A new promising phylogenetic marker to study the diversity of fungal communities: the GLYCOSIDE HYDROLASE 63 gene L Pérez‐Izquierdo, E Morin, JP Maurice, F Martin, A Rincón, M Buée. Molecular Ecology Resources
In molecular ecology, the development of efficient molecular markers for fungi remains an important research domain. Nuclear ribosomal internal transcribed spacer (ITS) region was proposed as universal DNA barcode marker for Fungi, but this marker was criticized for idel-induced alignment problems and its potential lack of phylogenetic resolution. Our main aim was to develop a new phylogenetic gene and a putative functional marker, from single-copy gene, to describe fungal diversity. Thus, we developed a series of primers to amplify a polymorphic region of the Glycoside Hydrolase GH63 gene, encoding exo-acting α-glucosidases, in Basidiomycetes. These primers were validated on 125 different fungal genomic DNAs and GH63 amplification yield was compared with that of already published functional markers targeting genes coding for laccases, N-acetylhexosaminidases, cellobiohydrolases and class II Peroxidases. Specific amplicons were recovered for 95% of the fungal species tested and GH63 amplification success was strikingly higher than rates obtained with other functional genes. We downloaded the GH63 sequences from 483 fungal genomes publicly available at the JGI MycoCosm database. GH63 was present in 461 fungal genomes belonging to all phyla, except Microsporidia and Neocallimastigomycotadivisions. Moreover, the phylogenetic trees built with both GH63 and Rpb1 protein sequences revealed that GH63 is also a promising phylogenetic marker. Finally, a very high proportion of GH63 proteins was predicted to be secreted. This molecular tool could be a new phylogenetic marker of fungal species as well as potential indicator of functional diversity of Basidiomycotes fungal communities in term of secretory capacities.
Low effect of phenanthrene bioaccessibility on its biodegradation in diffusely contaminated soil M Crampon, A Cébron, F Portet-Koltalo, S Uroz, F Le Derf, J Bodilis Environmental Pollution
This study focused on the role of bioaccessibility in the phenanthrene (PHE) biodegradation in diffusely contaminated soil, by combining chemical and microbiological approaches. First, we determined PHE dissipation rates and PHE sorption/desorption isotherms for two soils (PPY and Pv) presenting similar chronic PAH contamination, but different physico-chemical properties. Our results revealed that the PHE dissipation rate was significantly higher in the Pv soil compared to the PPY soil, while PHE sorption/desorption isotherms were similar. Interestingly, increases of PHE desorption and potentially of PHE bioaccessibility were observed for both soils when adding rhamnolipids (biosurfactants produced by Pseudomonas aeruginosa). Second, using 13C-PHE incubated in the same soils, we analyzed the PHE degrading bacterial communities. The combination of stable isotope probing (DNA-SIP) and 16S rRNA gene pyrosequencing revealed that Betaproteobacteria were the main PHE degraders in the Pv soil, while a higher bacterial diversity (Alpha-, Beta-, Gammaproteobacteria and Actinobacteria) was involved in PHE degradation in the PPY soil. The amendment of biosurfactants commonly used in biostimulation methods (i.e. rhamnolipids) to the two soils clearly modified the PHE sorption/desorption isotherms, but had no significant impact on PHE degradation rates and PHE-degraders identity. These results demonstrated that increasing the bioaccessibility of PHE has a low impact on its degradation and on the functional populations involved in this degradation.
