C supply. In particular, N supply induced global transcriptional changes, whereas responses to P supply seemed to be independent from it. Symbiosis development with poplar is characterized by transcriptional waves. Basidiocarp development shares transcriptional signatures with other basidiomycetes. Overlaps in transcriptional responses of L. bicolor hyphae to a host plant and N/C supply next to co-regulation of genes in basidiocarps and mature mycorrhiza were detected. Few genes are induced in a single condition only, but functional and morphological differentiation rather involves fine tuning of larger gene sets. Overall, this transcriptomic atlas builds a reference to study the function and stability of EcM symbiosis in distinct conditions using L. bicolor as a model and indicates both similarities and differences with other ectomycorrhizal fungi, allowing researchers to distinguish conserved processes such as basidiocarp development from nutrient homeostasis.

While there has been significant progress characterizing the 'symbiotic tool kit' of ectomycorrhizal (ECM) fungi, how host specificity may be encoded into ECM fungal genomes remains poorly understood. We conducted a comparative genomic analysis of ECM fungal host specialists and generalists, focusing on the specialist genus Suillus. Global analyses of genome dynamics across 46 species were assessed, along with targeted analyses of three classes of molecules previously identified as important determinants of host specificity: small secreted proteins (SSPs), secondary metabolites (SMs), and G-protein coupled receptors (GPCRs). Relative to other ECM fungi, including other host specialists, Suillus had highly dynamic genomes including numerous rapidly evolving gene families and many domain expansions and contractions. Targeted analyses supported a role for SMs but not SSPs or GPCRs in Suillus host specificity. Phylogenomic-based ancestral state reconstruction identified Larix as the ancestral host of Suillus, with multiple independent switches between white and red pine hosts. These results suggest that like other defining characteristics of the ECM lifestyle, host specificity is a dynamic process at the genome level. In the case of Suillus, both SMs and pathways involved in the deactivation of reactive oxygen species appear to be strongly associated with enhanced host specificity.

Trees are able to colonize, establish and survive in a wide range of soils through associations with ectomycorrhizal (EcM) fungi. Proper functioning of EcM fungi implies the differentiation of structures within the fungal colony. A symbiotic structure is dedicated to nutrient exchange and the extramatricular mycelium explores soil for nutrients. Eventually, basidiocarps develop to assure last stages of sexual reproduction. The aim of this study is to understand how an EcM fungus uses its gene set to support functional differentiation and development of specialized morphological structures. We examined the transcriptomes of Laccaria bicolor under a series of experimental setups, including the growth with Populus tremula x alba at different developmental stages, basidiocarps and free-living mycelium, under various conditions of N, P and C supply. In particular, N supply induced global transcriptional changes, whereas responses to P supply seemed to be independent from it. Symbiosis development with poplar is characterized by transcriptional waves. Basidiocarp development shares transcriptional signatures with other basidiomycetes. Overlaps in transcriptional responses of L. bicolor hyphae to a host plant and N/C supply next to co-regulation of genes in basidiocarps and mature mycorrhiza were detected. Few genes are induced in a single condition only, but functional and morphological differentiation rather involves fine tuning of larger gene sets. Overall, this transcriptomic atlas builds a reference to study the function and stability of EcM symbiosis in distinct conditions using L. bicolor as a model and indicates both similarities and differences with other ectomycorrhizal fungi, allowing researchers to distinguish conserved processes such as basidiocarp development from nutrient homeostasis. This research study was supported by the Laboratory of Excellence ARBRE (ANR-11- LABX-0002-01), the Region Lorraine, the European Regional Development Fund and the Plant– Microbe Interfaces Scientific Focus Area in the Genomic Science Program, the Office of Biological and Environmental Research in the US DOE Office of Science. J.R. is an Agreenskills Marie Sklodowska Curie postdoctoral fellow co-funded by the European Union (CoFUND-FP7-267196). The authors greatly acknowledge Yohann Daguerre and Clément Pellegrin for assistance in setting up Laccaria–Populus co-cultures and Maarten Ottaway for English language editing of the manuscript

