Phytoplanktonic species in the haloalkaline Lake Dziani Dzaha select their archaeal microbiome.

Microorganisms are key contributors of aquatic biogeochemical cycles but their microscale ecology remains largely unexplored, especially interactions occurring between phytoplankton and microorganisms in the phycosphere, that is the region immediately surrounding phytoplankton cells. The current study aimed to provide evidence of the phycosphere taking advantage of a unique hypersaline, hyperalkaline ecosystem, Lake Dziani Dzaha (Mayotte), where two phytoplanktonic species permanently co-dominate: a cyanobacterium, Arthrospira fusiformis, and a green microalga, Picocystis salinarum. To assay phycospheric microbial diversity from in situ sampling, we set up a flow cytometry cell-sorting methodology for both phytoplanktonic populations, coupled with metabarcoding and comparative microbiome diversity. We focused on archaeal communities as they represent a non-negligible part of the phycospheric diversity, however their role is poorly understood. This work is the first which successfully explores in situ archaeal diversity distribution showing contrasted phycospheric compositions, with P. salinarum phycosphere notably enriched in Woesearchaeales OTUs while A. fusiformis phycosphere was enriched in methanogenic lineages affiliated OTUs such as Methanomicrobiales or Methanofastidiosales. Most archaeal OTUs, including Woesearchaeales considered in literature as symbionts, were either ubiquitous or specific of the free-living microbiome (i.e. present in the 3-0.2 µm fraction). Seminally, several archaeal OTUs were enriched from the free-living microbiome to the phytoplankton phycospheres, suggesting (i) either the inhibition or decrease of other OTUs, or (ii) the selection of specific OTUs resulting from the physical influence of phytoplanktonic species on surrounding Archaea.

Prokaryotic, Microeukaryotic, and Fungal Composition in a Long-Term Polychlorinated Biphenyl-Contaminated Brownfield.

Polychlorinated biphenyls (PCBs) are recognized as persistent organic pollutants and accumulate in organisms, soils, waters, and sediments, causing major health and ecological perturbations. Literature reported PCB bio-transformation by fungi and bacteria in vitro, but data about the in situ impact of those compounds on microbial communities remained scarce while being useful to guide biotransformation assays. The present work investigated for the first time microbial diversity from the three-domains-of-life in a long-term contaminated brownfield (a former factory land). Soil samples were ranked according to their PCB concentrations, and a significant increase in abundance was shown according to increased concentrations. Microbial communities structure showed a segregation from the least to the most PCB-polluted samples. Among the identified microorganisms, Bacteria belonging to Gammaproteobacteria class, as well as Fungi affiliated to Saccharomycetes class or Pleurotaceae family, including some species known to transform some PCBs were abundantly retrieved in the highly polluted soil samples.

Metabarcoding of the Three Domains of Life in Aquatic Saline Ecosystems.

High-throughput amplicon sequencing, known as metabarcoding, is a powerful technique to decipher exhaustive microbial diversity considering specific gene markers. While most of the studies investigating ecosystem functioning through microbial diversity targeted only one domain of life, either bacteria, or archaea or microeukaryotes, the remaining challenge in microbial ecology is to uncover the integrated view of microbial diversity occurring in ecosystems. Indeed, interactions occurring between the different microbial counterparts are now recognized having a great impact on stability and resilience of ecosystems. Here, we summarize protocols describing sampling, molecular, and simultaneous metabarcoding of bacteria, archaea, and microeukaryotes, as well as a bioinformatic pipeline allowing the study of exhaustive microbial diversity in natural aquatic saline samples.

Strong reorganization of multi-domain microbial networks associated with primary producers sedimentation from oxic to anoxic conditions in an hypersaline lake.

