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François Le Tacon - One of the best experts on this subject based on the ideXlab platform.
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SCAR markers to detect mycorrhizas of an American Laccaria bicolor strain inoculated in European Douglas-fir plantations
Mycorrhiza, 2002Co-Authors: Jean Weber, Marc-andré Selosse, Jesús Díez, François Le TaconAbstract:The American strain S238N of the ectomycorrhizal fungus Laccaria bicolor (Maire) Orton has been used to inoculate Douglas-fir [Pseudotsuga menziesii (Mir.) Franco] plantations in France over the last two decades. Laccaria fruit bodies are scarce in mature plantations, which precludes further assessment of its persistence by fruit body surveys. Our objective was to develop new markers to identify this strain and its eventual non-fruiting progeny on root tips. We converted nine random amplified polymorphic DNA markers into sequence characterized amplified region (SCAR) markers. Two of these SCAR markers enabled us to detect S238N on roots of seedlings and mature trees. No amplification of non-fungal (host plant, bacterial, etc.) DNA was observed. Moreover, both SCARs were amplified from Laccaria-like mycorrhizas in a Douglas-fir plantation inoculated 14 years ago, demonstrating the long-term persistence of the inoculant strain. We also obtained a SCAR marker to detect one strain of European origin (L. bicolor 81306), indicating that SCARs are potential markers to type the naturally occurring genets. Thus, SCAR markers are of great value in studying the persistence of inoculant strains and the effects on local populations of introducing foreign strains.
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Survival after outplanting of the ectomycorrhizal fungus Laccaria bicolor S238N inoculated on Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) cuttings
Annals of Forest Science, 2002Co-Authors: Céline Di Battista, D. Bouchard, Benoit Généré, Jean-michel Amirault, François Le TaconAbstract:Selected strains of ectomycorrhizal fungi can be inoculated in forest nurseries to improve survival and growth of seedlings or cuttings after field transplantation. The survival of the American strain Laccaria bicolorS238N on Douglas fir cuttings was evaluated in nursery and field conditions three years after outplanting using morphological and PCR/RFLP of nuclear rDNA spacers. The compari- son of the mycorrhizal status of Douglas fir cuttings at the end of the nursery phase and two years after outplanting shown several beha- viours among the ectomycorrhizal fungi naturally occurring in the nursery or artificially introduced. The naturally occurring Rhizopogon type disappeared after outplanting, while the inoculated strain Laccaria bicolorS238N and an unknown type (1/2 ITS ribotype) survived and competed with the naturally occurring fungi of the outplanting site. Only one indigenous type (1/3 ITS ribotype) seemed occurring in the outplanting site where Cenococcum geophilumwas almost completely absent.
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Effect of Laccaria bicolor strains inoculated on Douglas-fir (Pseudotsuga menziesii) several years after nursery inoculation
Canadian Journal of Forest Research, 2000Co-Authors: Marc-andré Selosse, D. Bouchard, F. Martin, François Le TaconAbstract:In the Saint-Brisson experiment conducted in central France, the American strain of the ectomycorrhizal fungus Laccaria bicolor (Maire) P.D. Orton S238N and the French strain L. bicolor 81306 inoculated on containerized Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings increased by 60% the total volume of wood produced 8 years after outplanting as compared with uninoculated but naturally mycorrhizal trees. The two strains introduced 10 years before in the inoculated plots are still present and dominant; they did not prevent the colonization of Douglas-fir roots by naturally occurring ectomycorrhizal fungi but allowed for the establishment of a very diversified symbiotic microflora. Eight to 12 years after outplanting, all the Douglas-fir plots were colonized by Laccaria laccata (Scop.:Fr.) Cooke or L. bicolor strains, as well as some other species, independently of the nursery treatments. With one exception in one plot, the presence of indigenous genets in the control treatments may have preven...
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Survival of an introduced ectomycorrhizal Laccaria bicolor strain in a European forest plantation monitored by mitochondrial ribosomal DNA analysis
New Phytologist, 1998Co-Authors: Marc-andré Selosse, Francis Martin, François Le TaconAbstract:Mitochondrial and nuclear genes have different inheritance, thus studies of fungal populations should use both mitochondrial and nuclear markers. Using nuclear markers, the S238N strain of the ectomycorrhizal basidiomycete Laccaria bicolor ((Maire) Orton) has been previously shown to persist for at least 10 yr after outplanting in a plantation of Douglas fir (Pseudotsuga menziesii (Mir.) Franco) inoculated with this strain. In the present study, we have sampled 539 sporophores of Laccaria spp. from this plantation, some of which had the S238N nuclear genotype, to study mitochondrial DNA polymorphism and persistence of the inoculated S238N mitochondrial genome. Length polymorphism in fragments of the large subunit of mitochondrial ribosomal DNA (LrDNA) allowed distinction of the haplotypes present in the plantation at the species level. In addition, heteroduplex analysis and sequencing revealed intraspecific polymorphism of LrDNA among the L. bicolor sporophores and enabled specific identification of S238N LrDNA. This haplotype was only retained in sporophores carrying the S238N nuclear genome, confirming the survival of this introduced strain in a natural population.
