Metatranscriptomics sheds light on the links between the functional traits of fungal guilds and ecological processes in forest soil ecosystems.

Soil fungi belonging to different functional guilds, such as saprotrophs, pathogens, and mycorrhizal symbionts, play key roles in forest ecosystems. To date, no study has compared the actual gene expression of these guilds in different forest soils. We used metatranscriptomics to study the competition for organic resources by these fungal groups in boreal, temperate, and Mediterranean forest soils. Using a dedicated mRNA annotation pipeline combined with the JGI MycoCosm database, we compared the transcripts of these three fungal guilds, targeting enzymes involved in C- and N mobilization from plant and microbial cell walls. Genes encoding enzymes involved in the degradation of plant cell walls were expressed at a higher level in saprotrophic fungi than in ectomycorrhizal and pathogenic fungi. However, ectomycorrhizal and saprotrophic fungi showed similarly high expression levels of genes encoding enzymes involved in fungal cell wall degradation. Transcripts for N-related transporters were more highly expressed in ectomycorrhizal fungi than in other groups. We showed that ectomycorrhizal and saprotrophic fungi compete for N in soil organic matter, suggesting that their interactions could decelerate C cycling. Metatranscriptomics provides a unique tool to test controversial ecological hypotheses and to better understand the underlying ecological processes involved in soil functioning and carbon stabilization.

Stable functional structure despite high taxonomic variability across fungal communities in soils of old-growth montane forests.

BACKGROUND: Major advances over the past decade in molecular ecology are providing access to soil fungal diversity in forest ecosystems worldwide, but the diverse functions and metabolic capabilities of this microbial community remain largely elusive. We conducted a field survey in montane old-growth broadleaved and conifer forests, to investigate the relationship between soil fungal diversity and functional genetic traits. To assess the extent to which variation in community composition was associated with dominant tree species (oak, spruce, and fir) and environmental variations in the old-growth forests in the Jade Dragon Snow Mountain in Yunnan Province, we applied rDNA metabarcoding. We also assessed fungal gene expression in soil using mRNA sequencing and specifically assessed the expression of genes related to organic matter decomposition and nutrient acquisition in ectomycorrhizal and saprotrophic fungi. RESULTS: Our taxonomic profiling revealed striking shifts in the composition of the saprotrophic and ectomycorrhizal guilds among the oak-, fir-, and spruce-dominated forests. The core fungal microbiome comprised only ~ 20% of the total OTUs across all soil samples, although the overlap between conifer-associated communities was substantial. In contrast, seasonality and soil layer explained only a small proportion of the variation in community structure. However, despite their highly variable taxonomic composition, fungal guilds exhibited remarkably similar functional traits for growth-related and core metabolic pathways across forest associations, suggesting ecological redundancy. However, we found that the expression profiles of genes related to polysaccharide and protein degradation and nutrient transport notably varied between and within the fungal guilds, suggesting niche adaptation. CONCLUSIONS: Overall, our metatranscriptomic analyses revealed the functional potential of soil fungal communities in montane old-growth forests, including a suite of specialized genes and taxa involved in organic matter decomposition. By linking genes to ecological traits, this study provides insights into fungal adaptation strategies to biotic and environmental factors, and sheds light on the importance of understanding functional gene expression patterns in predicting ecosystem functioning. Video Abstract.

Ectomycorrhizal symbiosis prepares its host locally and systemically for abiotic cue signaling.

Tree growth and survival are dependent on their ability to perceive signals, integrate them, and trigger timely and fitted molecular and growth responses. While ectomycorrhizal symbiosis is a predominant tree-microbe interaction in forest ecosystems, little is known about how and to what extent it helps trees cope with environmental changes. We hypothesized that the presence of Laccaria bicolor influences abiotic cue perception by Populus trichocarpa and the ensuing signaling cascade. We submitted ectomycorrhizal or non-ectomycorrhizal P. trichocarpa cuttings to short-term cessation of watering or ozone fumigation to focus on signaling networks before the onset of any physiological damage. Poplar gene expression, metabolite levels, and hormone levels were measured in several organs (roots, leaves, mycorrhizas) and integrated into networks. We discriminated the signal responses modified or maintained by ectomycorrhization. Ectomycorrhizas buffered hormonal changes in response to short-term environmental variations systemically prepared the root system for further fungal colonization and alleviated part of the root abscisic acid (ABA) signaling. The presence of ectomycorrhizas in the roots also modified the leaf multi-omics landscape and ozone responses, most likely through rewiring of the molecular drivers of photosynthesis and the calcium signaling pathway. In conclusion, P. trichocarpa-L. bicolor symbiosis results in a systemic remodeling of the host's signaling networks in response to abiotic changes. In addition, ectomycorrhizal, hormonal, metabolic, and transcriptomic blueprints are maintained in response to abiotic cues, suggesting that ectomycorrhizas are less responsive than non-mycorrhizal roots to abiotic challenges.

