Category Archives: Academic productions

Article: Metallomics

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Common and metal-specific proteomic responses to cadmium and zinc in the metal tolerant ericoid mycorrhizal fungus Oidiodendron maius Zn  M Chiapello, E Martino, S Perotto Metallomics

Abstract

Although adaptive metal tolerance may arise in fungal populations in polluted soils, the mechanisms underlying metal-specific tolerance are poorly understood. Comparative proteomics is a powerful tool to identify variation in protein profiles caused by changing environmental conditions, and was used to investigate protein accumulation in a metal tolerant isolate of the ericoid mycorrhizal fungus Oidiodendron maius exposed to zinc and cadmium. Two-dimensional gel electrophoresis and shotgun proteomics followed by mass spectrometry lead to the identification of common and metal-specific proteins and pathways. Proteins selectively induced by cadmium exposure were molecular chaperons of the Hsp90 family, cytoskeletal proteins and components of the translation machinery. Zinc significantly up-regulated metabolic pathways related to energy production and carbohydrates metabolism, likely mirroring zinc adaptation of this fungal isolate. Common proteins induced by the two metal ions were the antioxidant enzyme Cu/Zn superoxide dismutase and ubiquitin. In mycelia exposed to zinc and cadmium, both proteomic techniques also identified agmatinase, an enzyme involved in polyamine biosynthesis. This novel finding suggests that, like plants, polyamines may have important functions in response to abiotic environmental stress in fungi. Genetic evidence also suggests that the biosynthesis of polyamines via an alternative metabolic pathway may be widespread in fungi.

Article: Journal of Experimental Botany

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Function of glutathione peroxidases in legume root nodules

MA Matamoros, A Saiz, M Peñuelas, P Bustos-Sanmamed, JM Mulet, …
Journal of Experimental Botany, erv066

Abstract

Glutathione peroxidases (Gpxs) are antioxidant enzymes not studied so far in legume nodules, despite the fact that reactive oxygen species are produced at different steps of the symbiosis. The function of two Gpxs that are highly expressed in nodules of the model legume Lotus japonicuswas examined. Gene expression analysis, enzymatic and nitrosylation assays, yeast cell complementation, in situ mRNA hybridization, immunoelectron microscopy, and LjGpx-green fluorescent protein (GFP) fusions were used to characterize the enzymes and to localize each transcript and isoform in nodules. The LjGpx1 and LjGpx3 genes encode thioredoxin-dependent phospholipid hydroperoxidases and are differentially regulated in response to nitric oxide (NO) and hormones. LjGpx1 and LjGpx3 are nitrosylated in vitro or in plants treated with S-nitrosoglutathione (GSNO). Consistent with the modification of the peroxidatic cysteine of LjGpx3, in vitro assays demonstrated that this modification results in enzyme inhibition. The enzymes are highly expressed in the infected zone, but the LjGpx3 mRNA is also detected in the cortex and vascular bundles. LjGpx1 is localized to the plastids and nuclei, and LjGpx3 to the cytosol and endoplasmic reticulum. Based on yeast complementation experiments, both enzymes protect against oxidative stress, salt stress, and membrane damage. It is concluded that both LjGpxs perform major antioxidative functions in nodules, preventing lipid peroxidation and other oxidative processes at different subcellular sites of vascular and infected cells. The enzymes are probably involved in hormone and NO signalling, and may be regulated through nitrosylation of the peroxidatic cysteine essential for catalytic function.

Article: Environmental Microbiology

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Metagenome sequence of Elaphomyces granulatus from sporocarp tissue reveals Ascomycota ectomycorrhizal fingerprints of genome expansion and a Proteobacteria rich microbiome

CA Quandt, A Kohler, C Hesse, T Sharpton, F Martin, JW Spatafora
Environmental Microbiology

Abstract

Many obligate symbiotic fungi are difficult to maintain in culture, and there is a growing need for alternative approaches to obtaining tissue and subsequent genomic assemblies from such species. In this study, the genome of Elaphomyces granulatus was sequenced from sporocarp tissue. The genome assembly remains on many contigs, but gene space is estimated to be mostly complete. Phylogenetic analyses revealed that the Elaphomyces lineage is most closely related to Talaromyces and Trichocomaceae s.s. The genome of E. granulatus is reduced in CAZymes, despite a large expansion in genome size, both of which are consistent with what is seen in T. melanosporum, the other sequenced ectomycorrhizal ascomycete. A large number of transposable elements are predicted in the E. granulatus genome, especially Gypsy-like long terminal repeats (LTR), and there has also been an expansion in helicases. The metagenome is a complex community dominated by bacteria in Bradyrhizobiaceae, and there is evidence to suggest that the community may be reduced in functional capacity as estimated by KEGG pathways. Through the sequencing of sporocarp tissue this study has provided insights into Elaphomyces phylogenetics, genomics, metagenomics, and the evolution of the ectomycorrhizal association.