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Meiotic segregation and recombination of the intergenic spacer of the ribosomal DNA in the ectomycorrhizal basidiomycete Laccaria bicolor
Current Genetics, 1996Co-Authors: Marc-andré Selosse, François Le Tacon, Céline Di Battista, G. Costa, F. MartinAbstract:The aim of this study was to clarify the inheritance of the nuclear ribosomal DNA (rDNA) in the ectomycorrhizal basidiomycete Laccaria bicolor S238N in order to resolve inter- and within-strain relationships in forest ecosystems. PCR amplification of the intergenic spacer (IGS) was carried out in the dikaryotic mycelium and its haploid progeny. In the dikaryotic mycelium, multiple amplification products were produced for the 25s/5s (IGS1) and 5s/17s (IGS2) intergenic spacers. The 4.5- and 4.0-kb fragments of IGS2 (haplotypes α and β, respectively) were observed to occur in a 1:1 ratio within the haploid progeny as a result of divergent IGS haplotypes in the two separate nuclei. Recombinant monokaryons having both types of IGS2 occurred at a low frequency (6.5%; 60 kb per centimorgan) during meiosis. Haplotypes α and β of IGS1 cross-hybridized forming heteroduplexes during the PCR temperature cycle. The two IGS1 haplotypes differed only by the repeat number of a TA2C3 motif and co-segregated with the IGS2 haplotypes. Heteroduplex formation and IGS polymorphism provide information that is helpful in distinguishing between introduced exotic L. bicolor S238N and indigenous populations of Laccaria spp. in forest ecosystems.
Marc-andré Selosse - One of the best experts on this subject based on the ideXlab platform.
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SCAR markers to detect mycorrhizas of an American Laccaria bicolor strain inoculated in European Douglas-fir plantations
Mycorrhiza, 2002Co-Authors: Jean Weber, Marc-andré Selosse, Jesús Díez, François Le TaconAbstract:The American strain S238N of the ectomycorrhizal fungus Laccaria bicolor (Maire) Orton has been used to inoculate Douglas-fir [Pseudotsuga menziesii (Mir.) Franco] plantations in France over the last two decades. Laccaria fruit bodies are scarce in mature plantations, which precludes further assessment of its persistence by fruit body surveys. Our objective was to develop new markers to identify this strain and its eventual non-fruiting progeny on root tips. We converted nine random amplified polymorphic DNA markers into sequence characterized amplified region (SCAR) markers. Two of these SCAR markers enabled us to detect S238N on roots of seedlings and mature trees. No amplification of non-fungal (host plant, bacterial, etc.) DNA was observed. Moreover, both SCARs were amplified from Laccaria-like mycorrhizas in a Douglas-fir plantation inoculated 14 years ago, demonstrating the long-term persistence of the inoculant strain. We also obtained a SCAR marker to detect one strain of European origin (L. bicolor 81306), indicating that SCARs are potential markers to type the naturally occurring genets. Thus, SCAR markers are of great value in studying the persistence of inoculant strains and the effects on local populations of introducing foreign strains.
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Effect of Laccaria bicolor strains inoculated on Douglas-fir (Pseudotsuga menziesii) several years after nursery inoculation
Canadian Journal of Forest Research, 2000Co-Authors: Marc-andré Selosse, D. Bouchard, F. Martin, François Le TaconAbstract:In the Saint-Brisson experiment conducted in central France, the American strain of the ectomycorrhizal fungus Laccaria bicolor (Maire) P.D. Orton S238N and the French strain L. bicolor 81306 inoculated on containerized Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings increased by 60% the total volume of wood produced 8 years after outplanting as compared with uninoculated but naturally mycorrhizal trees. The two strains introduced 10 years before in the inoculated plots are still present and dominant; they did not prevent the colonization of Douglas-fir roots by naturally occurring ectomycorrhizal fungi but allowed for the establishment of a very diversified symbiotic microflora. Eight to 12 years after outplanting, all the Douglas-fir plots were colonized by Laccaria laccata (Scop.:Fr.) Cooke or L. bicolor strains, as well as some other species, independently of the nursery treatments. With one exception in one plot, the presence of indigenous genets in the control treatments may have preven...