Atypical protein disulfide isomerases (PDI): Comparison of the molecular and catalytic properties of poplar PDI-A and PDI-M with PDI-L1A B Selles, F Zannini, J Couturier, JP Jacquot, N Rouhier PloS one 12 (3), e0174753
Protein disulfide isomerases are overwhelmingly multi-modular redox catalysts able to perform the formation, reduction or isomerisation of disulfide bonds. We present here the biochemical characterization of three different poplar PDI isoforms. PDI-A is characterized by a single catalytic Trx module, the so-called a domain, whereas PDI-L1a and PDI-M display an a-b-b’-a’ and a°-a-b organisation respectively. Their activities have been tested in vitro using purified recombinant proteins and a series of model substrates as insulin, NADPH thioredoxin reductase, NADP malate dehydrogenase (NADP-MDH), peroxiredoxins or RNase A. We demonstrated that PDI-A exhibited none of the usually reported activities, although the cysteines of the WCKHC active site signature are able to form a disulfide with a redox midpoint potential of -170 mV at pH 7.0. The fact that it is able to bind a [Fe2S2] cluster upon Escherichia coli expression and anaerobic purification might indicate that it does not have a function in dithiol-disulfide exchange reactions. The two other proteins were able to catalyze oxidation or reduction reactions, PDI-L1a being more efficient in most cases, except that it was unable to activate the non-physiological substrate NADP-MDH, in contrast to PDI-M. To further evaluate the contribution of the catalytic domains of PDI-M, the dicysteinic motifs have been independently mutated in each a domain. The results indicated that the two a domains seem interconnected and that the a° module preferentially catalyzed oxidation reactions whereas the a module catalyzed reduction reactions, in line with the respective redox potentials of -170 mV and -190 mV at pH 7.0. Overall, these in vitro results illustrate that the number and position of aand b domains influence the redox properties and substrate recognition (both electron donors and acceptors) of PDI which contributes to understand why this protein family expanded along evolution.
Populus trichocarpa encodes small, effector-like secreted proteins that are highly induced during mutualistic symbiosis JM Plett, H Yin, R Mewalal, R Hu, T Li, P Ranjan, S Jawdy, HC De Paoli, … Scientific Reports 7 (1), 382
During symbiosis, organisms use a range of metabolic and protein-based signals to communicate. Of these protein signals, one class is defined as ‘effectors’, i.e., small secreted proteins (SSPs) that cause phenotypical and physiological changes in another organism. To date, protein-based effectors have been described in aphids, nematodes, fungi and bacteria. Using RNA sequencing of Populus trichocarpa roots in mutualistic symbiosis with the ectomycorrhizal fungus Laccaria bicolor, we sought to determine if host plants also contain genes encoding effector-like proteins. We identified 417 plant-encoded putative SSPs that were significantly regulated during this interaction, including 161 SSPs specific to P. trichocarpa and 15 SSPs exhibiting expansion in Populus and closely related lineages. We demonstrate that a subset of these SSPs can enter L. bicolor hyphae, localize to the nucleus and affect hyphal growth and morphology. We conclude that plants encode proteins that appear to function as effector proteins that may regulate symbiotic associations.
A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy. CM Guennoc, C Rose, F Guinnet, I Miquel, J Labbé, A Deveau. JoVE (Journal of Visualized Experiments), e54771-e54771
Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.
Cora Miquel-Guennoc défendra publiquement sa thèse Lundi 6 mars à 14h30 (amphi 7 FST).
“Etude de l’interaction physique entre le champignon ectomycorhizien Laccaria bicolor S238N et la bactérie auxiliaire de la mycorhization Pseudomonas fluorescens BBc6R8”
Dans les sols, les champignons ectomycorhiziens (ECM) forment une symbiose très répandue avec les racines des arbres et contribuent ainsi à leur croissance et à leur santé. Des études antérieures ont montré que certaines bactéries pouvaient influencer positivement la symbiose entre les ECM et les arbres, appelées BAM pour Bactéries Auxiliaires de la Mycorhization. Les mécanismes de l’effet auxiliaire des BAM sont encore peu connus. En amont de cette thèse, il avait été montré in vitro que la BAM Pseudomonas fluorescens BBc6 formait des structures similaires à des biofilms sur les hyphes de l’ECM Laccaria bicolor. Dans ce contexte, afin d’enrichir les connaissances concernant les interactions entre les ECM et les BAM, cette thèse a porté sur l’interaction physique entre ces deux organismes. L’étude a en partie été réalisée via une méthode d’analyse par microscopie confocale, développée durant cette thèse. Les résultats obtenus ont montré que cette bactérie formait des biofilms localisés préférentiellement sur la région apicale des colonies de l’ECM ce qui pourrait indiquer une interaction trophique. L’existence d’une telle interaction a d’ailleurs par la suite été confirmée. Les résultats ont également montré que l’interaction physique entre L. bicolor et BBc6 n’est pas spécifique puisque l’ensemble des treize autres souches bactériennes testées a formé des biofilms sur les hyphes de L. bicolor. En revanche, BBc6 s’est montrée incapable de former des biofilms sur certains champignons appartenant aux Ascomycètes, suggérant des mécanismes d’inhibition. De plus, l’étude de la matrice des biofilms formés par BBc6R8 a révélé la présence de réseaux de filaments constitués d’ADN qui semblent structurer ces biofilms et qui ont aussi été observés chez l’ensemble des souches bactériennes testées. Ces résultats révèlent un rôle structural de la molécule d’ADN qui, bien qu’il semble répandu, n’a que peu été reporté jusqu’à présent. Enfin, il a été montré que des mutants de BBc6 qui ont perdu leur effet auxiliaire forment des biofilms différents de la souche sauvage sur une surface abiotique suggérant un lien potentiel entre l’effet auxiliaire et la formation de biofilms.
