Author Archives: eric

PhD Defense: J. Galet

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The defense will be held the 16th September 2014 at 14H00 in Amphitheater 7, Faculté des Sciences, Boulevard des Aiguillettes, Vandoeuvre

“Vers la compréhension des dialogues microbiens dans l’écosystème sol : étude de l’interaction entre Streptomyces et Pseudomonas”

 

Résumé: 

Les Streptomyces sont des bactéries communes des écosystèmes forestiers tempérés. Elles sont présentes au sein de différentes niches écologiques telles que la rhizosphère des plantes, la mycorhizosphère ou encore la minéralosphère. Dans ces niches, elles interagissent avec de nombreux autres genres bactériens et différents champignons symbiotiques, pathogènes et saprophytes. Bien que les membres du genre Streptomyces soient bien connus pour leur capacité à produire une grande variété de métabolites secondaires, le rôle écologique de ces produits naturels reste largement inconnu. Si leur rôle en tant que molécules antagonistes est probable, ils pourraient également fournir des signaux chimiques dans la communication intercellulaire et jouer un rôle majeur dans la structuration des communautés microbiennes du sol. La question se pose alors de savoir si en retour, l’environnement biotique de Streptomyces module les voies de biosynthèse de métabolites secondaires chez ces bactéries et s’il impacte leur comportement au sein des communautés microbiennes du sol.

Afin de répondre à ces questions, nous avons entrepris une étude sur l’interaction entre deux souches bactériennes modèles du sol Streptomyces ambofaciens ATCC23877 et Pseudomonas fluorescens BBc6R8. S. ambofaciens ATCC23877 est connue pour produire au moins cinq molécules antimicrobiennes et deux sidérophores et l’analyse de son génome a révélé la présence de 16 clusters de gènes supplémentaires impliqués dans la synthèse de métabolites secondaires. P. fluorescens BBc6R8 est une bactérie auxiliaire de mycorhization isolée de fructifications du champignon Laccaria bicolor. Des expériences de cocultures sur différents milieux gélosés ont révélé que chaque souche bactérienne affecte la production de métabolites secondaires chez l’autre partenaire. Ainsi, P. fluorescens inhibe la capacité de S. ambofaciens à produire la kinamycine, un « effet ping-pong » a été mis en évidence. D’autre part, sur un milieu déficient en fer, P. fluorescens est capable d’utiliser les desferrioxamines et la coelicheline produites par S. ambofaciens en tant que xénosiderophores et ne produit alors plus ses propres sidérophores, la pyoverdine et l’énantio-pyochéline. La protéine FoxA de P. fluorescens BBc6R8, un récepteur TonB-dépendant, est impliquée dans la détection et l’utilisation de ces deux sidérophores de S. ambofaciens ATCC 23877. Enfin, au cours des études d’interactions, P. fluorescens BBc6R8 s’est révélée capable d’inhiber la production du pigment bleu diffusible gamma-actinorhodine chez Streptomyces coelicolor A3(2) M145 en acidifiant le milieu par la production d’acide gluconique. D’autres souches de Pseudomonas fluorescent et la souche opportuniste Pseudomonas aeruginosa PAO1 empêche, de la même manière, la production de gamma-actinorhodine. L’ensemble de ces résultats nous permet de proposer différentes hypothèses sur l’importance écologique de ces trois interactions en les replaçant au sein du contexte de l’écosystème sol.