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Temporal persistence and spatial distribution of an American inoculant strain of the ectomycorrhizal basidiomycete Laccaria bicolor in a French forest plantation
Molecular Ecology, 1998Co-Authors: Marc-andré Selosse, D. Bouchard, D. Jacquot, F. MartinAbstract:Selected strains of ectomycorrhizal fungi, such as the basidiomycete Laccaria bicolor, are currently being used as inoculants in nurseries to improve growth of forest trees after outplanting. Information is needed on the survival of these introduced strains in forests and their impact on indigenous biodiversity. Dissemination and persistence of an American strain, L. bicolor S238N, were studied 10 years after outplanting in a Douglas fir plantation located at Saint-Brisson (Morvan, France). About 430 Laccaria spp. sporophores were collected over 3 years. Inheritance of nuclear ribosomal DNA, as well as RAPD markers, was characterized in L. bicolor S238N, using a haploid progeny set of 91 monokaryons. More than 50 markers were identified (19 heterozygous and 33 homozygous or cytoplasmic markers), which unambiguously confirmed that the introduced strain was still present in the inoculated plots. Neither selfing (P < 0.0008) nor introgression with indigenous strains was detected although in vitro interfertility between the American strain and indigenous L. bicolor was identified. No ingress of the introduced genet into adjacent uninoculated plots colonized by various local Laccaria genets was detected. It is proposed that the spatial distributions identified have developed through mycelial propagation of the introduced strain and intraspecific competition with native genets. Although longer-term data is still lacking, the stability of the inoculant strain and the limited disturbance to indigenous populations described support large-scale nursery production of this host-fungal combination.
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Survival of an introduced ectomycorrhizal Laccaria bicolor strain in a European forest plantation monitored by mitochondrial ribosomal DNA analysis
New Phytologist, 1998Co-Authors: Marc-andré Selosse, Francis Martin, François Le TaconAbstract:Mitochondrial and nuclear genes have different inheritance, thus studies of fungal populations should use both mitochondrial and nuclear markers. Using nuclear markers, the S238N strain of the ectomycorrhizal basidiomycete Laccaria bicolor ((Maire) Orton) has been previously shown to persist for at least 10 yr after outplanting in a plantation of Douglas fir (Pseudotsuga menziesii (Mir.) Franco) inoculated with this strain. In the present study, we have sampled 539 sporophores of Laccaria spp. from this plantation, some of which had the S238N nuclear genotype, to study mitochondrial DNA polymorphism and persistence of the inoculated S238N mitochondrial genome. Length polymorphism in fragments of the large subunit of mitochondrial ribosomal DNA (LrDNA) allowed distinction of the haplotypes present in the plantation at the species level. In addition, heteroduplex analysis and sequencing revealed intraspecific polymorphism of LrDNA among the L. bicolor sporophores and enabled specific identification of S238N LrDNA. This haplotype was only retained in sporophores carrying the S238N nuclear genome, confirming the survival of this introduced strain in a natural population.
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Meiotic segregation and recombination of the intergenic spacer of the ribosomal DNA in the ectomycorrhizal basidiomycete Laccaria bicolor
Current Genetics, 1996Co-Authors: Marc-andré Selosse, François Le Tacon, Céline Di Battista, G. Costa, F. MartinAbstract:The aim of this study was to clarify the inheritance of the nuclear ribosomal DNA (rDNA) in the ectomycorrhizal basidiomycete Laccaria bicolor S238N in order to resolve inter- and within-strain relationships in forest ecosystems. PCR amplification of the intergenic spacer (IGS) was carried out in the dikaryotic mycelium and its haploid progeny. In the dikaryotic mycelium, multiple amplification products were produced for the 25s/5s (IGS1) and 5s/17s (IGS2) intergenic spacers. The 4.5- and 4.0-kb fragments of IGS2 (haplotypes α and β, respectively) were observed to occur in a 1:1 ratio within the haploid progeny as a result of divergent IGS haplotypes in the two separate nuclei. Recombinant monokaryons having both types of IGS2 occurred at a low frequency (6.5%; 60 kb per centimorgan) during meiosis. Haplotypes α and β of IGS1 cross-hybridized forming heteroduplexes during the PCR temperature cycle. The two IGS1 haplotypes differed only by the repeat number of a TA2C3 motif and co-segregated with the IGS2 haplotypes. Heteroduplex formation and IGS polymorphism provide information that is helpful in distinguishing between introduced exotic L. bicolor S238N and indigenous populations of Laccaria spp. in forest ecosystems.