Environmental pollution and scarcity of natural resources lead to an increased interest in developing more sustainable materials. For example, the traditional construction industry, which is largely based on the extraction of fossil fuels and raw materials, is called into question. A solution can be found in biologically augmented materials that are made by growing mycelium-forming fungal microorganisms on natural fibres rich in cellulose, hemicellulose and lignin. In this way, organic waste streams, such as agricultural waste, are valorised while creating a material that is biodegradable at the end of its life cycle - a process that fits in the spirit of circular economy. Mycelium-based materials have properties that are promising for a wide range of applications, including the use as construction materials. Despite this promise, the applicability and the practicality of these materials are largely unexplored and moreover, individual studies use a wide range of different experimental approaches and non-standardized procedures. In this review, we critically evaluate existing data on the composition of mycelium-based materials and process variables with the aim of providing a comprehensive framework of the production process. The framework illustrates the many input factors during the production that have an impact on the final characteristics of the material, and the unique potential to deploy more tuneable levels in the fabrications process that can serve to prototype a diversity of new unprecedented applications. Furthermore, we determine the applicability of existing data and identify knowledge gaps. This framework is valuable in identifying standardized approaches for future studies and in informing the design and process of new applications of mycelium-based materials.

The basidiomycete Suillus luteus is an important member of the ectomycorrhizal community that thrives in heavy metal polluted soils covered with pioneer pine forests. This study aimed to identify potential heavy metal chelators in S. luteus. Two metallothionein (MT) coding genes, SlMTa and SlMTb, were identified. When heterologously expressed in yeast, both SlMTa and SlMTb can rescue the Cu sensitive mutant from Cu toxicity. In S. luteus, transcription of both SlMTa and SlMTb is induced by Cu but not Cd or Zn. Several putative Cu-sensing and metal-response elements are present in the promoter sequences. These results indicate that SlMTa and SlMTb function as Cu-thioneins. Homologs of the S. luteus MTs are present in 49 species belonging to 10 different orders of the subphylum Agaricomycotina and are remarkably conserved. The length of the proteins, number and distribution of cysteine residues indicate a novel family of fungal MTs. The ubiquitous and highly conserved features of these MTs suggest that they are important for basic cellular functions in species in the subphylum Agaricomycotina.

Human-altered environments can shape the evolution of organisms. Fungi are no exception, although little is known about how they withstand anthropogenic pollution. Here, we document adaptation in the mycorrhizal fungus Suillus luteus driven by soil heavy metal contamination. Genome scans across individuals from recently polluted and nearby unpolluted soils in Belgium revealed low divergence across isolates and no evidence of population structure based on soil type. However, we detected single nucleotide polymorphism divergence and gene copy-number variation, with different genetic combinations potentially conferring the ability to persist in contaminated soils. Variants were shared across the population but found to be under selection in isolates exposed to pollution and located across the genome, including in genes involved in metal exclusion, storage, immobilization and reactive oxygen species detoxification. Together, our results point to S. luteus undergoing the initial steps of adaptive divergence and contribute to understanding the processes underlying local adaptation under strong environmental selection.

Human-altered environments can shape the evolution of organisms. Fungi are no exception, though little is known about how they withstand anthropogenic pollution. Here, we document incipient polygenic local adaptation in the mycorrhizal fungus Suillus luteus driven by recent soil heavy metal contamination. Genome scans across individuals from recently polluted and nearby unpolluted soils in Belgium revealed no evidence of population structure but detected allelic divergence and gene copy number variation in genes involved in metal exclusion, storage, immobilization, and reactive oxygen species detoxification. Standing genetic variation included multiple alleles of small effects contributing to heavy metal tolerance, suggesting the existence of different strategies to withstand contamination. These variants were shared across the whole population but under selection in isolates exposed to pollution. Together, our results point to S. luteus undergoing the initial steps of adaptive divergence and contribute to understanding the processes underlying local adaptation under strong environmental selection.