Article: Applied and Environmental Microbiology

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Pseudomonas fluorescens pirates both ferrioxamine and ferri-coelichelin siderophores from Streptomyces ambofaciens. J Galet, A Deveau, L Hôtel, P Frey-Klett, P Leblond, B Aigle. Applied and Environmental Microbiology, AEM. 03520-14

ABSTRACT

Iron is essential in many biological processes. However, its bioavailability is reduced in aerobic environments such as soil. To overcome this limitation, microorganisms have developed different strategies, such as iron chelation by siderophores. Some bacteria have even gained the ability to detect and utilize xenosiderophores, i.e. siderophores produced by other organisms. We illustrate an example of such an interaction between two soil bacteria, Pseudomonas fluorescens strain BBc6R8 and Streptomyces ambofaciens ATCC23877, which produce the siderophores pyoverdine and enantio-pyochelin and the siderophores desferrioxamines B, E and coelichelin, respectively. During pairwise cultures on iron-limiting agar medium, no induction of siderophore synthesis byP. fluorescens BBc6R8 was observed in presence of S. ambofaciens ATCC23877. Co-cultures with a Streptomyces mutant strain that produced either coelichelin or desferrioxamines, as well as culture in a medium supplemented with desferrioxamine B, resulted in the absence of pyoverdine production; however, culture with a double mutant deficient in desferrioxamines and coelichelin production did not. This strongly suggests that P. fluorescens BBbc6R8 utilizes the ferrioxamines and ferri-coelichelin produced by S. ambofaciens as xenosiderophores, and therefore no longer activates the production of its own siderophores. A screening of a library of P. fluorescens BBc6R8 mutants highlighted the involvement of the TonB-dependent receptor FoxA in this process: the expression of foxA and genes involved in the regulation of its biosynthesis was induced in presence of S. ambofaciens. In a competitive environment such as soil, siderophore piracy could well be one of the driving forces that determines the outcome of microbial competition.

Article: Fungal Genetics and Biology

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Evidence for homoploid speciation in Phytophthora alni supports taxonomic reclassification in this species complex

C Husson, J Aguayo, C Revellin, P Frey, R Ioos, B Marçais
Fungal Genetics and Biology

Abstract

Alder decline has been a problem along European watercourses since the early 1990s. Hybridization was identified as the main cause of this emerging disease. Indeed, the causal agent, a soil-borne pathogen named Phytophthora alni subsp. alni (Paa) is the result of interspecific hybridization between two taxa, Phytophthora alni subsp. multiformis (Pam) and Phytophthora alni subsp. uniformis (Pau), initially identified as subspecies of Paa. The aim of this work was to characterize the ploidy level within the P. alni complex that is presently poorly understood. For that, we used two complementary approaches for a set of 31 isolates of Paa, Pam and Pau: (i) quantification of allele copy number of three single-copy nuclear genes using allele-specific real-time PCR and (ii) comparison of the genome size estimated by flow cytometry. Relative quantification of alleles of the three single-copy genes showed that the copy number of a given allele in Paa was systematically half that of its parents Pau or Pam. Moreover, DNA content estimated by flow cytometry in Paa was equal to half the sum of those in Pam and Pau. Our results therefore suggest that the hybrid Paa is an allotriploid species, containing half of the genome of each of its parents Pam and Pau, which in turn are considered to be allotetraploid and diploid, respectively. Paa thus results from a homoploid speciation process. Based on published data and on results from this study, a new formal taxonomic name is proposed for the three taxa Paa, Pam and Pau which are raised to species status and renamed P. ×alni, P. ×multiformis and P. uniformis, respectively.

Article: Aerobiologica

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Relationship between the genetic characteristics of Botrytis sp. airborne inoculum and meteorological parameters, seasons and the origin of air masses C Leyronas, F Halkett, PC Nicot. Aerobiologia, 1-14

Abstract

Grey mould is a worldwide disease on many economically important crops. It is caused by two fungal species, Botrytis cinerea and B. pseudocinerea, which are mainly airborne dispersed. Although several studies have considered the abundance of airborne inoculum of B. cinerea in models forecasting the risk of grey mould epidemics, the genetic characteristics of this inoculum are poorly known. In the present study, airborne inoculum of B. cinerea and B. pseudocinerea was collected on 29 dates over a 2.5-year period on a site in south-eastern France. The 683 sampled isolates were genotyped with nine microsatellites markers, and 616 were identified as B. cinerea. The genetic structure of B. cinerea airborne inoculum was inferred with Bayesian assignment tests. Eight genetic clusters were identified. Cluster abundance showed temporal variation and was statistically linked to the season (P = 0.0009) and the origin of air masses (P < 0.0001). The proportion of isolates belonging to the species B. pseudocinerea was equal to 9.8 % on average, but it showed temporal variation; it tended to be higher in winter. This study is the first to provide information about the genetic characteristics of airborne inoculum of B. cinerea and B. pseudocinerea and to bring evidence of relationship with seasons, meteorological parameters and with the origin of air masses.

