Trees are able to colonize, establish and survive in a wide range of soils through associations w... more 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 de...
To elucidate the genetic bases of mycorrhizal lifestyle evolution, we sequenced new fungal genome... more 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
Human-altered environments can shape the evolution of organisms. Fungi are no exception,<br>... more Human-altered environments can shape the evolution of organisms. Fungi are no exception,<br>though little is known about how they withstand anthropogenic pollution. Here, we document<br>incipient polygenic local adaptation in the mycorrhizal fungus <i>Suillus luteus</i> driven by recent soil<br>heavy metal contamination. Genome scans across individuals from recently polluted and nearby<br>unpolluted soils in Belgium revealed no evidence of population structure but detected allelic<br>divergence and gene copy number variation in genes involved in metal exclusion, storage,<br>immobilization, and reactive oxygen species detoxification. Standing genetic variation included<br>multiple alleles of small effects contributing to heavy metal tolerance, suggesting the existence<br>of different strategies to withstand contamination. These variants were shared across the whole<br>population but under selection in isolates exposed ...
Zinc (Zn) is a plant essential micronutrient involved in a wide range of cellular processes. Ecto... more 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.
Quantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the expres... more 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 expressio...
Human-altered environments can shape the evolution of organisms. Fungi are no exception, though l... more 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 dive...
Abiotic changes due to anthropogenic activities affect selection regimes for organisms. How trees... more Abiotic 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 luteus is an excellent ectomycorrhizal model to study evolutionary dynamics of local adaptation. We assessed pathways of homeostasis and detoxification in S. luteus isolates, 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 of SlZnT2, enc...
The ectomycorrhizal symbiosis is a predominant tree-microbe interaction in forest ecosystems sust... more 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 genes, required to set up the symbiosis by modifying the host metabolism and/or building the symbiotic interfaces. Since L. bicolor MiSSP8 is the third most highly induced MiSSPs in symbiotic tissues and it is expressed in fruiting bodies, we report its functional study by analysing phenotypes of L. bicolor MiSSP8-RNAi mutants and MiSSP8 in planta subcellular localization. The MiSSP8-RNAi knockdown mutants are impaired in their mycorrhization ability with Populus, with the lack of fungal mantle and Hartig net development. When expressed heterologously in planta, MiSSP8 shows a punctate localization that partially colocalize with plasmode...
Zinc (Zn) is an essential micronutrient but may become toxic when present in excess. In Zn-contam... more 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 prote...
Two genes, SlZnT1 and SlZnT2, encoding Cation Diffusion Facilitator (CDF) family transporters wer... more 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...
Trees are able to colonize, establish and survive in a wide range of soils through associations w... more 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 de...
To elucidate the genetic bases of mycorrhizal lifestyle evolution, we sequenced new fungal genome... more 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
Human-altered environments can shape the evolution of organisms. Fungi are no exception,<br>... more Human-altered environments can shape the evolution of organisms. Fungi are no exception,<br>though little is known about how they withstand anthropogenic pollution. Here, we document<br>incipient polygenic local adaptation in the mycorrhizal fungus <i>Suillus luteus</i> driven by recent soil<br>heavy metal contamination. Genome scans across individuals from recently polluted and nearby<br>unpolluted soils in Belgium revealed no evidence of population structure but detected allelic<br>divergence and gene copy number variation in genes involved in metal exclusion, storage,<br>immobilization, and reactive oxygen species detoxification. Standing genetic variation included<br>multiple alleles of small effects contributing to heavy metal tolerance, suggesting the existence<br>of different strategies to withstand contamination. These variants were shared across the whole<br>population but under selection in isolates exposed ...
Zinc (Zn) is a plant essential micronutrient involved in a wide range of cellular processes. Ecto... more 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.
Quantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the expres... more 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 expressio...
Human-altered environments can shape the evolution of organisms. Fungi are no exception, though l... more 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 dive...
Abiotic changes due to anthropogenic activities affect selection regimes for organisms. How trees... more Abiotic 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 luteus is an excellent ectomycorrhizal model to study evolutionary dynamics of local adaptation. We assessed pathways of homeostasis and detoxification in S. luteus isolates, 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 of SlZnT2, enc...
The ectomycorrhizal symbiosis is a predominant tree-microbe interaction in forest ecosystems sust... more 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 genes, required to set up the symbiosis by modifying the host metabolism and/or building the symbiotic interfaces. Since L. bicolor MiSSP8 is the third most highly induced MiSSPs in symbiotic tissues and it is expressed in fruiting bodies, we report its functional study by analysing phenotypes of L. bicolor MiSSP8-RNAi mutants and MiSSP8 in planta subcellular localization. The MiSSP8-RNAi knockdown mutants are impaired in their mycorrhization ability with Populus, with the lack of fungal mantle and Hartig net development. When expressed heterologously in planta, MiSSP8 shows a punctate localization that partially colocalize with plasmode...
Zinc (Zn) is an essential micronutrient but may become toxic when present in excess. In Zn-contam... more 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 prote...
Two genes, SlZnT1 and SlZnT2, encoding Cation Diffusion Facilitator (CDF) family transporters wer... more 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...
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Papers by Joske Ruytinx