SummaryAbiotic changes due to anthropogenic activities affect selection regimes for organisms. How trees and their mycorrhizal symbionts adapt to altered environments in heterogeneous landscapes is of great interest. With a global distribution and multiple adaptive phenotypes available,Suillus luteusis an excellent ectomycorrhizal model to study evolutionary dynamics of local adaptation. We assessed pathways of homeostasis and detoxification inS. luteusisolates, displaying contrasting Zn tolerance phenotypes to identify mechanisms underlying adaptive Zn tolerance. Using 30 randomly selected isolates sampled at metal contaminated and control sites, we documented Zn tolerance phenotypes, assessed the link with identified candidate genes and explored its genetic basis via targeted amplicon sequencing and qPCR. Zn tolerance phenotypes covering a continuum from Zn sensitive to hypertolerant were identified and inversely correlate with cellular Zn accumulation. Gene expression ofSlZnT2, encoding a putative Zn transporter explains 72% of the observed phenotypic variation.SlZnT2copy number varies among isolates and different promotor genotypes were identified. Rapid adaptation in this species is supported by the cumulative effect of gene copy number variation and differences in cis-regulation and might be triggered by environmental stress rather than being the result of standing variation.Originality - significance statementTo the best of our knowledge, this is the first study linking genotypes to adaptive phenotypes in mycorrhizal fungi. It is unique in the way it combines evolutionary and functional genetics to allow a significant advance in the understanding of responses to environmental stress factors in general and, to soil metal pollution in particular. A better understanding of adaptive tolerance mechanisms in keystone symbiotic fungi is paramount for developing impactful phyto and mycoremediation strategies for metal polluted waste land and to predict the impact of future environmental change on mycorrhizal diversity and ecosystem functioning.

Ectomycorrhizal (ECM) fungi are important root symbionts of trees, as they can have significant effects on the nutrient status of plants. In polluted environments, particular ECM fungi can protect their host tree from Zn toxicity by restricting the transfer of Zn while securing supply of essential nutrients. However, mechanisms and regulation of cellular Zn homeostasis in ECM fungi are largely unknown, and it remains unclear how ECM fungi affect the Zn status of their host plants. This study focuses on the characterization of a ZIP (Zrt/IrtT-like protein) transporter, SlZRT2, in the ECM fungus Suillus luteus, a common root symbiont of young pine trees. SlZRT2 is predicted to encode a plasma membrane-located Zn importer. Heterologous expression of SlZRT2 in yeast mutants with impaired Zn uptake resulted in a minor impact on cellular Zn accumulation and growth. The SlZRT2 gene product showed a dual localization and was detected at the plasma membrane and perinuclear region. S. luteus ZIP-family Zn uptake transporters did not show the potential to induce trehalase activity in yeast and to function as Zn sensors. In response to excess environmental Zn, gene expression analysis demonstrated a rapid but minor and transient decrease in SlZRT2 transcript level. In ECM root tips, the gene is upregulated. Whether this regulation is due to limited Zn availability at the fungal-plant interface or to developmental processes is unclear. Altogether, our results suggest a function for SlZRT2 in cellular Zn redistribution from the ER next to a putative role in Zn uptake in S. luteus. Funding was provided by the Research Foundation Flanders (FWO project G079213N). LC holds a Flanders Innovation & Entrepreneurships Ph.D. fellowship (IWT project 141461). Her research visit at INRA Grand Est Nancy was funded by the Laboratory of Excellence Advanced Research on the Biology of Tree and Forest Ecosystems (ARBRE; grant No. ANR-11-LABX-0002-01).

Mycorrhizas are mutually beneficial associations between soil-borne fungi and plant roots. Mycorrhizal fungi provide their host plant with essential nutrients in exchange for sugars and/or lipids. Traditionally, transport and translocation of macronutrients, including nitrogen and phosphorus, throughout the fungal mycelium and towards the host plant are well studied. However, the regulation of nutrient exchange and their contribution in the morphogenesis and development of mycorrhizas remains unclear. In this Opinion, we argue that adding micronutrients in the current models of symbiotic transport is essential to fully understand the establishment, maintenance, and functioning of mycorrhizal associations. Homeostatic mechanisms at the cellular level and the first transport proteins involved have been recently documented for zinc (Zn) in arbuscular mycorrhizal, ectomycorrhizal, and ericoid mycorrhizal fungi. Mycorrhizal plants benefit from an improved Zn status in control conditions and are better protected when environmental Zn availability fluctuates. These recent progresses are paving the way for a better understanding of micronutrient allocation in mycorrhizas. Revising our vision on the role of micronutrients, particularly of Zn, in these interactions will allow a better use of mycorrhizal fungi in sustainable agriculture and forestry, and will increase management practices in waste land, as well as in agricultural and natural ecosystems.