Understanding the role of microbial interactions in the functioning of natural systems is often impaired by the levels of complexity they encompass. In this study, we used the relative simplicity of an hypersaline crater lake hosting only microbial organisms (Dziani Dzaha) to provide a detailed analysis of the microbial networks including the three domains of life. We identified two main ecological zones, one euphotic and oxic zone in surface, where two phytoplanktonic organisms produce a very high biomass, and one aphotic and anoxic deeper zone, where this biomass slowly sinks and undergoes anaerobic degradation. We highlighted strong differences in the structure of microbial communities from the two zones and between the microbial consortia associated with the two primary producers. Primary producers sedimentation was associated with a major reorganization of the microbial network at several levels: global properties, modules composition, nodes and links characteristics. We evidenced the potential dependency of Woesearchaeota to the primary producers' exudates in the surface zone, and their disappearance in the deeper anoxic zone, along with the restructuration of the networks in the anoxic zone toward the decomposition of the organic matter. Altogether, we provided an in-depth analysis of microbial association network and highlighted putative changes in microbial interactions supporting the functioning of the two ecological zones in this unique ecosystem.

Eukaryotic Cell Capture by Amplified Magnetic in situ Hybridization Using Yeast as a Model.

A non-destructive approach based on magnetic in situ hybridization (MISH) and hybridization chain reaction (HCR) for the specific capture of eukaryotic cells has been developed. As a prerequisite, a HCR-MISH procedure initially used for tracking bacterial cells was here adapted for the first time to target eukaryotic cells using a universal eukaryotic probe, Euk-516R. Following labeling with superparamagnetic nanoparticles, cells from the model eukaryotic microorganism Saccharomyces cerevisiae were hybridized and isolated on a micro-magnet array. In addition, the eukaryotic cells were successfully targeted in an artificial mixture comprising bacterial cells, thus providing evidence that HCR-MISH is a promising technology to use for specific microeukaryote capture in complex microbial communities allowing their further morphological characterization. This new study opens great opportunities in ecological sciences, thus allowing the detection of specific cells in more complex cellular mixtures in the near future.

Datamining and functional environmental genomics reassess the phylogenetics and functional diversity of fungal monosaccharide transporters.

Sugar transporters are essential components of carbon metabolism and have been extensively studied to control sugar uptake by yeasts and filamentous fungi used in fermentation processes. Based on published information on characterized fungal sugar porters, we show that this protein family encompasses phylogenetically distinct clades. While several clades encompass transporters that seemingly specialized on specific "sugar-related" molecules (e.g., myo-inositol, charged sugar analogs), others include mostly either mono- or di/oligosaccharide low-specificity transporters. To address the issue of substrate specificity of sugar transporters, that protein primary sequences do not fully reveal, we screened "multi-species" soil eukaryotic cDNA libraries for mannose transporters, a sugar that had never been used to select transporters. We obtained 19 environmental transporters, mostly from Basidiomycota and Ascomycota. Among them, one belonged to the unusual "Fucose H+ Symporter" family, which is only known in Fungi for a rhamnose transporter in Aspergillus niger. Functional analysis of the 19 transporters by expression in yeast and for two of them in Xenopus laevis oocytes for electrophysiological measurements indicated that most of them showed a preference for D-mannose over other tested D-C6 (glucose, fructose, galactose) or D-C5 (xylose) sugars. For the several glucose and fructose-negative transporters, growth of the corresponding recombinant yeast strains was prevented on mannose in the presence of one of these sugars that may act by competition for the binding site. Our results highlight the potential of environmental genomics to figure out the functional diversity of key fungal protein families and that can be explored in a context of biotechnology. KEY POINTS: • Most fungal sugar transporters accept several sugars as substrates. • Transporters, belonging to 2 protein families, were isolated from soil cDNA libraries. • Environmental transporters featured novel substrate specificities.

Protection from metal toxicity by Hsp40-like protein isolated from contaminated soil using functional metagenomic approach.

