Langues du site :

Partenaires

CNRS
UCBL
INRA
ENVL
FR41
IDEX




Rechercher

Sur ce site


Accueil > Equipes de Recherche > Adaptation des Microorganismes Eucaryotes à leur Environnement (AMEE) > En - Adaptation des Microorganismes Eucaryotes à leur Environnement (AMEE)

En - Adaptation des Microorganismes Eucaryotes à leur Environnement (AMEE)

Team leader : MARMEISSE Roland

Co-Team leader:LUIS Patricia

Permanent members

DORE Jeanne Assistant Engineer (AI), UCB
FRAISSINET-TACHET Laurence Lecturer (MCF), UCB
HUGONI Mylène Lecturer (MCF), UCB
LUIS Patricia Lecturer (MCF), UCB
MARMEISSE Roland Senior Research Scientist (DR), CNRS
MELAYAH Delphine Lecturer (MCF), UCB
VALLON Laurent Technician (T), UCB

Non permanent members

BARBI Florian PhD Student (2012-2015)
BRAGALINI Claudia PhD Student (2012-2013 co-tutelle university of Turin, Italy)
ZILLER Antoine PhD Student (2013-2016)

Presentation :

Eukaryotic microorganisms (fungi, "protists") are major players in soil biology. They represent the main decomposers of plant organic matter (saprotrophic fungi), they regulate bacterial populations and biomass (phagotrophic protists) and many of them are either beneficial (eg. mycorrhizal symbiotic fungi) or on the contrary damaging (pathogenic) partners of macroorganisms, either plants or animals. Although their contributions to C and N terrestrial cycles are important, the level of functional diversity of the soil eukaryotic microflora remains under evaluated. Indeed, soil encompasses at different spatial scales a complex microflora whose taxonomic composition varies with time and for which only a small fraction is accessible to laboratory studies as numerous microbial species remain uncultivable.
Research activities of the AMEE team aim at better evaluating the roles of soil microbial eukaryotes. Two main programmes are developed.

Research programmes : 

Theme 1:Genomics and genetics of ectomycorrhizal symbiotic fungi

Most temperate forest trees (oak, beech, poplar, pine, fir…) and numerous fungal species (amanitas, boletes, truffles…) associate to each other to form the so-called ectomycorrhizal root symbiosis. This beneficial symbiosis leads to bidirectional fluxes of nutrients between the partners (plant sugars in exchange for soil-derived N, P and other minerals) and contributes to host plant fitness and optimal use of limiting and poorly accessible forest soil nutrients.
Although it is now well established that the capacity of forming ectomycorrhizas has appeared several times independently during fungal evolution, the fungal functions implicated in the establishment of a functional symbiotic association remain largely unknown.
Our research project aims at identifying experimentally these functions using the basidiomycete species Hebeloma cylindrosporum associated to Pinus pinaster (Fig. 1). We developed an insertional mutagenesis programme, which leads to the random insertion, within the fungal genome, of mutagenic T-DNA from the bacterium Agrobacterium tumefaciens. We thus obtained for the first time non symbiotic mutants of an ectomycorrhizal fungus.
Characterisation of the mutated genes will give us information on the fungal functions necessary for symbiosis establishment, as well as on the potential implication of homologous fungal genes in other plant/fungal interactions either symbiotic or pathogenic.
Since the public release of the full genomic sequence of H. cylindrosporum (http://genome.jgi-psf.org/Hebcy2/Hebcy2.home.htm), we also try to decipher globally, using transcriptomic and proteomic approaches, the genomic programs leading to a functional symbiosis. Targeted analysis of secreted proteins (i.e ; the secretome) is also carried out to evaluate the controversial "saprotrophic potential" of symbiotic fungi and their capacity to mobilise nutrients from forest soils.

Figure 1. The basidiomycete fungus Hebeloma cylindrosporum associated with Pinus pinaster as a model to infer the genetic and molecular bases of differentiation and functioning of the ectomycorrhizal symbiosis. Its entire life cycle, from spore to spore, can be obtained under laboratory conditions (Debaud & Gay, 1987, New Phytol 105 : 429-435) ; it can be easily transformed using Agrobacterium tumefaciens (Combier et al. 2003 FEMS Microbiol lett 220 : 141-148) and a collection of mutant strains is available, including non mycorrhizal ones (Combier et al, 2004 Mol Plant Microbe Interact 17 : 1029-1038). Its genome has been sequenced and annotated at the Joint Genome Institute (http://genome.jgi-psf.org/Hebcy2/Hebcy2.home.htm) in association with the AMEE team.