The ectomycorrhizal symbiosis is a predominant tree–microbe interaction in forest ecosystems sustaining tree growth and health. Its establishment and functioning implies a long‐term and intimate relationship between the soil‐borne fungi and the roots of trees. Mycorrhiza‐induced Small‐Secreted Proteins (MiSSPs) are hypothesized as keystone symbiotic proteins, required to set up the symbiosis by modifying the host metabolism and/or building the symbiotic interfaces. L. bicolor MiSSP8 is the third most highly induced MiSSPs in symbiotic tissues and it is also expressed in fruiting bodies. The MiSSP8‐RNAi knockdown mutants are strongly impaired in their mycorrhization ability with Populus, with the lack of fungal mantle and Hartig net development due to the lack of hyphal aggregation. MiSSP8 C‐terminus displays a repetitive motif containing a kexin cleavage site, recognized by KEX2 in vitro. This suggests MiSSP8 protein might be cleaved into small peptides. Moreover, the MiSSP8 repetitive motif is found in other proteins predicted secreted by both saprotrophic and ectomycorrhizal fungi. Thus, our data indicate that MiSSP8 is a small‐secreted protein involved at early stages of ectomycorrhizal symbiosis, likely by regulating hyphal aggregation and pseudoparenchyma formation. Fil: Pellegrin, Clément. université de Lorraine; Francia Fil: Pellegrin, Clément. Fil: Ruytinx, Joske. Fil: Guinet, Frédéric. Fil: Kemppainen, Minna. Fil: Frei dit Frey, Nicolas. Fil: Puech Pagès, Virginie. Fil: Hecker, Arnaud. Fil: Pardo, Alejandro Guillermo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Micología Molecular; Argentina Fil: Martin, Francis M.. Fil: Veneault Fourrey, Claire.

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Hydrophobins are small secreted proteins that are present as several gene copies in most fungal genomes. Their properties are now well understood: they are amphiphilic and assemble at hydrophilic/hydrophobic interfaces. However, their physiological functions remain largely unexplored, especially within mycorrhizal fungi. In this study, we identified hydrophobin genes and analysed their distribution in eight mycorrhizal genomes. We then measured their expression levels in three different biological conditions (mycorrhizal tissue vs. free-living mycelium, organic vs. mineral growth medium and aerial vs. submerged growth). Results confirmed that the size of the hydrophobin repertoire increased in the terminal orders of the fungal evolutionary tree. Reconciliation analysis predicted that in 41% of the cases, hydrophobins evolved from duplication events. Whatever the treatment and the fungal species, the pattern of expression of hydrophobins followed a reciprocal function, with one gene much more expressed than others from the same repertoire. These most-expressed hydrophobin genes were also among the most expressed of the whole genome, which suggests that they play a role as structural proteins. The fine-tuning of the expression of hydrophobin genes in each condition appeared complex because it differed considerably between species, in a way that could not be explained by simple ecological traits. Hydrophobin gene regulation in mycorrhizal tissue as compared with free-living mycelium, however, was significantly associated with a calculated high exposure of hydrophilic residues. The authors are thankful to the Mycorrhizal Genomics Initiative for providing access to genomic and transcriptomic data. Hafida Lmalem and Francois Rineau are grateful to the BOF (Special Research Fund) from Hasselt University for financing their research.