Article: Nature communications

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Volatile signalling by sesquiterpenes from ectomycorrhizal fungi reprogrammes root architecture.

FA Ditengou, A Müller, M Rosenkranz, J Felten, H Lasok, MM van Doorn, …
Nature communications 6, 6279

Abstract

The mutualistic association of roots with ectomycorrhizal fungi promotes plant health and is a hallmark of boreal and temperate forests worldwide. In the pre-colonization phase, before direct contact, lateral root (LR) production is massively stimulated, yet little is known about the signals exchanged during this step. Here, we identify sesquiterpenes (SQTs) as biologically active agents emitted by Laccaria bicolor while interacting with Populus or Arabidopsis. We show that inhibition of fungal SQT production by lovastatin strongly reduces LR proliferation and that (-)-thujopsene, a low-abundance SQT, is sufficient to stimulate LR formation in the absence of the fungus. Further, we show that the ectomycorrhizal ascomycote, Cenococcum geophilum, which cannot synthesize SQTs, does not promote LRs. We propose that the LR-promoting SQT signal creates a win-win situation by enhancing the root surface area for plant nutrient uptake and by improving fungal access to plant-derived carbon via root exudates.

Article: Nature Genetics

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Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists A Kohler, A Kuo, LG Nagy, E Morin, KW Barry, F Buscot, B Canbäck, …Nature Genetics

Abstract

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.

Article: Plant Physiology

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Arabidopsis glutaredoxin S17 and its partner NF-YC11/NC2α contribute to maintenance of the shoot apical meristem under long-day photoperiod

J Knuesting, C Riondet, C Maria, I Kruse, N Becuwe, N König, C Berndt, …
Plant Physiology, pp. 00049.2015

Abstract

Glutaredoxins (GRXs) catalyse the reduction of protein disulfide bonds using glutathione as a reductant. Certain GRXs are able to transfer iron-sulfur (Fe-S) clusters to other proteins. To investigate the function of Arabidopsis thaliana GRXS17, we applied a strategy combining biochemical, genetic and physiological approaches. GRXS17 was localized in the nucleus and cytosol, and its expression was elevated in the shoot meristems and reproductive tissues. Recombinant GRXS17 bound Fe2S2 clusters, a property likely contributing to its ability to complement the defects of a yeast strain lacking the mitochondrial GRX5. However, a grxs17 knock-out Arabidopsis mutant exhibited only a minor decrease in the activities of Fe-S enzymes, suggesting that its primary function is as a disulfide oxidoreductase. The grxS17 plants were sensitive to high temperature and long-day photoperiod, resulting in elongated leaves, compromised shoot apical meristem, and delayed bolting. Both environmental conditions applied simultaneously led to a growth arrest. Using affinity chromatography and split-YFP methods, a nuclear transcriptional regulator termed NF-YC11/NC2α was identified as a GRXS17 interacting partner. A mutant deficient in NF-YC11/NC2α exhibited similar phenotypes to grxs17 in response to photoperiod. Therefore, we propose that GRXS17 interacts with NF-YC11/NC2α to relay a redox signal generated by photoperiod to maintain meristem function.

Article: Molecular Ecology Resources

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A survey of genome-wide single nucleotide polymorphisms through genome re-sequencing in the Périgord black truffle (Tuber melanosporum Vittad.)

Thibaut Payen, Claude Murat, Anaïs Gigant, Emmanuelle Morin, Stéphane De Mita and Francis Martin

Abstract

The Périgord black truffle (Tuber melanosporum Vittad.), considered a gastronomic delicacy worldwide, is an ectomycorrhizal filamentous fungus that is ecologically important in Mediterranean French, Italian, and Spanish woodlands. In this study, we developed a novel resource of single nucleotide polymorphisms (SNPs) for T. melanosporum using Illumina high-throughput re-sequencing. The genome from six T. melanosporum geographic accessions was sequenced to a depth of ~20×. These geographic accessions were selected from different populations within the northern and southern regions of the geographical species distribution. Approximately 80% of the reads for each of the six re-sequenced geographic accessions mapped against the reference T. melanosporum genome assembly, estimating the core genome size of this organism to be ~110 Mbp. A total of 442,326 SNPs corresponding to 3,540 SNPs/Mbps were identified as being included in all seven genomes. The SNPs occurred more frequently in repeated sequences (85%), although 4,501 SNPs were also identified in the coding regions of 2,587 genes. Using the ratio of non-synonymous mutations per non-synonymous site (pN) to synonymous mutations per synonymous site (pS) and Tajima’s D index scanning the whole genome, we were able to identify genomic regions and genes potentially subjected to positive or purifying selection. The SNPs identified represent a valuable resource for future population genetics and genomics studies.