Desert truffle genomes reveal their reproductive modes and new insights into plant-fungal interaction and ectendomycorrhizal lifestyle.

Desert truffles are edible hypogeous fungi forming ectendomycorrhizal symbiosis with plants of Cistaceae family. Knowledge about the reproductive modes of these fungi and the molecular mechanisms driving the ectendomycorrhizal interaction is lacking. Genomes of the highly appreciated edible desert truffles Terfezia claveryi Chatin and Tirmania nivea Trappe have been sequenced and compared with other Pezizomycetes. Transcriptomes of T. claveryi × Helianthemum almeriense mycorrhiza from well-watered and drought-stressed plants, when intracellular colonizations is promoted, were investigated. We have identified the fungal genes related to sexual reproduction in desert truffles and desert-truffles-specific genomic and secretomic features with respect to other Pezizomycetes, such as the expansion of a large set of gene families with unknown Pfam domains and a number of species or desert-truffle-specific small secreted proteins differentially regulated in symbiosis. A core set of plant genes, including carbohydrate, lipid-metabolism, and defence-related genes, differentially expressed in mycorrhiza under both conditions was found. Our results highlight the singularities of desert truffles with respect to other mycorrhizal fungi while providing a first glimpse on plant and fungal determinants involved in ecto to endo symbiotic switch that occurs in desert truffle under dry conditions.

Dual transcriptomics and proteomics analyses of the early stage of interaction between Caballeronia mineralivorans PML1(12) and mineral.

Minerals and rocks represent essential reservoirs of nutritive elements for the long-lasting functioning of forest ecosystems developed on nutrient-poor soils. While the presence of effective mineral weathering bacteria was evidenced in the rhizosphere of different plants, the molecular mechanisms involved remain uncharacterized. To fill this gap, we combined transcriptomic, proteomics, geo-chemical and physiological analyses to decipher the potential molecular mechanisms explaining the mineral weathering effectiveness of strain PML1(12) of Caballeronia mineralivorans. Considering the early-stage of the interaction between mineral and bacteria, we identified the genes and proteins differentially expressed when: (i) the environment is depleted of certain essential nutrients (i.e., Mg and Fe), (ii) a mineral is added and (iii) the carbon source (i.e., glucose vs mannitol) differs. The integration of these data demonstrates that strain PML1(12) is capable of (i) mobilizing iron through the production of a non-ribosomal peptide synthetase-independent siderophore, (ii) inducing chemotaxis and motility in response to nutrient availability and (iii) strongly acidifying its environment in the presence of glucose using a suite of GMC oxidoreductases to weather mineral. These results provide new insights into the molecular mechanisms involved in mineral weathering and their regulation and highlight the complex sequence of events triggered by bacteria to weather minerals.

Transcriptome Analysis of Apple Leaves Infected by the Rust Fungus Gymnosporangium yamadae at Two Sporulation Stages.

Apple rust disease caused by Gymnosporangium yamadae is one of the major threats to apple orchards. In this study, dual RNA-seq analysis was conducted to simultaneously monitor gene expression profiles of G. yamadae and infected apple leaves during the formation of rust spermogonia and aecia. The molecular mechanisms underlying this compatible interaction at 10 and 30 days postinoculation (dpi) indicate a significant reaction from the host plant and comprise detoxication pathways at the earliest stage and the induction of secondary metabolism pathways at 30 dpi. Such host reactions have been previously reported in other rust pathosystems and may represent a general reaction to rust infection. G. yamadae transcript profiling indicates a conserved genetic program in spermogonia and aecia that is shared with other rust fungi, whereas secretome prediction reveals the presence of specific secreted candidate effector proteins expressed during apple infection. Unexpectedly, the survey of fungal unigenes in the transcriptome assemblies of inoculated and mock-inoculated apple leaves reveals that G. yamadae infection may modify the fungal community composition in the apple phyllosphere at 30 dpi. Collectively, our results provide novel insights into the compatible apple-G. yamadae interaction and advance the knowledge of this heteroecious demicyclic rust fungus.