Article: Fungal Genetics and Biology

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OmGOGAT disruption in the ericoid mycorrhizal fungus Oidiodendron maius induces reorganization of the N pathway and reduces tolerance to heavy-metals. HR Khouja, S Daghino, S Abbà, F Boutaraa, M Chalot, D Blaudez, …Fungal Genetics and Biology

Abstract

Mycorrhizal fungi are key mediators of soil-to-plant movement of mineral nutrients, including essential and non-essential metals. In soil conditions that facilitate mobilization of metal ions, potentially toxic metals can interfere with nitrogen metabolism in both plants and microorganisms. Less is known about possible relationships between nitrogen metabolism and responses to heavy metals. Aim of this study was to investigate this aspect in the ericoid mycorrhizal fungus Oidiodendron maius strain Zn, a metal tolerant ascomycete. Growth of O. maius Zn on zinc and cadmium containing media was significantly affected by the nitrogen source. Screening of a library of O. maius Zn random genetic transformants for sensitivity to heavy metals (zinc and cadmium) and oxidative stress (menadione) yielded a mutant strain that carried a partial deletion of the glutamate synthase (NADH-GOGAT EC 1.4.1.14) gene and its adjacent gene, the APC15subunit of the anaphase promoting complex. Comparison of WT and OmGOGAT-OmAPC15 mutant strains indicated an impaired N-metabolism and altered stress tolerance, and assays on the OmAPC15-recomplemented strains ascribed the observed phenotypes to the deletion in the OmGOGAT gene.OmGOGAT disruption modified the nitrogen pathway, with a strong reduction of the associated glutamine synthetase (GS, EC 6.3.1.2) activity and an up-regulation of the alternative NADP-glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4) pathway for glutamate biosynthesis. Unless they were supplemented with glutamine, O. maius Zn transformants lacking OmGOGAT were very sensitive to zinc. These results highlight the importance of nitrogen metabolism not only for nitrogen assimilation and transformation, but also for stress tolerance. For mycorrhizal fungi, such as O. maius, this may bear consequences not only to the fungus, but also to the host plant.

Modification du statut des ECs

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Pour votre information, le décret modifiant les dispositions statutaires applicables aux enseignants-chercheurs est paru au JO du 4 septembre 2014.

Décret n° 2014-997 du 2 septembre 2014 modifiant le décret n° 84-431 du 6 juin 1984 fixant les dispositions statutaires communes applicables aux enseignants-chercheurs et portant statut particulier du corps des professeurs des universités et du corps des maîtres de conférences

En résumé, voici les principaux changements introduits dans le décret statutaire (source syndicale)

•  possibilité de mutation et de détachement des collègues pour rapprochement de conjoint ou handicap sans examen par un comité de sélection

•  fixation par chaque établissement d’un nombre d’emplois à pourvoir exclusivement par la voie de la mutation

•  encadrement strict de la modulation de service qui devient facultative et qui nécessite d’obtenir un accord écrit de l’intéressé

•  remplacement de l’évaluation par un suivi de carrière quinquennal qui prend en compte l’ensemble des activités

•  précision des modalités de demande d’examen pour les dossiers de qualification relevant de plusieurs sections CNU

•  possibilité de demander le réexamen d’un refus de rattachement à une équipe de recherche

•  élargissement de la liste des fonctions ouvrant droit à concours réservé pour les MC HDR particulièrement impliqués (dans la limite de 1/9e des emplois PR mis aux concours dans l’ensemble des disciplines)

•  expérimentation du dé-contingentement des emplois de professeurs ouverts au recrutement au titre de l’agrégation externe dans les disciplines économiques et de gestion

•  création de l’éméritat pour les maîtres de conférences

•  maintien de la parité PR/MC dans les conseils siégeant en formation restreinte et les comités de sélection 

•  composition des comités de sélection avec 40% de membres de chaque sexe sauf dérogation pour certaines disciplines

•  dispositions spécifiques pour les personnes en situation de handicap et précision des modalités de leur titularisation

•  suppression de l’impossibilité de participer à plus de 3 comités de sélection : pour contourner les difficultés de parité dans certaines disciplines

•  instauration d’une possibilité de mise en situation professionnelle lors de l’audition des candidats par un comité de sélection, sous forme de leçon ou de séminaire de présentation des travaux de recherche. Cette mise en situation peut être publique.