Theme 2 : Adaptation of soil eukaryotic microbial communities to their environment

This research subject does not target a single species but aims at identifying the roles played by the different members of a soil microbial community. We aim at establishing what are the different functions really expressed in situ in the soils by the different eukaryotic organisms, cultivable or not. To reach this objective, we developed an environmental genomic approach called metatranscriptomics. A metatranscriptome represents the sum of the different genes expressed by the different soil eukaryotic organisms (i.e., the sum of their transcriptomes). From an experimental point of view, RNA synthesized by all organisms are directly extracted from soil samples and eukaryotic polyadenylated mRNA are specifically converted into cDNA which can be directly sequenced or cloned to constitute environmental cDNA libraries (Figure 2). The analysis of expressed genes reflects the activities performed in situ by microorganisms directly in soil. Genes of interest are selected by different approaches such as their expression in yeast or high-throughput systematic sequencing. This approach which goes from "soil RNA" to the expression of environmental functional gene in yeast has been validated in the laboratory. In parallel, an analysis of the taxonomic diversity of eukaryotes is also performed (Figure 2). In soils, some eukaryotic phyla are notoriously under-estimated and under-studied, this is the case for example of Foraminifera (Rhizaria) that we detected almost systematically in various soils and which were initially only known from the marine environment.

The metatranscriptomic approach is being developed in two different contexts :
-  In a context of ecotoxicology to understand the adaptive responses of eukaryotic microbial communities to heavy metal pollutions. This project led us to characterise novel gene families, from the soil metatranscriptomes, which confer a cadmium or zinc resistant phenotype to yeast metal-sensitive mutants.
-  In the context of global changes to understand how changes in land use or climate (change in the level of precipitations) affect the process of soil organic matter degradation, which is essential for nutrient cycling in soils. These projects involve high-throughput sequencing of total eukaryotic metatranscriptomes from different sites, as well as the specific study of selected families of genes coding lignocellulolytic enzymes and transporters of sugars and nitrogenous molecules.


Figure 2. The metatranscriptomic approach : from nucleic acids extracted from environmental samples to eukaryotic genes expressed in yeast. This experimental approach, validated in the laboratory, allows the analysis of all genes expressed by all eukaryotic microorganisms, cultivable or not, present in an environmental sample.

Experimental approaches and technical skills

Members of the AMEE team have developed, acquired and optimised a number of original techniques in the field of fungal genetics, molecular biology and environmental genomics. The main ones, which can be used for other projects are :
-  Agrotransformation and insertional mutagenesis in filamentous fungi.
-  Extraction and purification of intracellular and excreted fungal proteins for proteomic analyses
-  Extraction of soil RNA and from tree roots
-  Construction of environmental cDNA libraries enriched in full-length genes for phenotypic screening
-  Phenotypic screening of cDNA libraries in yeast (S. cerevisiae and others) and phenotyping of transformants (Omnilog and Bioscreen)
-  High-throughput sequencing (HiSeq & MiSeq technologies) of fungal or soil-extracted RNA and of PCR products.

 

Bibliography :

2017



  • Doré J, et al. 2017. The ectomycorrhizal basidiomycete Hebeloma cylindrosporum undergoes early waves of transcriptional reprogramming prior to symbiotic structures differentiation. Environmental Microbiology. n/a-n/a. doi: 10.1111/1462-2920.13670.


  • Hugoni M, Vellet A, Debroas D. 2017. Unique and highly variable bacterial communities inhabiting the surface microlayer of an oligotrophic lake. Aquatic Microbial Ecology. 79:115-125. doi: 10.3354/ame01825.

2016



  • Barbi F, et al. 2016. Tree species select diverse soil fungal communities expressing different sets of lignocellulolytic enzyme-encoding genes. Soil Biology and Biochemistry. 100:149-159. doi: 10.1016/j.soilbio.2016.06.008.


  • Marmeisse R, Girlanda M. 2016. 10 Mycorrhizal Fungi and the Soil Carbon and Nutrient Cycling. In: Environmental and Microbial Relationships. Druzhinina, IS & Kubicek, CP. Springer International Publishing: Cham p. 189-203. http://link.springer.com/10.1007/978-3-319-29532-9_10.


  • Yadav RK, Bragalini C, Fraissinet-Tachet L, Marmeisse R, Luis P. 2016. Metatranscriptomics of Soil Eukaryotic Communities. In: Microbial Environmental Genomics (MEG). Martin, F & Uroz, S. Vol. 1399 Springer New York: New York, NY p. 273-287. http://link.springer.com/10.1007/978-1-4939-3369-3_16.


  • Ziller A, et al. 2016. Metagenomics analysis reveals a new metallothionein family: Sequence and metal-binding features of new environmental cysteine-rich proteins. Journal of Inorganic Biochemistry. doi: 10.1016/j.jinorgbio.2016.11.017.

2015


  • Barbi F. 2015. Impact de l’essence forestière sur les processus de dégradation et d’assimilation des polysaccharides végétaux par la communauté fongique des sols forestiers. Lyon 1 http://www.theses.fr/2015LYO10347.


  • Doré J, et al. 2015. Comparative genomics, proteomics and transcriptomics give new insight into the exoproteome of the basidiomycete <i>Hebeloma cylindrosporum</i> and its involvement in ectomycorrhizal symbiosis. New Phytologist. n/a-n/a. doi: 10.1111/nph.13546.