Anaerobic lignocellulolytic microbial consortium derived from termite gut: enrichment, lignocellulose degradation and community dynamics.

BACKGROUND: Lignocellulose is the most abundant renewable carbon resource that can be used for biofuels and commodity chemicals production. The ability of complex microbial communities present in natural environments that are specialized in biomass deconstruction can be exploited to develop lignocellulose bioconversion processes. Termites are among the most abundant insects on earth and play an important role in lignocellulose decomposition. Although their digestive microbiome is recognized as a potential reservoir of microorganisms producing lignocellulolytic enzymes, the potential to enrich and maintain the lignocellulolytic activity of microbial consortia derived from termite gut useful for lignocellulose biorefinery has not been assessed. Here, we assessed the possibility of enriching a microbial consortium from termite gut and maintaining its lignocellulose degradation ability in controlled anaerobic bioreactors. RESULTS: We enriched a termite gut-derived consortium able to transform lignocellulose into carboxylates under anaerobic conditions. To assess the impact of substrate natural microbiome on the enrichment and the maintenance of termite gut microbiome, the enrichment process was performed using both sterilized and non-sterilized straw. The enrichment process was carried out in bioreactors operating under industrially relevant aseptic conditions. Two termite gut-derived microbial consortia were obtained from Nasutitermes ephratae by sequential batch culture on raw wheat straw as the sole carbon source. Analysis of substrate loss, carboxylate production and microbial diversity showed that regardless of the substrate sterility, the diversity of communities selected by the enrichment process strongly changed compared to that observed in the termite gut. Nevertheless, the community obtained on sterile straw displayed higher lignocellulose degradation capacity; it showed a high xylanase activity and an initial preference for hemicellulose. CONCLUSIONS: This study demonstrates that it is possible to enrich and maintain a microbial consortium derived from termite gut microbiome in controlled anaerobic bioreactors, producing useful carboxylates from raw biomass. Our results suggest that the microbial community is shaped both by the substrate and the conditions that prevail during enrichment. However, when aseptic conditions are applied, it is also affected by the biotic pressure exerted by microorganisms naturally present in the substrate and in the surrounding environment. Besides the efficient lignocellulolytic consortium enriched in this study, our results revealed high levels of xylanase activity that can now be further explored for enzyme identification and overexpression for biorefinery purposes.

Screening of Phytophagous and Xylophagous Insects Guts Microbiota Abilities to Degrade Lignocellulose in Bioreactor.

Microbial consortia producing specific enzymatic cocktails are present in the gut of phytophagous and xylophagous insects; they are known to be the most efficient ecosystems to degrade lignocellulose. Here, the ability of these consortia to degrade ex vivo lignocellulosic biomass in anaerobic bioreactors was characterized in term of bioprocess performances, enzymatic activities and bacterial community structure. In a preliminary screening, guts of Ergates faber (beetle), Potosia cuprea (chafer), Gromphadorrhina portentosa (cockroach), Locusta migratoria (locust), and Gryllus bimaculatus (cricket) were inoculated in anaerobic batch reactors, in presence of grounded wheat straw at neutral pH. A short duration fermentation of less than 8 days was observed and was related to a drop of pH from 7 to below 4.5, leading to an interruption of gas and metabolites production. Consistently, a maximum of 180 mgeq.COD of metabolites accumulated in the medium, which was related to a low degradation of the lignocellulosic biomass, with a maximum of 5 and 2.2% observed for chafer and locust gut consortia. The initial cell-bound and extracellular enzyme activities, i.e., xylanase and ß-endoglucanase, were similar to values observed in the literature. Wheat straw fermentation in bioreactors leads to an increase of cell-bounded enzyme activities, with an increase of 145% for cockroach xylanase activity. Bacterial community structures were insect dependent and mainly composed of Clostridia, Bacteroidia and Gammaproteobacteria. Improvement of lignocellulose biodegradation was operated in successive batch mode at pH 8 using the most interesting consortia, i.e., locust, cockroaches and chafer gut consortia. In these conditions, lignocellulose degradation increased significantly: 8.4, 10.5, and 21.0% of the initial COD were degraded for chafer, cockroaches and locusts, respectively in 15 days. Consistently, xylanase activity tripled for the three consortia, attesting the improvement of the process. Bacteroidia was the major bacterial class represented in the bacterial community for all consortia, followed by Clostridia and Gammaproteobacteria classes. This work demonstrates the possibility to maintain apart of insect gut biological activity ex vivo and shows that lignocellulose biodegradation can be improved by using a biomimetic approach. These results bring new insights for the optimization of lignocellulose degradation in bioreactors.