Zinc (Zn) is an essential micronutrient but may become toxic when present in excess. In Zn-contaminated environments, trees can be protected from Zn toxicity by their root-associated micro-organisms, in particular ectomycorrhizal fungi. The mechanisms of cellular Zn homeostasis in ectomycorrhizal fungi and their contribution to the host tree's Zn status are however not yet fully understood. The aim of this study was to identify and characterize transporters involved in Zn uptake in the ectomycorrhizal fungus Suillus luteus, a cosmopolitan pine mycobiont. Zn uptake in fungi is known to be predominantly governed by members of the ZIP (Zrt/IrtT-like protein) family of Zn transporters. Four ZIP transporter encoding genes were identified in the S. luteus genome. By in silico and phylogenetic analysis, one of these proteins, SlZRT1, was predicted to be a plasma membrane located Zn importer. Heterologous expression in yeast confirmed the predicted function and localization of the protein. A gene expression analysis via RT-qPCR was performed in S. luteus to establish whether SlZRT1 expression is affected by external Zn concentrations. SlZRT1 transcripts accumulated almost immediately, though transiently upon growth in the absence of Zn. Exposure to elevated concentrations of Zn resulted in a significant reduction of SlZRT1 transcripts within the first hour after initiation of the exposure. Altogether, the data support a role as cellular Zn importer for SlZRT1 and indicate a key role in cellular Zn uptake of S. luteus. Further research is needed to understand the eventual contribution of SlZRT1 to the Zn status of the host plant. This work was financially supported by the Research Foundation Flanders (FWO Project G079213N). LC holds a Flanders Innovation & Entrepreneurships Ph.D. fellowship (IWT Project 141461) and her research visit at INRA Grand Est Nancy was funded by the Laboratory of Excellence Advanced Research on the Biology of Tree and Forest Ecosystems (ARBRE; grant No. ANR-11-LABX-0002-01). Part of the computations were performed at the INRA Grand Est-Nancy Ecogenomics facilities. The Mycorrhizal Genomics Initiative is supported by the French National Institute for Agricultural Research (INRA), the US Department of Energy (DOE) Joint Genome Institute (JGI; Office of Science of the US Department of Energy), the Region Lorraine Research Council and the European Commission [European Regional Development Fund (ERDF)].

Quantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the expression analysis of a set of chosen genes of interest. The accuracy and reliability of qRT-PCR measurements strongly depends on the normalization with appropriate endogenous reference genes. In this study a set of candidate reference genes for the use in gene expression studies of a basidiomycete fungus, Suillus luteus, exposed to toxic concentrations of zinc or cadmium was identified, evaluated and validated. Seven candidate genes were selected from cDNA-AFLP as stably expressed and the algorithms geNorm and Normfinder were used to evaluate these genes alongside the traditionally used housekeeping genes (actin, tubulin) in different S. luteus isolates. The use of several S. luteus isolates revealed that each isolate has its own most stably expressed set of reference genes, regardless of the metal treatments, in casu metal exposures. Metal treatments had only a minor impact on the expression of the candidate reference genes. The validated reference genes outperform the in fungal research commonly used single, arbitrary chosen (“housekeeping”) genes in terms of reliability, and have the potential to be suitable reference genes when studying the effect of other environmental factors. A relatively high number of reference genes is required to correct for intraspecific variability when studying natural populations.

cellular homeostasis; ectomycorrhizal fungi; fungusroot interface; molecular mycorrhizal symbiosis; plant nutrient uptake system; reactive oxygen species; trace element acquisition; transcriptome analysis

The early antioxidative defence mechanisms were studied in Arabidopsis thaliana by applying a range of realistic Cd concentrations. Our data suggest that a 24 h exposure to 20 μM CdSO 4 is already too toxic to study moderate toxicity, whereas a highly coordinated oxidative stress-related defence response could be observed after Cd application of 5 and 10 μM. Significant differences in transcript abundance of several genes involved in antioxidative defence were observed. The generation of superoxide seems the main cause of oxidative stress in the roots, whereas in the leaves hydrogen peroxide appears to be an important player. Furthermore, an increased transcript level of lipoxygenase, a potential inducer of oxidative stress, suggests a central role of this gene in causing the Cd-related redox imbalance. Our results show that Cd as a non-redox-active metal induces oxidative stress and indicate that the antioxidative defence system is moderated by the activation of different genes in different organs and cellular compartments.