In soil ecosystems, ectomycorrhizal fungi (ECM) form a widespread symbiosis with roots of trees, contributing to tree growth and health. It has been shown that some bacteria, called mycorrhization helper bacteria (MHB), stimulate mycorrhizal symbiosis. The mechanisms of this helper effect are poorly understood. Previous studies have shown that the MHB Pseudomonas fluorescens BBc6 formed biofilm-like structures around the hyphae of the ECM Laccaria bicolor during their in vitro interaction. In this context, in order to increase knowledge concerning MHB / ECM interactions, the work presented here focuses on the physical interaction between these two organisms. To this purpose, a method of analysis based on confocal microscopy was developed. The results showed that the bacteria formed biofilms preferentially localized on the apical region of the ECM colonies, which could indicate a trophic interaction. Such an interaction has been subsequently confirmed. The results also showed that the physical interaction between L. bicolor and BBc6 is not specific since all thirteen other bacterial strains tested formed biofilms on the hyphae of L. bicolor. On the other hand, BBc6 was unable to form biofilms on some fungi belonging to Ascomycetes, suggesting the existence of inhibition mechanisms. Moreover, the study of the BBc6 biofilm matrix revealed networks of DNA-containing filaments which seem to structure these biofilms and which have also been observed in all the bacterial strains tested. These results reveal a structural role of the DNA molecule, a role that has been rarely reported so far despite its probable high occurence. Finally, it has been shown that BBc6R8 mutants having lost their helper effect presented a modified phenotype concerning their biofilm formation on abiotic surface, suggesting a potential link between the helper effect and the biofilms formation.
Post-translational modifications of Medicago truncatula glutathione peroxidase 1 induced by nitric oxide C Castella, I Mirtziou, A Seassau, A Boscari, F Montrichard, … Nitric Oxide
Plant glutathione peroxidases (Gpx) catalyse the reduction of various peroxides, such as hydrogen peroxide (H2O2), phospholipid hydroperoxides and peroxynitrite, but at the expense of thioredoxins rather than glutathione. A main function of plant Gpxs is the protection of biological membranes by scavenging phospholipid hydroperoxides, but some Gpxs have also been associated with H2O2 sensing and redox signal transduction. Nitric oxide (NO) is not only known to induce the expression of Gpx family members, but also to inhibit Gpx activity, presumably through the S-nitrosylation of conserved cysteine residues. In the present study, the effects of NO-donors on both the activity and S-nitrosylation state of purified Medicago truncatula Gpx1 were analyzed using biochemical assay measurements and a biotin-switch/mass spectrometry approach. MtGpx1 activity was only moderately inhibited by the NO-donors diethylamine-NONOate and S-nitrosoglutathione, and the inhibition may be reversed by DTT. The three conserved Cys of MtGpx1 were found to be modified through S-nitrosylation and S-glutathionylation, although to different extents, by diethylamine-NONOate and S-nitrosoglutathione, or by a combination of diethylamine-NONOate and reduced glutathione. The regulation of MtGpx1 and its possible involvement in the signaling process is discussed in the light of these results.