Alejandro G. Pardo - One of the best experts on this subject based on the ideXlab platform.
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phg psilbaγ vector system for efficient gene silencing in homobasidiomycetes optimization of ihprna triggering in the mycorrhizal fungus laccaria bicolor
Microbial Biotechnology, 2010Co-Authors: Minna Kemppainen, Alejandro G. PardoAbstract:pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy oriented two‐step PCR cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300‐based binary vector carrying a hygromycin resistance cassette, the pHg/pSILBAγ plasmid is used for Agrobacterium‐mediated transformation. The pHg/pSILBAγ system results in predominantly single integrations of RNA silencing triggering T‐DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. pSILBAγ construct and two other pSILBA plasmid variants (pSILBA and pSILBAα) were evaluated for their capacity to silence Laccaria nitrate reductase gene. While all pSILBA variants tested resulted in up to 65–76% of transformants with reduced growth on nitrate, pSILBAγ produced the highest number (65%) of strongly affected fungal strains. The strongly silenced phenotype was shown to correlate with T‐DNA integration in transcriptionally active genomic sites. pHg/pSILBAγ was shown to produce T‐DNAs with minimum CpG methylation in transgene promoter regions which assures the maximum silencing trigger production in Laccaria. Methylation of the target endogene was only slight in RNA silencing triggered with constructs carrying an intronic spacer hairpin sequence. The silencing capacity of the pHg/pSILBAγ was further tested with Laccaria inositol‐1,4,5‐triphosphate 5‐phosphatase gene. Besides its use in silencing triggering, the herein described plasmid system can also be used for transgene expression in Laccaria. pHg/pSILBAγ silencing system is optimized for L. bicolor but it should be highly useful also for other homobasidiomycetes, group of fungi currently lacking molecular tools for RNA silencing.
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nitrogen metabolism in ectomycorrhizal fungi fhant ac gene regulation in laccaria bicolor
2010Co-Authors: Minna Kemppainen, Alejandro G. Pardo, Lab Oratorio De Micologia MolecularAbstract:The mycorrhizal symbiosis is an ancient mutualistic association between fungi and the roots of the vast majority of terrestrial plant species. In natural ecosystems the plant nutrient uptake (N, P and several micronutrients) from soil happens via the extraradical mycelia of these symbiotic fungi. This association also improves plant water acquisition, heavy metal tolerance and resistance to pathogens. While most herbaceous plants and tropical trees are engaged in endomycorrhiza-type interactions, forest trees of boreal and temperate zones are typically ectomycorrhizal (ECM) plants. These species include the majority of economically important trees and the fungal symbionts are predominantly filamentous basidiomycetes. In order to understand how an ECM fungus exploits soil N resources the expression profile of Laccaria fHANT-AC, a gene cluster responsible for growth on nitrate and containing Lbnrt, Lbnr and Lbnir, was analyzed on variable N regimens and using the RNA silencing technology. As a result a novel regulatory mechanism, not previously described for fungal nitrate acquisition, was discovered. The repression of Laccaria fHANT-AC on ammonium seems not to be mediated via L-glutamine as in ascomycete fungi. Growth on different organic N sources, these including also L-glutamine, results in high transcript levels of Laccaria fHANT-AC and suggest a direct ammonium repression effect. Also uptake of nitrate could be observed when growing on organic N. This finding indicates that the symbiotic fungus, differing from its saprotrophic competitors in soil, has the capacity for efficient simultaneous utilization of both inorganic and organic soil N resources. This also suggests that Laccaria can maintain active nitrate uptake from soil despite the known high hyphal free L-glutamine concentration in ECM fungi destined for N translocation towards the host plant. This novel fHANT-AC regulation pattern reveals an elegant adaptive response of an ECM fungus for maximizing its N acquisition especially in soil-horizons rich in organic N and with spatial and temporal fluctuation of nitrate.
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fHANT-AC genes of the ectomycorrhizal fungus Laccaria bicolor are not repressed by l-glutamine allowing simultaneous utilization of nitrate and organic nitrogen sources.