The basidiomycete Suillus luteus is an important member of the ectomycorrhizal community that thrives in heavy metal polluted soils covered with pioneer pine forests. This study aimed to identify potential heavy metal chelators in S. luteus. Two metallothionein (MT) coding genes, SlMTa and SlMTb, were identified. When heterologously expressed in yeast, both SlMTa and SlMTb can rescue the Cu sensitive mutant from Cu toxicity. In S. luteus, transcription of both SlMTa and SlMTb is induced by Cu but not Cd or Zn. Several putative Cu-sensing and metal-response elements are present in the promoter sequences. These results indicate that SlMTa and SlMTb function as Cu-thioneins. Homologs of the S. luteus MTs are present in 49 species belonging to 10 different orders of the subphylum Agaricomycotina and are remarkably conserved. The length of the proteins, number and distribution of cysteine residues indicate a novel family of fungal MTs. The ubiquitous and highly conserved features of these MTs suggest that they are important for basic cellular functions in species in the subphylum Agaricomycotina.

The impact of soil metal pollution on plant communities has been studied extensively in the past. However, very little is known about the fungal species that co-occur with these plant communities on metal polluted soils. We characterized the belowground fungal community in a heavy metal polluted and a non-polluted soil using 454 pyrosequencing. The fungal communities at both study sites were shown to consist mainly of the same ectomycorrhizal species, but a consistent shift in the relative abundances of these species was observed, whereas no differences in fungal diversity were found. In metal polluted soil, root tips of young pines were initially largely colonized by stress-tolerant dark Ascomycota that were mostly replaced by metal-tolerant Basidiomycota within 2 years. Compared to older forests, a low belowground fungal diversity was observed in the two pioneer stands. (C) 2015 Elsevier Ltd. All rights reserved. The authors would like to thank Marc Missoorten for granting access to the study sites and for assistance during fieldwork. We would also like to thank Michael Waud for performing the pyrosequencing runs. Op De Beeck M. is a PhD student funded by the Research Foundation Flanders (FWO). Smits M. is a post-doc funded by the FWO. The FWO had no involvement in the study design, in the collection, analysis or interpretation of the data, in the writing of the report nor in the decision to submit the article for publication

To elucidate the genetic bases of mycorrhizal lifestyle evolution, we sequenced new fungal genomes, including 13 ectomycorrhizal (ECM), orchid (ORM) and ericoid (ERM) species, and five saprotrophs, which we analyzed along with other fungal genomes. Ectomycorrhizal fungi have a reduced complement of genes encoding plant cell wall–degrading enzymes (PCWDEs), as compared to their ancestral wood decayers. Nevertheless, they have retained a unique array of PCWDEs, thus suggesting that they possess diverse abilities to decompose lignocellulose. Similar functional categories of nonorthologous genes are induced in symbiosis. Of induced genes, 7–38% are orphan genes, including genes that encode secreted effector-like proteins. Convergent evolution of the mycorrhizal habit in fungi occurred via the repeated evolution of a ‘symbiosis toolkit’, with reduced numbers of PCWDEs and lineage-specific suites of mycorrhiza-induced genes. This material is based on work conducted by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, supported under contract no. DE-AC02-05CH11231. This work was also supported by the Laboratory of Excellence ARBRE (ANR-11-LABX-0002-01), the Genomic Science Program (Plant-Microbe Interactions project) funded by the US Department of Energy, Office of Science, Biological and Environmental Research (contract DE-AC05-00OR22725), the Lorraine Region Council (to F.M.), US National Science Foundation grants DEB-1208719 and DEB-0933081 (both to D.S.H. and DEB-1021606 (to A.P.)), the German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig (DFG FTZ 118) and the German Science Foundation (DFG, grant BU941/20-1) (to F.B.) and the Swedish Research Council (to A. Tunlid). This work was partly supported by the Laboratory of Excellence TULIP (ANR-10-LABX-41 and ANR-11-IDEX-0002-02). F.M. would like to acknowledge M.A. Selosse and B. Lindahl for helpful discussions.

