Fungal heavy metal adaptation through single nucleotide polymorphisms and copy-number variation.

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.

SlZRT2 Encodes a ZIP Family Zn Transporter With Dual Localization in the Ectomycorrhizal Fungus Suillus luteus.

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.

The SlZRT1 Gene Encodes a Plasma Membrane-Located ZIP (Zrt-, Irt-Like Protein) Transporter in the Ectomycorrhizal Fungus Suillus luteus.

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.

Identification, evolution and functional characterization of two Zn CDF-family transporters of the ectomycorrhizal fungus Suillus luteus.

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 Δzrc1 and localized to the vacuolar membrane. Transformation of Δzrc1 yeast cells with SlZnT1 resulted in an increased accumulation of Zn compared to empty vector transformed Δzrc1 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.

Genetic stability of ectomycorrhizal fungi is not affected by cryopreservation at -130 °C or cold storage with repeated sub-cultivations over a period of 2 years.

Cryopreservation is considered the most reliable method for storage of filamentous fungi including ectomycorrhizal (ECM) fungi. A number of studies, however, have reported genetic changes in fungus cultures following cryopreservation. In the present study, the genetic stability of six ECM fungus isolates was analyzed using amplified fragment length polymorphism (AFLP). The isolates were preserved for 2 years either by cryopreservation (at -130 °C) or by storage at 4 °C with regular sub-cultivation. A third preservation treatment consisting of isolates maintained on Petri dishes at 22-23 °C for 2 years (i.e., without any sub-cultivation) was included and used as a control. The differences observed in AFLP patterns between the three preservation methods remained within the range of the total error generated by the AFLP procedure (6.85%). Therefore, cryopreservation at -130 °C and cold storage with regular sub-cultivation did not affect the genetic stability of the ECM fungus isolates, and both methods can be used for the routine storage of ECM fungus isolates over a period of 2 years.

A novel, highly conserved metallothionein family in basidiomycete fungi and characterization of two representative SlMTa and SlMTb genes in the ectomycorrhizal fungus Suillus luteus.

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.

Ectomycorrhizal Fungal Protein Degradation Ability Predicted by Soil Organic Nitrogen Availability.

In temperate and boreal forest ecosystems, nitrogen (N) limitation of tree metabolism is alleviated by ectomycorrhizal (ECM) fungi. As forest soils age, the primary source of N in soil switches from inorganic (NH4 (+) and NO3 (-)) to organic (mostly proteins). It has been hypothesized that ECM fungi adapt to the most common N source in their environment, which implies that fungi growing in older forests would have greater protein degradation abilities. Moreover, recent results for a model ECM fungal species suggest that organic N uptake requires a glucose supply. To test the generality of these hypotheses, we screened 55 strains of 13 Suillus species with different ecological preferences for their in vitro protein degradation abilities. Suillus species preferentially occurring in mature forests, where soil contains more organic matter, had significantly higher protease activity than those from young forests with low-organic-matter soils or species indifferent to forest age. Within species, the protease activities of ecotypes from soils with high or low soil organic N content did not differ significantly, suggesting resource partitioning between mineral and organic soil layers. The secreted protease mixtures were strongly dominated by aspartic peptidases. Glucose addition had variable effects on secreted protease activity; in some species, it triggered activity, but in others, activity was repressed at high concentrations. Collectively, our results indicate that protease activity, a key ectomycorrhizal functional trait, is positively related to environmental N source availability but is also influenced by additional factors, such as carbon availability.

Impact of metal pollution on fungal diversity and community structures.

The impact of metal pollution on plant communities has been studied extensively in the past, but little is known about the effects of metal pollution on fungal communities that occur in metal-polluted soils. Metal-tolerant ecotypes of the ectomycorrhizal fungus Suillus luteus are frequently found in pioneer pine forests in the Campine region in Belgium on metal-polluted soils. We hypothesized that metal pollution would play an important role in shaping below-ground fungal communities that occur in these soils and that Suillus luteus would be a dominant player. To test these hypotheses, the fungal communities in a young pine plantation in soil polluted with zinc, and cadmium were studied using 454 amplicon pyrosequencing. Results show that zinc, cadmium and soil organic matter content were strongly correlated with the fungal community composition, but no effects on fungal diversity were observed. As hypothesized, S. luteus was found to be a dominant member of the studied fungal communities. However, other dominant fungal species, such as Sistotrema sp., Wilcoxina mikolae and Cadophora finlandica were found as well. Their presence in metal-polluted sites is discussed.