Pollution in the environment due to accumulation of potentially toxic metals results in deterioration of soil and water quality, thus impacting health of all living organisms including microbes. In the present investigation, a functional metagenomics approach was adopted to mine functional genes involved in metal tolerance from potentially toxic metal contaminated site. Eukaryotic cDNA library (1.0-4.0 kb) was screened for the genes providing tolerance to cadmium (Cd) toxicity through a functional complementation assay using Cd-sensitive Saccharomyces cerevisiae mutant ycf1Δ. Out of the 98 clones able to recover growth on Cd-supplemented selective medium, one clone designated as PLCc43 showed more tolerance to Cd along with some other clones. Sequence analysis revealed that cDNA PLCc43 encodes a 284 amino acid protein harbouring four characteristic zinc finger motif repeats (CXXCXGXG) and showing partial homology with heat shock protein (Hsp40) of Acanthamoeba castellanii. qPCR analysis revealed the induction of PLCc43 in the presence of Cd, which was further supported by accumulation of Cd in ycf1Δ/PLCc43 mutant. Cu-sensitive (cup1Δ), Zn-sensitive (zrc1Δ) and Co-sensitive (cot1Δ) yeast mutant strains were rescued from sensitivity when transformed with cDNA PLCc43 indicating its ability to confer tolerance to various potentially toxic metals. Oxidative stress tolerance potential of PLCc43 was also confirmed in the presence of H2O2. Present study results suggest that PLCc43 originating from a functional eukaryotic gene of soil community play an important role in detoxification of potentially toxic metals and may be used as biomarker in various contaminated sites.

The pentose catabolic pathway of the rice-blast fungus Magnaporthe oryzae involves a novel pentose reductase restricted to few fungal species.

A gene (MoPRD1), related to xylose reductases, was identified in Magnaporthe oryzae. Recombinant MoPRD1 displays its highest specific reductase activity toward L-arabinose and D-xylose. Km and Vmax values using L-arabinose and D-xylose are similar. MoPRD1 was highly overexpressed 2-8h after transfer of mycelium to D-xylose or L-arabinose, compared to D-glucose. Therefore, we conclude that MoPDR1 is a novel pentose reductase, which combines the activities and expression patterns of fungal L-arabinose and D-xylose reductases. Phylogenetic analysis shows that PRD1 defines a novel family of pentose reductases related to fungal D-xylose reductases, but distinct from fungal L-arabinose reductases. The presence of PRD1, L-arabinose and D-xylose reductases encoding genes in a given species is variable and likely related to their life style.

The dominant Hc.Sdh (R) carboxin-resistance gene of the ectomycorrhizal fungus Hebeloma cylindrosporum as a selectable marker for transformation.

In an attempt to get a marker gene suitable for genetical transformation of the ectomycorrhizal fungus Hebeloma cylindrosporum, the gene Hc.Sdh (R) that confers carboxin-resistance was isolated from a UV mutant of this fungus. It encodes a mutant allele of the Fe-S subunit of the succinate dehydrogenase gene that carries a single amino acid substitution known to confer carboxin-resistance. This gene was successfully used as the selective marker to transform, via Agrobacterium tumefaciens, monokaryotic and dikaryotic strains of H. cylindrosporum. We also successfully transformed hygromycin-resistant insertional mutants. Transformation yielded mitotically stable carboxin-resistant mycelia. This procedure produced transformants, the growth of which was not affected by 2 microg l(-1) carboxin, whereas wild-type strains were unable to grow in the presence of 0.1 microg l(-1) of this fungicide. This makes the carboxin-resistance cassette much more discriminating than the hygromycin-resistance one. PCR amplification and Southern blot hybridisation indicated that more than 90% of the tested carboxin-resistant mycelia contained the Hc.Sdh (R) cassette, usually as a single copy. The AGL-1 strain of A. tumefaciens was a much less efficient donor than LBA 1126; the former yielded ca. 0-30% transformation frequency, depending on fungal strain and resistance cassette used, whereas the latter yielded ca. 60-95%.

Monitoring of Venturia inaequalis harbouring the QoI resistance G143A mutation in French orchards as revealed by PCR assays.