On Zn-polluted soils, populations of the ectomycorrhizal basidiomycete Suillus bovinus exhibit an elevated Zn tolerance when compared to populations on non-polluted sites. To elucidate the mechanism of Zn tolerance, the time-course of Zn uptake was studied in isolates with contrasting Zn tolerance. Unidirectional fluxes and subcellular compartmentation of Zn were investigated through radiotracer flux analyses. Fluorescence imaging was used to support the subcellular Zn compartmentation. After 2 h of exposure to 200 μM Zn, significantly more Zn was accumulated in Zn-sensitive isolates compared to tolerant isolates, despite similar short-term uptake kinetics and similar extracellular Zn sequestration in cell walls. In Zn-sensitive isolates twice as much Zn accumulated in the cytoplasm and 12 times more Zn in the vacuole. (65)Zn efflux analyses revealed a considerably faster Zn export in the Zn-tolerant isolate. The adaptive Zn tolerance in S. bovinus is therefore achieved by a preferential removal of Zn out of the cytoplasm, back into the apoplast, instead of the usual transfer of Zn into the vacuole. Zn exclusion in the fungal symbiont eventually contributes to a lower Zn influx in host plants.

Two genes, SlZnT1 and SlZnT2, encoding Cation Diffusion Facilitator (CDF) family transporters were isolated from Suillus luteus mycelium by genome walking. Both gene models are very similar and phylogenetic analysis indicates that they are most likely the result of a recent gene duplication event. Comparative sequence analysis of the deduced proteins predicts them to be Zn transporters. This function was confirmed by functional analysis in yeast for SlZnT1. SlZnT1 was able to restore growth of the highly Zn sensitive yeast mutant zrc1 and localized to the vacuolar membrane. Transformation of zrc1 yeast cells with SlZnT1 resulted in an increased accumulation of Zn compared to empty vector transformed zrc1 yeast cells and equals Zn accumulation in wild type yeast cells. We were not able to express functional SlZnT2 in yeast. In S. luteus, both SlZnT genes are constitutively expressed whatever the external Zn concentrations. A labile Zn pool was detected in the vacuoles of S. luteus free-living mycelium. Therefore we conclude that SlZnT1 is indispensable for maintenance of Zn homeostasis by transporting excess Zn into the vacuole. This research was financially supported by the Research Foundation - Flanders (FWO-project G.0925.10 and G.0792.13) and a Methusalem project (08M03). The authors declare that they have no competing interest. LC's research visit at INRA Grand Est Nancy was funded by the Laboratory of Excellence Advanced Research on the Biology of Tree and Forest Ecosystems (ARBRE; grant ANR-11-LABX-0002-01). Part of the computations were performed at the INRA Grand Est-Nancy Ecogenomics facilities. The Mycorrhizal Genomics Initiative is supported by the French National Institute for Agricultural Research (INRA), the US Department of Energy (DOE) Joint Genome Institute (JGI; Office of Science of the US Department of Energy), the Region Lorraine Research Council and the European Commission (European Regional Development Fund (ERDF)).

The cellular redox state is an important determinant of metal phytotoxicity. In this study we investigated the influence of cadmium (Cd) and copper (Cu) stress on the cellular redox balance in relation to oxidative signalling and damage in Arabidopsis thaliana. Both metals were easily taken up by the roots, but the translocation to the aboveground parts was restricted to Cd stress. In the roots, Cu directly induced an oxidative burst, whereas enzymatic ROS (reactive oxygen species) production via NADPH oxidases seems important in oxidative stress caused by Cd. Furthermore, in the roots, the glutathione metabolism plays a crucial role in controlling the gene regulation of the antioxidative defence mechanism under Cd stress. Metal-specific alterations were also noticed with regard to the microRNA regulation of CuZnSOD gene expression in both roots and leaves. The appearance of lipid peroxidation is dual: it can be an indication of oxidative damage as well as an indication of oxidative signalling as lipoxygenases are induced after metal exposure and are initial enzymes in oxylipin biosynthesis. In conclusion, the metal-induced cellular redox imbalance is strongly dependent on the chemical properties of the metal and the plant organ considered. The stress intensity determines its involvement in downstream responses in relation to oxidative damage or signalling.