Comparison and validation of some ITS primer pairs useful for fungal metabarcoding studies.

Current metabarcoding studies aiming to characterize microbial communities generally rely on the amplification and sequencing of relatively short DNA regions. For fungi, the internal transcribed spacer (ITS) region in the ribosomal RNA (rRNA) operon has been accepted as the formal fungal barcode. Despite an increasing number of fungal metabarcoding studies, the amplification efficiency of primers is generally not tested prior to their application in metabarcoding studies. Some of the challenges that metabarcoding primers should overcome efficiently are the amplification of target DNA strands in samples rich in non-target DNA and environmental pollutants, such as humic acids, that may have been co-extracted with DNA. In the current study, three selected primer pairs were tested for their suitability as fungal metabarcoding primers. The selected primer pairs include two primer pairs that have been frequently used in fungal metabarcoding studies (ITS1F/ITS2 and ITS3/ITS4) and a primer pair (ITS86F/ITS4) that has been shown to efficiently amplify the ITS2 region of a broad range of fungal taxa in environmental soil samples. The selected primer pairs were evaluated in a 454 amplicon pyrosequencing experiment, real-time PCR (qPCR) experiments and in silico analyses. Results indicate that experimental evaluation of primers provides valuable information that could aid in the selection of suitable primers for fungal metabarcoding studies. Furthermore, we show that the ITS86F/ITS4 primer pair outperforms other primer pairs tested in terms of in silico primer efficiency, PCR efficiency, coverage, number of reads and number of species-level operational taxonomic units (OTUs) obtained. These traits push the ITS86F/ITS4 primer pair forward as highly suitable for studying fungal diversity and community structures using DNA metabarcoding.

Soil metatranscriptomics for mining eukaryotic heavy metal resistance genes.

Heavy metals are pollutants which affect all organisms. Since a small number of eukaryotes have been investigated with respect to metal resistance, we hypothesize that many genes that control this phenomenon remain to be identified. This was tested by screening soil eukaryotic metatranscriptomes which encompass RNA from organisms belonging to the main eukaryotic phyla. Soil-extracted polyadenylated mRNAs were converted into cDNAs and 35 of them were selected for their ability to rescue the metal (Cd or Zn) sensitive phenotype of yeast mutants. Few of the genes belonged to families known to confer metal resistance when overexpressed in yeast. Several of them were homologous to genes that had not been studied in the context of metal resistance. For instance, the BOLA ones, which conferred cross metal (Zn, Co, Cd, Mn) resistance may act by interfering with Fe homeostasis. Other genes, such as those encoding 110- to 130-amino-acid-long, cysteine-rich polypeptides, had no homologues in databases. This study confirms that functional metatranscriptomics represents a powerful approach to address basic biological processes in eukaryotes. The selected genes can be used to probe new pathways involved in metal homeostasis and to manipulate the resistance level of selected organisms.

Zinc export results in adaptive zinc tolerance in the ectomycorrhizal basidiomycete Suillus bovinus.

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.

Cryopreservation of ectomycorrhizal fungi has minor effects on root colonization of Pinus sylvestris plantlets and their subsequent nutrient uptake capacity.