Article: Acta Crystallographica

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X-ray structures of Nfs2, the plastidial cysteine desulfurase from Arabidopsis thaliana

T Roret, H Pégeot, J Couturier, G Mulliert, N Rouhier, C Didierjean
Acta Crystallographica Section F: Structural Biology Communications 70 (9 …

Abstract:

The chloroplastic Arabidopsis thaliana Nfs2 (AtNfs2) is a group II pyridoxal 5′-phosphate-dependent cysteine desulfurase that is involved in the initial steps of iron-sulfur cluster biogenesis. The group II cysteine desulfurases require the presence of sulfurtransferases such as SufE proteins for optimal activity. Compared with group I cysteine desulfurases, proteins of this group contains a smaller extended lobe harbouring the catalytic cysteine and have a [beta]-hairpin constraining the active site. Here, two crystal structures of AtNfs2 are reported: a wild-type form with the catalytic cysteine in a persulfide-intermediate state and a C384S variant mimicking the resting state of the enzyme. In both structures the well conserved Lys241 covalently binds pyridoxal 5′-phosphate, forming an internal aldimine. Based on available homologous bacterial complexes, a model of a complex between AtNfs2 and the SufE domain of its biological partner AtSufE1 is proposed, revealing the nature of the binding sites.

Article: Fungal Genetics and Biology

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Genomic and transcriptomic analysis of Laccaria bicolor CAZome reveals insights into polysaccharides remodelling during symbiosis establishment C Veneault-Fourrey, C Commun, A Kohler, E Morin, R Balestrini, J Plett, …

Fungal Genetics and Biology

Abstract

Ectomycorrhizal fungi, living in soil forests, are required microorganisms to sustain tree growth and productivity. The establishment of mutualistic interaction with roots to form ectomycorrhiza (ECM) is not well known at the molecular level. In particular, how fungal and plant cell walls are rearranged to establish a fully functional ectomycorrhiza is poorly understood. Nevertheless, it is likely that Carbohydrate Active enZymes (CAZyme) produced by the fungus participate in this process.

Genome-wide transcriptome profiling during ECM development was used to examine how the CAZome of L. bicolor is regulated during symbiosis establishment.

CAZymes active on fungal cell wall were upregulated during ECM development in particular after 4 weeks of contact when the hyphae are surrounding the root cells and start to colonize the apoplast. We demonstrated that one expansin-like protein, whose expression is specific to symbiotic tissues, localizes within fungal cell wall.

Whereas L. bicolor genome contained a constricted repertoire of CAZymes active on cellulose and hemicellulose, these CAZymes were expressed during the first steps of root cells colonization. L. bicolorretained the ability to use homogalacturonan, a pectin-derived substrate, as carbon source. CAZymes likely involved in pectin hydrolysis were mainly expressed at the stage of a fully mature ECM.

All together, our data suggest an active remodelling of fungal cell wall with a possible involvement of expansin during ECM development. By contrast, a soft remodelling of the plant cell wall likely occurs through the loosening of the cellulose microfbrils by AA9 or GH12 CAZymes and middle lamella smooth remodelling through pectin (homogalacturonan) hydrolysis likely by GH28, GH12 CAZymes.

Article: Frontiers in Plant Science

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Genome-wide patterns of segregation and linkage disequilibrium: the construction of a linkage genetic map of the poplar rust fungus Melampsora larici-populina