FROGS: Find, Rapidly, OTUs with Galaxy Solution.

Motivation: Metagenomics leads to major advances in microbial ecology and biologists need user friendly tools to analyze their data on their own. Results: This Galaxy-supported pipeline, called FROGS, is designed to analyze large sets of amplicon sequences and produce abundance tables of Operational Taxonomic Units (OTUs) and their taxonomic affiliation. The clustering uses Swarm. The chimera removal uses VSEARCH, combined with original cross-sample validation. The taxonomic affiliation returns an innovative multi-affiliation output to highlight databases conflicts and uncertainties. Statistical results and numerous graphical illustrations are produced along the way to monitor the pipeline. FROGS was tested for the detection and quantification of OTUs on real and in silico datasets and proved to be rapid, robust and highly sensitive. It compares favorably with the widespread mothur, UPARSE and QIIME. Availability and implementation: Source code and instructions for installation: https://github.com/geraldinepascal/FROGS.git. A companion website: http://frogs.toulouse.inra.fr. Contact: geraldine.pascal@inra.fr. Supplementary information: Supplementary data are available at Bioinformatics online.

Analysis of large 16S rRNA Illumina data sets: Impact of singleton read filtering on microbial community description.

Next-generation sequencing technologies give access to large sets of data, which are extremely useful in the study of microbial diversity based on 16S rRNA gene. However, the production of such large data sets is not only marred by technical biases and sequencing noise but also increases computation time and disc space use. To improve the accuracy of OTU predictions and overcome both computations, storage and noise issues, recent studies and tools suggested removing all single reads and low abundant OTUs, considering them as noise. Although the effect of applying an OTU abundance threshold on α- and ß-diversity has been well documented, the consequences of removing single reads have been poorly studied. Here, we test the effect of singleton read filtering (SRF) on microbial community composition using in silico simulated data sets as well as sequencing data from synthetic and real communities displaying different levels of diversity and abundance profiles. Scalability to large data sets is also assessed using a complete MiSeq run. We show that SRF drastically reduces the chimera content and computational time, enabling the analysis of a complete MiSeq run in just a few minutes. Moreover, SRF accurately determines the actual community diversity: the differences in α- and ß-community diversity obtained with SRF and standard procedures are much smaller than the intrinsic variability of technical and biological replicates.

Uncovering the Potential of Termite Gut Microbiome for Lignocellulose Bioconversion in Anaerobic Batch Bioreactors.

Termites are xylophages, being able to digest a wide variety of lignocellulosic biomass including wood with high lignin content. This ability to feed on recalcitrant plant material is the result of complex symbiotic relationships, which involve termite-specific gut microbiomes. Therefore, these represent a potential source of microorganisms for the bioconversion of lignocellulose in bioprocesses targeting the production of carboxylates. In this study, gut microbiomes of four termite species were studied for their capacity to degrade wheat straw and produce carboxylates in controlled bioreactors. All of the gut microbiomes successfully degraded lignocellulose and up to 45% w/w of wheat straw degradation was observed, with the Nasutitermes ephratae gut-microbiome displaying the highest levels of wheat straw degradation, carboxylate production and enzymatic activity. Comparing the 16S rRNA gene diversity of the initial gut inocula to the bacterial communities in lignocellulose degradation bioreactors revealed important changes in community diversity. In particular, taxa such as Spirochaetes and Fibrobacteres that were highly abundant in the initial gut inocula were replaced by Firmicutes and Proteobacteria at the end of incubation in wheat straw bioreactors. Overall, this study demonstrates that termite-gut microbiomes constitute a reservoir of lignocellulose-degrading bacteria that can be harnessed in artificial conditions for biomass conversion processes that lead to the production of useful molecules.

CAZyChip: dynamic assessment of exploration of glycoside hydrolases in microbial ecosystems.

