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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.

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).

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 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