The use of ectomycorrhizal (ECM) fungi for afforestation, bioremediation, and timber production requires their maintenance over long periods under conditions that preserve their genetic, phenotypic, and physiological stability. Cryopreservation is nowadays considered as the most suitable method to maintain the phenotypic and genetic stability of a large number of filamentous fungi including the ECM fungi. Here, we compared the ability of eight ECM fungal isolates to colonize Pinus sylvestris roots and to transport inorganic phosphate (Pi) and NH4 (+) from the substrate to the plant after cryopreservation for 6 months at -130 °C or after storage at 4 °C. Overall, the mode of preservation had no significant effect on the colonization rates of P. sylvestris, the concentrations of ergosterol in the roots and substrate, and the uptake of Pi and NH4 (+). Comparing the isolates, differences were sometimes observed with one or the other method of preservation. Suillus bovinus exhibited a reduced ability to form mycorrhizas and to take up Pi following cryopreservation, while one Suillus luteus isolate exhibited a decreased ability to take up NH4 (+). Conversely, Hebeloma crustuliniforme, Laccaria bicolor, Paxillus involutus, and Pisolithus tinctorius exhibited a reduced ability to form mycorrhizas after storage at 4 °C, although this did not result in a reduced uptake of Pi and NH4 (+). Cryopreservation appeared as a reliable method to maintain important phenotypic characteristics (i.e., root colonization and nutrient acquisition) of most of the ECM fungal isolates studied. For 50 % of the ECM fungal isolates, the colonization rate was even higher with the cultures cryopreserved at -130 °C as compared to those stored at 4 °C.

Viability of ectomycorrhizal fungi following cryopreservation.

The use of ectomycorrhizal (ECM) fungi in biotechnological processes requires their maintenance over long periods under conditions that maintain their genetic, phenotypic, and physiological stability. Cryopreservation is considered as the most reliable method for long-term storage of most filamentous fungi. However, this technique is not widespread for ECM fungi since many do not survive or exhibit poor recovery after freezing. The aim of this study was to develop an efficient cryopreservation protocol for the long-term storage of ECM fungi. Two cryopreservation protocols were compared. The first protocol was the conventional straw protocol (SP). The mycelium of the ECM isolates was grown in Petri dishes on agar and subsequently collected by punching the mycelium into a sterile straw before cryopreservation. In the second protocol, the cryovial protocol (CP), the mycelium of the ECM isolates was grown directly in cryovials filled with agar and subsequently cryopreserved. The same cryoprotectant solution, freezing, and thawing process, and re-growth conditions were used in both protocols. The survival (positive when at least 60 % of the replicates showed re-growth) was evaluated before and immediately after freezing as well as after 1 week, 1 m, and 6 m of storage at -130 °C. Greater survival rate (80 % for the CP as compared to 25 % for the SP) and faster re-growth (within 10 d for the CP compared to the 4 weeks for the SP) were observed for most isolates with the CP suggesting that the preparation of the cultures prior to freezing had a significant impact on the isolates survival. The suitability of the CP for cryopreservation of ECM fungi was further confirmed on a set of 98 ECM isolates and displayed a survival rate of 88 % of the isolates. Only some isolates belonging to Suillus luteus, Hebeloma crustuliniforme, Paxillus involutus and Thelephora terrestris failed to survive. This suggested that the CP is an adequate method for the ultra-low cryopreservation of a large set of ECM fungi and that further studies are necessary for the more recalcitrant ones.

Transcriptome analysis by cDNA-AFLP of Suillus luteus Cd-tolerant and Cd-sensitive isolates.

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.

Zn pollution counteracts Cd toxicity in metal-tolerant ectomycorrhizal fungi and their host plant, Pinus sylvestris.

Adaptive Zn and Cd tolerance have evolved in populations of the ectomycorrhizal fungus Suillus luteus. When exposed to high concentrations of both metals in vitro, a one-sided antagonism was apparent in the Zn- and Cd-tolerant isolates. Addition of high Zn concentrations restored growth of Cd-stressed isolates, but not vice versa. The antagonistic effect was not detected in a S. luteus isolate from non-contaminated land and in Paxillus involutus. The fungi were inoculated on pine seedlings and subsequently exposed to ecologically relevant Zn and Cd concentrations in single and mixed treatments. The applied doses severely reduced nutrient acquisition of non-mycorrhizal pines and pines inoculated with metal-sensitive S. luteus. Highest translocation of Zn and Cd to shoots occurred in the same plants. Seedlings inoculated with fungi collected from the polluted site reduced metal transfer to their host and maintained nutrient acquisition under high metal exposure. The isolate showing highest tolerance in vitro also offered best protection in symbiosis. The antagonistic effect of high Zn on Cd toxicity was confirmed in the plant experiment. The results indicate that a Zn- and Cd-polluted soil has selected ectomycorrhizal fungi that are able to survive and protect their phytobiont from nutrient starvation and excessive metal uptake.