M Pernaci, S De_mita, A Andrieux, J Pétrowski, F Halkett, S Duplessis, …
Name: Frontiers in Plant Science 5, 454
Abstract
The poplar rust fungus Melampsora larici-populina causes significant yield reduction and severe economic losses in commercial poplar plantations. After several decades of breeding for qualitative resistance and subsequent breakdown of the released resistance genes, breeders now focus on quantitative resistance, perceived to be more durable. But quantitative resistance also can be challenged by an increase of aggressiveness in the pathogen. Thus it is of primary importance to better understand the genetic architecture of aggressiveness traits. To this aim, our goal is to build a genetic linkage map for M. larici-populina in order to map quantitative trait loci related to aggressiveness. First, a large progeny of M. larici-populina was generated through selfing of the reference strain 98AG31 (which genome sequence is available) on larch plants, the alternate host of the poplar rust fungus. The progeny’s meiotic origin was validated through a segregation analysis of 115 offspring with 14 polymorphic microsatellite markers, of which 12 segregated in the expected 1:2:1 Mendelian ratio. A microsatellite-based linkage disequilibrium analysis allowed us to identify one potential linkage group comprising two scaffolds. The whole genome of a subset of 47 offspring was resequenced using the Illumina HiSeq 2000 technology at a mean sequencing depth of 6X. The reads were mapped onto the reference genome of the parental strain and 144,566 SNPs were identified across the genome. Analysis of distribution and polymorphism of the SNPs along the genome led to the identification of 2580 recombination blocks. A second linkage disequilibrium analysis, using the recombination blocks as markers, allowed us to group 81 scaffolds into 23 potential linkage groups. These preliminary results showed that a high-density linkage map could be constructed by using high-quality SNPs based on low-coverage resequencing of a larger number of M. larici-populina offspring.

Article: Frontiers in Plant Science

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Patterns of genomic variation in the poplar rust fungus Melampsora larici-populina identify pathogenesis-related factors A Persoons, E Morin, C Delaruelle, T Payen, F Halkett, P Frey, S De Mita, … Plant-Microbe Interaction 5, 450

Abstract
Melampsora larici-populina is a fungal pathogen responsible for foliar rust disease on poplar trees, which causes damage to forest plantations worldwide, particularly in Northern Europe. The reference genome of the isolate 98AG31 was previously sequenced using a whole genome shotgun strategy, revealing a large genome of 101 megabases containing 16,399 predicted genes, which included secreted protein genes representing poplar rust candidate effectors. In the present study, the genomes of 15 isolates collected over the past 20 years throughout the French territory, representing distinct virulence profiles, were characterized by massively parallel sequencing to assess genetic variation in the poplar rust fungus. Comparison to the reference genome revealed striking structural variations. Analysis of coverage and sequencing depth identified large missing regions between isolates related to the mating type loci. More than 611,824 single-nucleotide polymorphism (SNP) positions were uncovered overall, indicating a remarkable level of polymorphism. Based on the accumulation of non-synonymous substitutions in coding sequences and the relative frequencies of synonymous and non-synonymous polymorphisms (i.e. PN/PS), we identify candidate genes that may be involved in fungal pathogenesis. Correlation between non-synonymous SNPs in genes encoding secreted proteins and pathotypes of the studied isolates revealed candidate genes potentially related to virulences 1, 6 and 8 of the poplar rust fungus.

Article: Frontiers in Pharmacology

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The still mysterious roles of cysteine-containing glutathione transferases in plants
P Lallement, B Brouwer, O Keech, A Hecker, N Rouhier
Experimental Pharmacology and Drug Discovery 5, 192

Abstract

Glutathione transferases (GSTs) represent a widespread multigenic enzyme family able to modify a broad range of molecules. These notably include secondary metabolites and exogenous substrates often referred to as xenobiotics, usually for their detoxification, subsequent transport or export. To achieve this, these enzymes can bind non-substrate ligands (ligandin function) and/or catalyze the conjugation of glutathione onto the targeted molecules, the latter activity being exhibited by GSTs having a serine or a tyrosine as catalytic residues. Besides, other GST members possess a catalytic cysteine residue, a substitution that radically changes enzyme properties. Instead of promoting GSH-conjugation reactions, cysteine-containing GSTs (Cys-GSTs) are able to perform deglutathionylation reactions similarly to glutaredoxins but the targets are usually different since glutaredoxin substrates are mostly oxidized proteins and Cys-GST substrates are metabolites. The Cys-GSTs are found in most organisms and form several classes. While Beta and Omega GSTs and chloride intracellular channel proteins (CLICs) are not found in plants, these organisms possess microsomal ProstaGlandin E-Synthase type 2, glutathionyl hydroquinone reductases, Lambda, Iota and Hemerythrin GSTs and dehydroascorbate reductases (DHARs); the four last classes being restricted to the green lineage. In plants, whereas the role of DHARs is clearly associated to the reduction of dehydroascorbate to ascorbate, the physiological roles of other Cys-GSTs remain largely unknown. In this context, a genomic and phylogenetic analysis of Cys-GSTs in photosynthetic organisms provides an updated classification that is discussed in the light of the recent literature about the functional and structural properties of Cys-GSTs. Considering the antioxidant potencies of phenolic compounds and more generally of secondary metabolites, the connection of GSTs with secondary metabolism may be interesting from a pharmacological perspective.