Environmental Microbiology Reports, 2009Co-Authors: Minna Kemppainen, Maria C. Alvarez Crespo, Alejandro G. PardoAbstract:Summary In boreal and temperate forest ectomycorrhizal fungi play a crucial role in nitrogen cycling by assimilating nitrogenous compounds from soil and transferring them to tree hosts. The expression profile of fHANT-AC genes, nitrate transporter (Lbnrt), nitrate reductase (Lbnr) and nitrite reductase (Lbnir), responsible for nitrate utilization in the ectomycorrhizal fungus Laccaria bicolor, was studied on variable N regimens. The three genes were shown to be under a common regulation: repressed in the presence of ammonium while growth on nitrate resulted in high transcripts accumulation. The presence of nitrate was shown not to be indispensable for activation of Laccaria fHANT-AC as also N starvation and growth on urea and l-asparagine resulted in high transcript levels. Equally high expression of Laccaria fHANT-AC genes was detected in mycelia grown on variable concentrations of l-glutamine. This finding shows that in L. bicolor N metabolite repression of fHANT-AC is not signalled via l-glutamine like described in ascomycetes. The expression patterns of Lbnrt and Lbnir were also studied in an Lbnr RNA-silenced Laccaria strain. No differences were observed on the N source regulation or the degree of transcript accumulation of these genes, indicating that the presence of high nitrate reductase activity is not a core regulator of L. bicolor fHANT-AC expression. The simultaneous utilization of nitrate and organic N sources, already suggested by high transcript levels of Laccaria fHANT-AC genes on organic N, was supported by the increase of culture medium pH as a result of nitrate transporter activity. The possible ecological and evolutionary significance of the herein reported high regulatory flexibility of Laccaria nitrate utilization pathway for ectomycorrizal fungi and the ectomycorrhizal symbiosis is discussed.
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T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor
Microbial Biotechnology, 2008Co-Authors: Minna Kemppainen, Sébastien Duplessis, Alejandro G. PardoAbstract:Ectomycorrhiza is a mutualistic symbiosis formed between fine roots of trees and the mycelium of soil fungi. This symbiosis plays a key role in forest ecosystems for the mineral nutrition of trees and the biology of the fungal communities associated. The characterization of genes involved in developmental and metabolic processes is important to understand the complex interactions that control the ectomycorrhizal symbiosis. Agrobacterium-mediated gene transfer (AMT) in fungi is currently opening a new era for fungal research. As whole genome sequences of several fungi are being released studies about T-DNA integration patterns are needed in order to understand the integration mechanisms involved and to evaluate the AMT as an insertional mutagenesis tool for different fungal species. The first genome sequence of a mycorrhizal fungus, the basidiomycete Laccaria bicolor, became public in July 2006. Release of Laccaria genome sequence and the availability of AMT makes this fungus an excellent model for functional genomic studies in ectomycorrhizal research. No data on the integration pattern in Laccaria genome were available, thus we optimized a plasmid rescue approach for this fungus. To this end the transformation vector (pHg/pBSk) was constructed allowing the rescue of the T-DNA right border (RB)–genomic DNA junctions in Escherichia coli. Fifty-one Agrobacterium-transformed fungal strains, picked up at random from a larger collection of T-DNA tagged strains (about 500), were analysed. Sixty-nine per cent were successfully rescued for the RB of which 87% were resolved for genomic integration sequences. Our results demonstrate that the plasmid rescue approach can be used for resolving T-DNA integration sites in Laccaria. The RB was well conserved during transformation of this fungus and the integration analysis showed no clear sequence homology between different genomic sites. Neither obvious sequence similarities were found between these sites and the T-DNA borders indicating non-homologous integration of the transgenes. Majority (75%) of the integrations were located in predicted genes. Agrobacterium-mediated gene transfer is a powerful tool that can be used for functional gene studies in Laccaria and will be helpful along with plasmid rescue in searching for relevant fungal genes involved in the symbiotic process.
Minna Kemppainen - One of the best experts on this subject based on the ideXlab platform.