BACKGROUND: Genetic resistance to QoI fungicides may account for recent failures to control Venturia inaequalis (Cooke) Winter in French orchards. Two PCR-based assays were developed to detect the G143A point mutation in the fungal mitochondrial cytochrome b gene. The mutation is known to confer a high level of resistance to QoI fungicides. Occurrence of the G143A mutation in French field isolates collected from 2004 to 2007 was monitored. RESULTS: The QoI-resistant cytochrome b allele was specifically detected either following the cleavage of the amplified marker by a restriction endonuclease (CAPS assay) or its amplification using an allele-specific PCR primer. Using either method, the G143A mutation was found in 42% of the 291 field samples originating from French orchards in which apple scab proved difficult to be controlled. Monitoring of the G143A mutation in orchards located in 15 French administrative regions indicated that the mutation was detected at least once in nine of the regions, and its presence ranged from 33% to 64% of the orchards analysed in 2004 and in 2007 respectively. CONCLUSION: The PCR-based methods developed in this study efficiently reveal the presence of the G143A mutation in French V. inaequalis field populations.

Distribution dynamics of the Tnt1 retrotransposon in tobacco.

Retrotransposons contribute significantly to the size, organization and genetic diversity of plant genomes. Although many retrotransposon families have been reported in plants, to this day, the tobacco Tnt1 retrotransposon remains one of the few elements for which active transposition has been shown. Demonstration that Tnt1 activation can be induced by stress has lent support to the hypothesis that, under adverse conditions, transposition can be an important source of genetic variability. Here, we compared the insertion site preference of a collection of newly transposed and pre-existing Tnt1 copies identified in plants regenerated from protoplasts or tissue culture. We find that newly transposed Tnt1 copies are targeted within or close to host gene coding sequences and that the distribution of pre-existing insertions does not vary significantly from this trend. Therefore, in spite of their potential to disrupt neighboring genes, insertions within or near CDS are not preferentially removed with age. Elimination of Tnt1 insertions within or near coding sequences may be relaxed due to the polyploid nature of the tobacco genome. Tnt1 insertions within or near CDS are thus better tolerated and can putatively contribute to the diversification of tobacco gene function.

Expression of Magnaporthe grisea avirulence gene ACE1 is connected to the initiation of appressorium-mediated penetration.

Magnaporthe grisea is responsible for a devastating fungal disease of rice called blast. Current control of this disease relies on resistant rice cultivars that recognize M. grisea signals corresponding to specific secreted proteins encoded by avirulence genes. The M. grisea ACE1 avirulence gene differs from others, since it controls the biosynthesis of a secondary metabolite likely recognized by rice cultivars carrying the Pi33 resistance gene. Using a transcriptional fusion between ACE1 promoter and eGFP, we showed that ACE1 is only expressed in appressoria during fungal penetration into rice and barley leaves, onion skin, and cellophane membranes. ACE1 is almost not expressed in appressoria differentiated on Teflon and Mylar artificial membranes. ACE1 expression is not induced by cellophane and plant cell wall components, demonstrating that it does not require typical host plant compounds. Cyclic AMP (cAMP) signaling mutants delta cpkA and delta mac1 sum1-99 and tetraspanin mutant delta pls1::hph differentiate melanized appressoria with normal turgor but are unable to penetrate host plant leaves. ACE1 is normally expressed in these mutants, suggesting that it does not require cAMP signaling or a successful penetration event. ACE1 is not expressed in appressoria of the buf1::hph mutant defective for melanin biosynthesis and appressorial turgor. The addition of hyperosmotic solutes to buf1::hph appressoria restores appressorial development and ACE1 expression. Treatments of young wild-type appressoria with actin and tubulin inhibitors reduce both fungal penetration and ACE1 expression. These experiments suggest that ACE1 appressorium-specific expression does not depend on host plant signals but is connected to the onset of appressorium-mediated penetration.

Nonmycorrhizal (myc-) mutants of Hebeloma cylindrosporum obtained through insertional mutagenesis.