Functional outcomes of fungal community shifts driven by tree genotype and spatial‐temporal factors in Mediterranean pine forests L Pérez‐Izquierdo, M Zabal‐Aguirre, D Flores‐Rentería, … Environmental Microbiology
Fungi provide relevant ecosystem services contributing to primary productivity and the cycling of nutrients in forests. These fungal inputs can be decisive for the resilience of Mediterranean forests under global change scenarios, making necessary an in-deep knowledge about how fungal communities operate in these ecosystems. By using high-throughput sequencing and enzymatic approaches, we studied the fungal communities associated with three genotypic variants of Pinus pinaster trees, in 45-yr-old common garden plantations. We aimed to determine the impact of biotic (i.e. tree genotype) and abiotic (i.e. season, site) factors on the fungal community structure, and to explore whether structural shifts triggered functional responses affecting relevant ecosystem processes. Tree genotype and spatial-temporal factors were pivotal structuring fungal communities, mainly by influencing their assemblage and selecting certain fungi. Diversity variations of total fungal community and of that of specific fungal guilds, together with edaphic properties and tree’s productivity, explained relevant ecosystem services such as processes involved in carbon turnover and phosphorous mobilization. A mechanistic model integrating relations of these variables and ecosystem functional outcomes is provided. Our results highlight the importance of structural shifts in fungal communities because they may have functional consequences for key ecosystem processes in Mediterranean forests. This article is protected by copyright. All rights reserved.
Antifungal activities of wood extractives N Valette, T Perrot, R Sormani, E Gelhaye, M Morel-Rouhier. Fungal Biology Reviews
Extractives are non-structural wood molecules that represent a minor fraction in wood. However, they are source of diverse molecules putatively bioactive. Inhibition of fungal growth is one of the most interesting properties of wood extractives in a context of wood preservation, crop protection or medical treatments. The antifungal effect of molecules isolated from wood extractives has been mainly attributed to various mechanisms such as metal and free radical scavenging activity, direct interaction with enzymes, disruption of membrane integrity and perturbation of ionic homeostasis. Lignolytic fungi, which are microorganisms adapted to wood substrates, have developed various strategies to protect themselves against this toxicity. A better knowledge of these strategies could help both developing new systems for extractive removal in biomass valorization processes and using these molecules as antifungal agents
The transcriptional landscape of basidiosporogenesis in mature Pisolithus microcarpus basidiocarp M de Freitas Pereira, AN da Rocha Campos, TC Anastacio, E Morin, …BMC genomics 18 (1), 157
Background: Pisolithus microcarpus (Cooke & Massee) G. Cunn is a gasteromycete that produces closed basidiocarps in symbiosis with eucalypts and acacias. The fungus produces a complex basidiocarp composed of peridioles at different developmental stages and an upper layer of basidiospores free of the hyphae and ready for wind dispersal upon the rupture of the basidiocarp pellis. During basidiosporogenesis, a process that takes place inside the basidiocarp peridioles, a conspicuous reserve of fatty acids is present throughout development. While several previous studies have described basidiosporogenesis inside peridioles, very little is known about gene expression changes that may occur during this part of the fungal life cycle. The objective of this work was to analyze gene transcription during peridiole and basidiospore development, while focusing specifically on cell cycle progression and lipid metabolism.
Results: Throughout different developmental stages of the peridioles we analyzed, 737 genes were regulated between adjacent compartments (>5 fold, FDR-corrected p-value < 0.05) corresponding to 3.49% of the genes present in the P. microcarpus genome. We identified three clusters among the regulated genes which showed differential expression between the peridiole developmental stages and the basidiospores. During peridiole development, transcripts for proteins involved in cellular processes, signaling, and information storage were detected, notably those for coding transcription factors, DNA polymerase subunits, DNA repair proteins, and genes involved in chromatin structure. For both internal embedded basidiospores (hereto referred to as “Internal spores”, IS) and external free basidiospores (hereto referred to as “Free spores”, FS), upregulated transcripts were found to involve primary metabolism, particularly fatty acid metabolism (FA). High expression of transcripts related to β-oxidation and the glyoxylate shunt indicated that fatty acids served as a major carbon source for basidiosporogenesis.
Conclusion: Our results show that basidiocarp formation in P. microcarpus involves a complex array of genes that are regulated throughout peridiole development. We identified waves of transcripts with coordinated regulation and identified transcription factors which may play a role in this regulation. This is the first work to describe gene expression patterns during basidiocarp formation in an ectomycorrhizal gasteromycete fungus and sheds light on genes that may play important roles in the developmental process.
Keywords: Gene expression, Peridiole development, Spores, Cell cycle, Fatty acid metabolism