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Fluorescent protein expression in the ectomycorrhizal fungus Laccaria bicolor: a plasmid toolkit for easy use of fluorescent markers in basidiomycetes
Current Genetics, 2020Co-Authors: Minna Kemppainen, Jamil Chowdhury, Judith Lundberg-felten, Alejandro PardoAbstract:For long time, studies on ectomycorrhiza (ECM) have been limited by inefficient expression of fluorescent proteins (FPs) in the fungal partner. To convert this situation, we have evaluated the basic requirements of FP expression in the model ECM homobasidiomycete Laccaria bicolor and established eGFP and mCherry as functional FP markers. Comparison of intron-containing and intronless FP-expression cassettes confirmed that intron-processing is indispensable for efficient FP expression in Laccaria . Nuclear FP localization was obtained via in-frame fusion of FPs between the intron-containing genomic gene sequences of Laccaria histone H2B, while cytosolic FP expression was produced by incorporating the intron-containing 5′ fragment of the glyceraldehyde-3-phosphate dehydrogenase encoding gene. In addition, we have characterized the consensus Kozak sequence of strongly expressed genes in Laccaria and demonstrated its boosting effect on transgene mRNA accumulation. Based on these results, an Agrobacterium -mediated transformation compatible plasmid set was designed for easy use of FPs in Laccaria . The four cloning plasmids presented here allow fast and highly flexible construction of C-terminal in-frame fusions between the sequences of interest and the two FPs, expressed either from the endogenous gene promoter, allowing thus evaluation of the native regulation modes of the gene under study, or alternatively, from the constitutive Agaricus bisporus gpdII promoter for enhanced cellular protein localization assays. The molecular tools described here for cell-biological studies in Laccaria can also be exploited in studies of other biotrophic or saprotrophic basidiomycete species susceptible to genetic transformation.
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phg psilbaγ vector system for efficient gene silencing in homobasidiomycetes optimization of ihprna triggering in the mycorrhizal fungus laccaria bicolor
Microbial Biotechnology, 2010Co-Authors: Minna Kemppainen, Alejandro G. PardoAbstract:pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy oriented two‐step PCR cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300‐based binary vector carrying a hygromycin resistance cassette, the pHg/pSILBAγ plasmid is used for Agrobacterium‐mediated transformation. The pHg/pSILBAγ system results in predominantly single integrations of RNA silencing triggering T‐DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. pSILBAγ construct and two other pSILBA plasmid variants (pSILBA and pSILBAα) were evaluated for their capacity to silence Laccaria nitrate reductase gene. While all pSILBA variants tested resulted in up to 65–76% of transformants with reduced growth on nitrate, pSILBAγ produced the highest number (65%) of strongly affected fungal strains. The strongly silenced phenotype was shown to correlate with T‐DNA integration in transcriptionally active genomic sites. pHg/pSILBAγ was shown to produce T‐DNAs with minimum CpG methylation in transgene promoter regions which assures the maximum silencing trigger production in Laccaria. Methylation of the target endogene was only slight in RNA silencing triggered with constructs carrying an intronic spacer hairpin sequence. The silencing capacity of the pHg/pSILBAγ was further tested with Laccaria inositol‐1,4,5‐triphosphate 5‐phosphatase gene. Besides its use in silencing triggering, the herein described plasmid system can also be used for transgene expression in Laccaria. pHg/pSILBAγ silencing system is optimized for L. bicolor but it should be highly useful also for other homobasidiomycetes, group of fungi currently lacking molecular tools for RNA silencing.
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nitrogen metabolism in ectomycorrhizal fungi fhant ac gene regulation in laccaria bicolor
2010Co-Authors: Minna Kemppainen, Alejandro G. Pardo, Lab Oratorio De Micologia MolecularAbstract:The mycorrhizal symbiosis is an ancient mutualistic association between fungi and the roots of the vast majority of terrestrial plant species. In natural ecosystems the plant nutrient uptake (N, P and several micronutrients) from soil happens via the extraradical mycelia of these symbiotic fungi. This association also improves plant water acquisition, heavy metal tolerance and resistance to pathogens. While most herbaceous plants and tropical trees are engaged in endomycorrhiza-type interactions, forest trees of boreal and temperate zones are typically ectomycorrhizal (ECM) plants. These species include the majority of economically important trees and the fungal symbionts are predominantly filamentous basidiomycetes. In order to understand how an ECM fungus exploits soil N resources the expression profile of Laccaria fHANT-AC, a gene cluster responsible for growth on nitrate and containing Lbnrt, Lbnr and Lbnir, was analyzed on variable N regimens and using the RNA silencing technology. As a result a novel regulatory mechanism, not previously described for fungal nitrate acquisition, was discovered. The repression of Laccaria fHANT-AC on ammonium seems not to be mediated via L-glutamine as in ascomycete fungi. Growth on different organic N sources, these including also L-glutamine, results in high transcript levels of Laccaria fHANT-AC and suggest a direct ammonium repression effect. Also uptake of nitrate could be observed when growing on organic N. This finding indicates that the symbiotic fungus, differing from its saprotrophic competitors in soil, has the capacity for efficient simultaneous utilization of both inorganic and organic soil N resources. This also suggests that Laccaria can maintain active nitrate uptake from soil despite the known high hyphal free L-glutamine concentration in ECM fungi destined for N translocation towards the host plant. This novel fHANT-AC regulation pattern reveals an elegant adaptive response of an ECM fungus for maximizing its N acquisition especially in soil-horizons rich in organic N and with spatial and temporal fluctuation of nitrate.