Article: Applied and Environmental Microbiology

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Transcriptomic responses of Phanerochaete chrysosporium to oak acetonic extracts: focus on a new glutathione transferase. A Thuillier, K Chibani, G Belli, E Herrero, S Dumarçay, P Gérardin, … Applied and Environmental Microbiology, AEM. 02103-14

Abstract

The first steps of wood degradation by fungi lead to the release of toxic compounds known as extractives. To better understand how lignolytic fungi cope with the toxicity of these molecules, a transcriptomic analysis of Phanerochaete chrysosporium genes was performed in presence of oak acetonic extracts. It reveals that in complement to the extracellular machinery of degradation, intracellular antioxidant and detoxification systems contribute to the lignolytic capabilities of fungi presumably by preventing cellular damages and maintaining fungal health. Focusing on these systems, a glutathione transferase (PcGTT2.1) has been selected for functional characterization. This enzyme, not characterized so far in basidiomycetes, has been first classified as a GTT2 in comparison to theSaccharomyces cerevisiae isoform. However, a deeper analysis shows that GTT2.1 isoform has functionally evolved to reduce lipid peroxidation by recognizing high-molecular weight peroxides as substrates. Moreover, the GTT2.1 gene has been lost in some non-wood decay fungi. This example suggests that the intracellular detoxification system could have evolved concomitantly with the extracellular ligninolytic machinery in relation to the capacity of fungi to degrade wood.

Article: Genome Biology

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The black truffle methylome: non-exhaustive DNA methylation-mediated transposon silencing in a complex fungal genome with massive repeat element content B Montanini, PY Chen, M Morselli, A Jaroszewicz, D Lopez, F Martin, … Genome Biology 15 (7), 411

Abstract

Background

We investigated how an extremely transposon element (TE)-rich organism such as the plant-symbiotic ascomycete truffle Tuber melanosporum exploits DNA methylation to cope with the more than 45,000 repeated elements that populate its genome.

Results

Whole-genome bisulfite sequencing performed on different developmental stages reveals a high fraction of methylated cytosines with a strong preference for CpG sites. The methylation pattern is highly similar among samples and selectively targets TEs rather than genes. A marked trend toward hypomethylation is observed for TEs located within a 1 kb distance from expressed genes, rather than segregated in TE-rich regions of the genome. Approximately 300 hypomethylated or unmethylated TEs are transcriptionally active, with higher expression levels in free-living mycelium compared to fruitbody. Indeed, multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs are found almost exclusively in free-living mycelium. A reduction of DNA methylation, restricted to non-CpG sites and accompanied by an increase in TE expression, is observed upon treatment of free-living mycelia with 5-azacytidine.

Conclusions

Evidence derived from analysis of the T. melanosporum methylome indicates that a non-exhaustive, partly reversible, methylation process operates in truffles. This allows for the existence of hypomethylated, transcriptionally active TEs that are associated with copy number variant regions of the genome. Non-exhaustive TE methylation may reflect a role of active TEs in promoting genome plasticity and the ability to adapt to sudden environmental changes.