The ectomycorrhizal basidiomycete Suillus luteus (L.:Fr.), a typical pioneer species which associates with young pine trees colonizing disturbed sites, is a common root symbiont found at heavy metal contaminated sites. Three Cd-sensitive and three Cd-tolerant isolates of S. luteus, isolated respectively from non-polluted and a heavy metal-polluted site in Limburg (Belgium), were used for a transcriptomic analysis. We identified differentially expressed genes by cDNA-AFLP analysis. The possible roles of some of the encoded proteins in heavy metal (Cd) accumulation and tolerance are discussed. Despite the high conservation of coding sequences in S. luteus, a large intraspecific variation in the transcript profiles was observed. This variation was as large in Cd-tolerant as in sensitive isolates and may help this pioneer species to adapt to novel environments.

Complementary DNA (cDNA)-amplified fragment-length polymorphism (AFLP) was applied to analyze transcript profiles of a Zn-tolerant and a Zn-sensitive isolate of the ectomycorrhizal basidiomycete Suillus luteus, both cultured with and without increased external zinc concentrations. From the obtained transcript profiles that covered approximately 2% of the total expected complement of genes in S. luteus, 144 nonredundant, differentially expressed transcript-derived fragments (TDFs), falling in different classes of expression pattern, were isolated and sequenced. Thirty-six of the represented genes showed homology to function-known genes, whereas 6 matched unknown protein coding sequences, and 102 were possibly novel. Although relatively few TDFs were found to be responsive to the different zinc treatments, their modulated expression levels may suggest a different transcriptional response to zinc treatments in both isolates. Among the identified genes that could be related to heavy-metal detoxification or the tolerance trait were genes encoding for homologues of a heat-shock protein, a putative metal transporter, a hydrophobin, and several proteins involved in ubiquitin-dependent proteolysis. © 2007 Springer-Verlag., SCOPUS: ar.j, info:eu-repo/semantics/published

Laccaria bicolor is an ectomycorrhizal fungus (ECM) which is able to make a mutualistic symbiosis with poplar. In exchange for carbohydrates, ECM fungi offer an improved mineral supply and stress protection for trees. So ECM play a major role in forestry ecosystem functionning. However the current knowledge about molecular mecanisms involved in symbiosis establishment has to be improved. We have studied the effect of plant diffusible molecules, rutin and quercetin (two root exudates) on biomasse, growth and morphology of Laccaria bicolor after 24h until 14 days of exposure. We were not able to detect any effect of rutin nor quercetin nor plant roots exudates on, biomass production, colonial growth and hyphal branching at the concentrations and exposure times tested. Moreover gene expression patterns of MiSSP7, MiSSP8,5 and MiSSP17 were also determined after 24h exposure of L.bicolor to rutin or quercetin or poplar roots by RTqPCR. We observed an upregulation of MiSSP7 after 24h exposure to poplar roots. Finally, data sets obtain during this internship contributes in understanding and modelling genes mecanisms involved in L. bicolor ECM development.; Laccaria bicolor est un champignon ectomycorhizien (ECM) capable d'établir une symbiose avec le peuplier. En échange de composés carbonés, les champignons ECM fournissent les composés minéraux et une protection aux stress biotiques et abiotiques. Les champignons ECM jouent donc un rôle majeur dans le fonctionnement des écosystèmes forestiers. Cependant les connaissances actuelles des mécanismes moléculaires impliqués dans l'établissement de la symbiose doivent être approfondies. Les effets des molécules diffusibles des plantes, de la rutine et la quercetine (deux exsudats racinaires) sur la biomasse, la croissance et la morphologie de Laccaria bicolor ont été étudiés après une exposition de 24h et jusqu' à 14jours. Aucun effet de la rutine, de la quercetine ou des exsudats racinaires n'ont pu être détectés sur la production de biomasse, la croissance des colonies et la ramification des hyphes du mycélium avec les concentrations et les temps d'exposition testés. De plus les profils d'expression de MiSSP7, MiSSP8,5 et MiSSP17 ont été déterminés après 24h d'exposition à la rutine, à la quercetine ou aux racines de peuplier par RTqPCR. Une surexpression de MiSSP7 après une exposition de 24h aux racines de peuplier à été constatée. Les données obtenues durant ce stage contribuent à comprendre et modéliser les mécanismes de régulation génétique impliqués dans le développement des ECM