Polyethylene glycol-mediated transformation of protoplasts was used as a method for insertional mutagenesis to obtain mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum impaired in symbiotic ability. Following restriction enzyme-mediated integration or conventional plasmid insertion, a library of 1,725 hygromycin-resistant monokaryotic transformants was generated and screened for the symbiotic defect, using Pinus pinaster seedlings as host plants. A total of 51 transformants displaying a dramatically reduced mycorrhizal ability were identified. Among them, 29 were nonmycorrhizal (myc-), but only 10 of them had integrated one or several copies of the transforming plasmid in their genome. Light and scanning electron microscopy observations of pine roots inoculated with myc- mutants suggested that we selected mutants blocked at early stages of interaction between partners or at the stage of Hartig net formation. Myc- mutants with plasmid insertions were crossed with a compatible wild-type monokaryon and allowed to fruit. Monokaryotic progenies were obtained in three independent crosses and were analyzed for symbiotic activity and plasmid insertion. In all three progenies, a 1:1 myc-:myc+ segregation ratio was observed, suggesting that each myc- phenotype resulted from a single gene mutation. However, for none of the three mutants, the myc- phenotype segregated with any of the plasmid insertions. Our results support the idea that master genes, the products of which are essential for symbiosis establishment, do exist in ectomycorrhizal fungi.

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum.

We transformed haploid mycelium of Hebeloma cylindrosporum via Agrobacterium tumefaciens and optimised the procedure to develop a new tool for insertional mutagenesis in this fungus. Southern blot analysis of 83 randomly selected transformants showed that they all contained plasmid inserts. Each of them showed a unique hybridisation pattern, suggesting that integration was random in the fungal genome. Sixty percent of transformants obtained in the presence of bacteria pre-treated with acetosyringone integrated a single transferred DNA copy. Thermal asymmetric interlaced polymerase chain reaction allowed us to recover the left border and the right border junctions in 85% and 15% of transformants analysed, respectively. Results show that A. tumefaciens-mediated transformation may be a powerful tool for insertional mutagenesis in H. cylindrosporum.

Analysis of molecular markers genetically linked to the Leptosphaeria maculans avirulence gene AvrLm1 in field populations indicates a highly conserved event leading to virulence on Rlm1 genotypes.

Map-based cloning of the avirulence gene AvrLm1 of Leptosphaeria maculans was initiated utilizing a genetic map of the fungus and a BAC library constructed from an AvrLm1 isolate. Seven polymorphic DNA markers closely linked to AvrLm1 were identified. Of these, two were shown to border the locus on its 5' end and were present, with size polymorphism, in both the virulent and the avirulent isolates. In contrast, three markers, J19-1.1, J53-1.3 (in coupling phase with avirulence), and Vir1 (in repulsion phase with avirulence), cosegregated with AvrLm1 in 312 progeny from five in vitro crosses. J19-1.1 and J53-1.3 were never amplified in the virulent parents or progeny, whereas Vir1 was never amplified in the avirulent parents or progeny. J19-1.1 and J53-1.3 were shown to be separated by 40 kb within a 184-kb BAC contig. In addition, the 1.6-cM genetic distance between J53-1.3 and the nearest recombinant marker corresponded to a 121-kb physical distance. When analyzing a European Union-wide collection of 192 isolates, J53-1.3, J19-1.1, and Vir1 were found to be closely associated with the AvrLm1 locus. The results of polymerase chain reaction amplification with primers for the three markers were in accordance with the interaction phenotype for 92.2% (J53-1.3), 90.6% (J19-1.1), and 88.0% (Vir1) of the isolates. In addition, genome organization of the AvrLm1 region was highly conserved in field isolates, because 89.1% of the avirulent isolates and 79.0% of the virulent isolates showed the same association of markers as that of the parents of in vitro crosses. The large-scale analysis of field isolates with markers originating from the genetic map therefore confirms (i) the physical proximity between the markers and the target locus and (ii) that AvrLm1 is located in (or close to) a recombination-deficient genome region. As a consequence, map-based markers provided us with high-quality markers for an overview of the occurrence of race "AvrLm1" at the field scale. These data were used to propose hypotheses on evolution towards virulence in field isolates.