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fHANT-AC genes of the ectomycorrhizal fungus Laccaria bicolor are not repressed by l-glutamine allowing simultaneous utilization of nitrate and organic nitrogen sources.
Environmental Microbiology Reports, 2009Co-Authors: Minna Kemppainen, Maria C. Alvarez Crespo, Alejandro G. PardoAbstract:Summary In boreal and temperate forest ectomycorrhizal fungi play a crucial role in nitrogen cycling by assimilating nitrogenous compounds from soil and transferring them to tree hosts. The expression profile of fHANT-AC genes, nitrate transporter (Lbnrt), nitrate reductase (Lbnr) and nitrite reductase (Lbnir), responsible for nitrate utilization in the ectomycorrhizal fungus Laccaria bicolor, was studied on variable N regimens. The three genes were shown to be under a common regulation: repressed in the presence of ammonium while growth on nitrate resulted in high transcripts accumulation. The presence of nitrate was shown not to be indispensable for activation of Laccaria fHANT-AC as also N starvation and growth on urea and l-asparagine resulted in high transcript levels. Equally high expression of Laccaria fHANT-AC genes was detected in mycelia grown on variable concentrations of l-glutamine. This finding shows that in L. bicolor N metabolite repression of fHANT-AC is not signalled via l-glutamine like described in ascomycetes. The expression patterns of Lbnrt and Lbnir were also studied in an Lbnr RNA-silenced Laccaria strain. No differences were observed on the N source regulation or the degree of transcript accumulation of these genes, indicating that the presence of high nitrate reductase activity is not a core regulator of L. bicolor fHANT-AC expression. The simultaneous utilization of nitrate and organic N sources, already suggested by high transcript levels of Laccaria fHANT-AC genes on organic N, was supported by the increase of culture medium pH as a result of nitrate transporter activity. The possible ecological and evolutionary significance of the herein reported high regulatory flexibility of Laccaria nitrate utilization pathway for ectomycorrizal fungi and the ectomycorrhizal symbiosis is discussed.
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T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor
Microbial Biotechnology, 2008Co-Authors: Minna Kemppainen, Sébastien Duplessis, Alejandro G. PardoAbstract:Ectomycorrhiza is a mutualistic symbiosis formed between fine roots of trees and the mycelium of soil fungi. This symbiosis plays a key role in forest ecosystems for the mineral nutrition of trees and the biology of the fungal communities associated. The characterization of genes involved in developmental and metabolic processes is important to understand the complex interactions that control the ectomycorrhizal symbiosis. Agrobacterium-mediated gene transfer (AMT) in fungi is currently opening a new era for fungal research. As whole genome sequences of several fungi are being released studies about T-DNA integration patterns are needed in order to understand the integration mechanisms involved and to evaluate the AMT as an insertional mutagenesis tool for different fungal species. The first genome sequence of a mycorrhizal fungus, the basidiomycete Laccaria bicolor, became public in July 2006. Release of Laccaria genome sequence and the availability of AMT makes this fungus an excellent model for functional genomic studies in ectomycorrhizal research. No data on the integration pattern in Laccaria genome were available, thus we optimized a plasmid rescue approach for this fungus. To this end the transformation vector (pHg/pBSk) was constructed allowing the rescue of the T-DNA right border (RB)–genomic DNA junctions in Escherichia coli. Fifty-one Agrobacterium-transformed fungal strains, picked up at random from a larger collection of T-DNA tagged strains (about 500), were analysed. Sixty-nine per cent were successfully rescued for the RB of which 87% were resolved for genomic integration sequences. Our results demonstrate that the plasmid rescue approach can be used for resolving T-DNA integration sites in Laccaria. The RB was well conserved during transformation of this fungus and the integration analysis showed no clear sequence homology between different genomic sites. Neither obvious sequence similarities were found between these sites and the T-DNA borders indicating non-homologous integration of the transgenes. Majority (75%) of the integrations were located in predicted genes. Agrobacterium-mediated gene transfer is a powerful tool that can be used for functional gene studies in Laccaria and will be helpful along with plasmid rescue in searching for relevant fungal genes involved in the symbiotic process.