Cd-tolerant Suillus luteus: a fungal insurance for pines exposed to Cd.

Soil metal pollution can trigger evolutionary adaptation in soil-borne organisms. An in vitro screening test showed cadmium adaptation in populations of Suillus luteus (L.: Fr.) Roussel, an ectomycorrhizal fungus of pine trees. Cadmium stress was subsequently investigated in Scots pine (Pinus sylvestris L.) seedlings inoculated with a Cd-tolerant S. luteus, isolated from a heavy metal contaminated site, and compared to plants inoculated with a Cd-sensitive isolate from a non-polluted area. A dose-response experiment with mycorrhizal pines showed better plant protection by a Cd-adapted fungus: more fungal biomass and a higher nutrient uptake at high Cd exposure. In addition, less Cd was transferred to aboveground plant parts. Because of the key role of the ectomycorrhizal symbiosis for tree fitness, the evolution of Cd tolerance in an ectomycorrhizal partner such as S. luteus can be of major importance for the establishment of pine forests on Cd-contaminated soils.

Genetic structure of Suillus luteus populations in heavy metal polluted and nonpolluted habitats.

The genetic structure of populations of the ectomycorrhizal basidiomycete Suillus luteus in heavy metal polluted and nonpolluted areas was studied. Sporocarps were collected at nine different locations and genotyped at four microsatellite loci. Six of the sampling sites were severely contaminated with heavy metals and were dominated by heavy metal-tolerant individuals. Considerable genetic diversity was found within the geographical subpopulations, but no reduction of the genetic diversity, current or historic, was observed in subpopulations inhabiting polluted soils. The genetic differentiation between the geographical subpopulations was low, and no evidence for clustering of subpopulations from polluted soils vs. subpopulations from nonpolluted soils was found. These results indicate that heavy metal pollution has a limited effect on the genetic structure of S. luteus populations, and this may be due to the high frequency of sexual reproduction and extensive gene flow in S. luteus, which allows rapid evolution of the tolerance trait while maintaining high levels of genetic diversity.

Zinc-tolerant Suillus bovinus improves growth of Zn-exposed Pinus sylvestris seedlings.

Scots pine (Pinus sylvestris L.) seedlings inoculated or not (NM) by a Zn-sensitive or a Zn-tolerant isolate of the ectomycorrhizal fungus Suillus bovinus (L. Fr.) Roussel were exposed to 0.1 or 150 muM Zn(2+) for 9 months. We hypothesized that inoculation with a Zn-tolerant S. bovinus isolate should result in added Zn resistance of the host plant. Plant and fungal growth as well as nutrient profiles and photosynthetic pigments in pine needles were quantified. In NM plants and in plants colonized by the Zn-sensitive isolate, plant growth, N, P, Mg and Fe assimilation were strongly inhibited under Zn stress and concurred with significantly reduced chlorophyll concentrations. In contrast, plants colonized by the Zn-tolerant isolate grew much better and remained physiologically healthier when exposed to elevated Zn. These results provide further evidence for the important role metal-adapted mycorrhizal fungi play as an effective biological barrier against metal toxicity in trees.

Ectomycorrhizas impede phytoremediation of polycyclic aromatic hydrocarbons (PAHs) both within and beyond the rhizosphere.

Exploitation of mycorrhizas to enhance phytoremediation of organic pollutants has received attention recently due to their positive effects on establishment of plants in polluted soils. Some evidence exist that ectomycorrhizas enhance the degradation of pollutants of low recalcitrance, while less easily degradable polyaromatic molecules have been degraded only by some of these fungi in vitro. Natural polyaromatic (humic) substances are degraded more slowly in soil where ectomycorrhizal fungi are present, thus phytoremediation of recalcitrant pollutants may not benefit from the presence of these fungi. Using a soil spiked with three polycyclic aromatic hydrocarbons (PAHs) and an industrially polluted soil (1 g kg(-1) of summation operator12 PAHs), we show that the ectomycorrhizal fungus Suillus bovinus, forming hydrophobic mycelium in soil that would easily enter into contact with hydrophobic pollutants, impedes rather than promotes PAH degradation. This result is likely to be a nutrient depletion effect caused by fungal scavenging of mineral nutrients.