  • Kohler A, et al. 2015. Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists. Nature Genetics. advance online publication. doi: 10.1038/ng.3223.


  • Reddy MS, et al. 2015. Metal induction of a <i>Pisolithus albus</i> metallothionein and its potential involvement in heavy metal tolerance during mycorrhizal symbiosis: <i>Pisolithus albus</i> metallothionein in metal tolerance. Environmental Microbiology. n/a-n/a. doi: 10.1111/1462-2920.13149.

2014



  • Barbi F, et al. 2014. PCR Primers to Study the Diversity of Expressed Fungal Genes Encoding Lignocellulolytic Enzymes in Soils Using High-Throughput Sequencing. PLoS ONE. 9:e116264. doi: 10.1371/journal.pone.0116264.


  • Belmondo S, et al. 2014. A dipeptide transporter from the arbuscular mycorrhizal fungus Rhizophagus irregularis is upregulated in the intraradical phase. Plant Traffic and Transport. 5:436. doi: 10.3389/fpls.2014.00436.


  • Bragalini C, et al. 2014. Solution Hybrid Selection Capture for the Recovery of Functional Full-Length Eukaryotic cDNAs From Complex Environmental Samples. DNA Research. doi: 10.1093/dnares/dsu030.


  • Bruto M, Prigent-Combaret C, Luis P, Moënne-Loccoz Y, Muller D. 2014. Frequent, independent transfers of a catabolic gene from bacteria to contrasted filamentous eukaryotes. Proceedings of the Royal Society B: Biological Sciences. 281:20140848. doi: 10.1098/rspb.2014.0848.


  • Doré J, Marmeisse R, Combier J-P, Gay G. 2014. A fungal conserved gene from the basidiomycete Hebeloma cylindrosporum is essential for efficient ectomycorrhiza formation. Molecular Plant-Microbe Interactions. doi: 10.1094/MPMI-03-14-0087-R.


  • Kellner H, et al. 2014. Widespread Occurrence of Expressed Fungal Secretory Peroxidases in Forest Soils. PLoS ONE. 9:e95557. doi: 10.1371/journal.pone.0095557.


  • Reddy SM, Prasanna L, Marmeisse R, Fraissinet-Tachet L. 2014. Differential expression of metallothioneins in response to heavy metals and their involvement in metal tolerance in the symbiotic basidiomycete Laccaria bicolor. Microbiology. mic.0.080218-0. doi: 10.1099/mic.0.080218-0.


  • Yadav RK, et al. 2014. Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA. BMC Biotechnology. 14:80. doi: 10.1186/1472-6750-14-80.

2013



  • Bruto M, et al. 2013. Horizontal Acquisition of Prokaryotic Genes for Eukaryote Functioning and Niche Adaptation. In: Evolutionary Biology: Exobiology and Evolutionary Mechanisms. Pontarotti, P. Springer Berlin Heidelberg p. 165-179. http://link.springer.com/chapter/10.1007/978-3-642-38212-3_11.


  • Fraissinet-Tachet L, Marmeisse R, Zinger L, Luis P. 2013. Metatranscriptomics of Soil Eukaryotic Communities. In: The Ecological Genomics of Fungi. Francisrtin,. John Wiley & Sons, Inc p. 305–323. http://onlinelibrary.wiley.com/doi/10.1002/9781118735893.ch14/summary.


  • Klaubauf S, et al. 2013. The pentose catabolic pathway of the rice-blast fungus Magnaporthe oryzae involves a novel pentose reductase restricted to few fungal species. FEBS Letters. 587:1346-1352. doi: 10.1016/j.febslet.2013.03.003.


  • Lehembre F, et al. 2013. Soil metatranscriptomics for mining eukaryotic heavy metal resistance genes. Environmental Microbiology. n/a–n/a. doi: 10.1111/1462-2920.12143.


  • Luis P, Gauthier A, Trouvelot S, Poinssot B, Frettinger P. 2013. The identification of Plasmopara viticola genes potentially involved in pathogenesis on grapevine suggests new similarities between oomycetes and true fungi. Phytopathology. 130501115210007. doi: 10.1094/PHYTO-06-12-0121-R.


  • Marmeisse R, Nehls U, Öpik M, Selosse MA, Pringle A. 2013. Bridging mycorrhizal genomics, metagenomics and forest ecology. New Phytologist. 198:343–346. doi: 10.1111/nph.12205.

  • Perraud M. 2013. Étude du dialogue moléculaire entre les partenaires de la symbiose ectomycorhizienne : implication d'une subtilase sécrétée par le champignon Hebeloma cylindrosporum. http://n2t.net/ark:/47881/m6d21vv2 (Consulté sans date).

  • Perrin A. 2013. Rôle des alpha-tubulines fongiques dans la symbiose ectomycorhizienne et dans les interactions champignons plantes. http://n2t.net/ark:/47881/m6p26w9p (Consulté sans date).