F. Martin - One of the best experts on this subject based on the ideXlab platform.
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Effect of Laccaria bicolor strains inoculated on Douglas-fir (Pseudotsuga menziesii) several years after nursery inoculation
Canadian Journal of Forest Research, 2000Co-Authors: Marc-andré Selosse, D. Bouchard, F. Martin, François Le TaconAbstract:In the Saint-Brisson experiment conducted in central France, the American strain of the ectomycorrhizal fungus Laccaria bicolor (Maire) P.D. Orton S238N and the French strain L. bicolor 81306 inoculated on containerized Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings increased by 60% the total volume of wood produced 8 years after outplanting as compared with uninoculated but naturally mycorrhizal trees. The two strains introduced 10 years before in the inoculated plots are still present and dominant; they did not prevent the colonization of Douglas-fir roots by naturally occurring ectomycorrhizal fungi but allowed for the establishment of a very diversified symbiotic microflora. Eight to 12 years after outplanting, all the Douglas-fir plots were colonized by Laccaria laccata (Scop.:Fr.) Cooke or L. bicolor strains, as well as some other species, independently of the nursery treatments. With one exception in one plot, the presence of indigenous genets in the control treatments may have preven...
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Temporal persistence and spatial distribution of an American inoculant strain of the ectomycorrhizal basidiomycete Laccaria bicolor in a French forest plantation
Molecular Ecology, 1998Co-Authors: Marc-andré Selosse, D. Bouchard, D. Jacquot, F. MartinAbstract:Selected strains of ectomycorrhizal fungi, such as the basidiomycete Laccaria bicolor, are currently being used as inoculants in nurseries to improve growth of forest trees after outplanting. Information is needed on the survival of these introduced strains in forests and their impact on indigenous biodiversity. Dissemination and persistence of an American strain, L. bicolor S238N, were studied 10 years after outplanting in a Douglas fir plantation located at Saint-Brisson (Morvan, France). About 430 Laccaria spp. sporophores were collected over 3 years. Inheritance of nuclear ribosomal DNA, as well as RAPD markers, was characterized in L. bicolor S238N, using a haploid progeny set of 91 monokaryons. More than 50 markers were identified (19 heterozygous and 33 homozygous or cytoplasmic markers), which unambiguously confirmed that the introduced strain was still present in the inoculated plots. Neither selfing (P < 0.0008) nor introgression with indigenous strains was detected although in vitro interfertility between the American strain and indigenous L. bicolor was identified. No ingress of the introduced genet into adjacent uninoculated plots colonized by various local Laccaria genets was detected. It is proposed that the spatial distributions identified have developed through mycelial propagation of the introduced strain and intraspecific competition with native genets. Although longer-term data is still lacking, the stability of the inoculant strain and the limited disturbance to indigenous populations described support large-scale nursery production of this host-fungal combination.
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Meiotic segregation and recombination of the intergenic spacer of the ribosomal DNA in the ectomycorrhizal basidiomycete Laccaria bicolor
Current Genetics, 1996Co-Authors: Marc-andré Selosse, François Le Tacon, Céline Di Battista, G. Costa, F. MartinAbstract:The aim of this study was to clarify the inheritance of the nuclear ribosomal DNA (rDNA) in the ectomycorrhizal basidiomycete Laccaria bicolor S238N in order to resolve inter- and within-strain relationships in forest ecosystems. PCR amplification of the intergenic spacer (IGS) was carried out in the dikaryotic mycelium and its haploid progeny. In the dikaryotic mycelium, multiple amplification products were produced for the 25s/5s (IGS1) and 5s/17s (IGS2) intergenic spacers. The 4.5- and 4.0-kb fragments of IGS2 (haplotypes α and β, respectively) were observed to occur in a 1:1 ratio within the haploid progeny as a result of divergent IGS haplotypes in the two separate nuclei. Recombinant monokaryons having both types of IGS2 occurred at a low frequency (6.5%; 60 kb per centimorgan) during meiosis. Haplotypes α and β of IGS1 cross-hybridized forming heteroduplexes during the PCR temperature cycle. The two IGS1 haplotypes differed only by the repeat number of a TA2C3 motif and co-segregated with the IGS2 haplotypes. Heteroduplex formation and IGS polymorphism provide information that is helpful in distinguishing between introduced exotic L. bicolor S238N and indigenous populations of Laccaria spp. in forest ecosystems.