BACKGROUND: Microorganisms constitute a reservoir of enzymes involved in environmental carbon cycling and degradation of plant polysaccharides through their production of a vast variety of Glycoside Hydrolases (GH). The CAZyChip was developed to allow a rapid characterization at transcriptomic level of these GHs and to identify enzymes acting on hydrolysis of polysaccharides or glycans. RESULTS: This DNA biochip contains the signature of 55,220 bacterial GHs available in the CAZy database. Probes were designed using two softwares, and microarrays were directly synthesized using the in situ ink-jet technology. CAZyChip specificity and reproducibility was validated by hybridization of known GHs RNA extracted from recombinant E. coli strains, which were previously identified by a functional metagenomic approach. The GHs arsenal was also studied in bioprocess conditions using rumen derived microbiota. CONCLUSIONS: The CAZyChip appears to be a user friendly tool for profiling the expression of a large variety of GHs. It can be used to study temporal variations of functional diversity, thereby facilitating the identification of new efficient candidates for enzymatic conversions from various ecosystems.

Efficient anaerobic transformation of raw wheat straw by a robust cow rumen-derived microbial consortium.

A rumen-derived microbial consortium was enriched on raw wheat straw as sole carbon source in a sequential batch-reactor (SBR) process under strict mesophilic anaerobic conditions. After five cycles of enrichment the procedure enabled to select a stable and efficient lignocellulolytic microbial consortium, mainly constituted by members of Firmicutes and Bacteroidetes phyla. The enriched community, designed rumen-wheat straw-derived consortium (RWS) efficiently hydrolyzed lignocellulosic biomass, degrading 55.5% w/w of raw wheat straw over 15days at 35°C and accumulating carboxylates as main products. Cellulolytic and hemicellulolytic activities, mainly detected on the cell bound fraction, were produced in the earlier steps of degradation, their production being correlated with the maximal lignocellulose degradation rates. Overall, these results demonstrate the potential of RWS to convert unpretreated lignocellulosic substrates into useful chemicals.

Comparison of gel-based methods for the detection of cerebrospinal fluid rhinorrhea.

Cerebrospinal fluid (CSF) rhinorrhea is a serious condition that may result in severe complications. Various laboratory tests, relying on the detection of CSF-specific proteins in nasal secretions, have been developed but diagnosis remains challenging. The aim of this study was to evaluate two new methods targeting either ß2-transferrin or beta-trace-protein. Rhinorrhea samples from patients suspected of CSF leakage (n=36) were analyzed using two-dimensional gel electrophoresis (2-DE) for CSF rhinorrhea diagnosis. Twelve patients with rhinorrhea strongly suggestive of a CSF leak also underwent a fluorescein test. The same cohort was retrospectively analyzed with a beta-trace protein immunoblot developed in-house (n=36) and a new commercial ß2-transferrin immunofixation assay (Sebia, Evry, France) (n=33). 2-DE was positive in 9 patients suffering from rhinorrhea following skull base fracture (n=3), post-surgery (n=4), or spontaneously (n=2). The 27 remaining cases were negative. These results were confirmed by the beta-trace protein immunoblot and ß2-transferrin immunofixation tests, except for one sample found negative with 2-DE but positive with the two other assays. Results from the three analytical methods were concordant with fluorescein tests. Beta-trace protein immunoblot and ß2-transferrin immunofixation assays are fast and reliable methods that allow detecting CSF leakage in nasal fluid with high sensitivity and specificity.

A biallelic gene polymorphism of CYP11B2 predicts increased aldosterone to renin ratio in selected hypertensive patients.

Altered control of aldosterone synthase (CYP11B2) gene expression may modulate aldosterone secretion, as suggested by a raised aldosterone to renin ratio (ARR) in some patients with essential hypertension. We compared the frequency of two linked CYP11B2 polymorphisms, one in the steroidogenic factor-1 (SF-1) binding site and the other an intronic conversion (Int2) in relation to ARR in 141 hypertensive patients. Patients were divided into groups with either normal or high supine ARR using a cut-off threshold of 145 pmol/liter per ng/liter. Supine ARR was normal in 104 patients and raised in 37 patients. The two polymorphisms were in strong linkage disequilibrium (chi(2) = 123.8; P < 0.0001). The SF-1 T and Int2 C alleles were more prevalent among patients with high ARR (46% and 43%, respectively) than with normal ARR (22% and 17%; P < 0.01 and P < 0.005, respectively). Odds ratios for raised ARR in subjects with a homozygous SF-1 T and Int2 C haplotype were 6.1 (95% confidence interval, 1.6-22.5; P < 0.005) when compared with the contrasting haplotype. Linear modeling of individual postural changes in renin and aldosterone showed a maximal achievable aldosterone increase of 110 pmol/liter with no mutated haplotype and 500 pmol/liter with two mutated haplotypes. These findings support the view of a molecular basis regulating aldosterone production.