Laurence Fraissinet-Tachet

Soil eukaryotic functional diversity, a metatranscriptomic approach

To appreciate the functional diversity of communities of soil eukaryotic micro-organisms we evaluated an experimental approach based on the construction and screening of a cDNA library using polyadenylated mRNA extracted from a forest soil. Such a library contains genes that are expressed by each of the different organisms forming the community and represents its metatranscriptome. The diversity of the organisms that contributed to this library was evaluated by sequencing a portion of the 18S rDNA gene amplified from either soil DNA or reverse-transcribed RNA. More than 70% of the sequences were from fungi and unicellular eukaryotes (protists) while the other most represented group was the metazoa. Calculation of richness estimators suggested that more than 180 species could be present in the soil samples studied. Sequencing of 119 cDNA identified genes with no homologues in databases (32%) and genes coding proteins involved in different biochemical and cellular processes. Surprisingly, the taxonomic distribution of the cDNA and of the 18S rDNA genes did not coincide, with a marked under-representation of the protists among the cDNA. Specific genes from such an environmental cDNA library could be isolated by expression in a heterologous microbial host, Saccharomyces cerevisiae. This is illustrated by the functional complementation of a histidine auxotrophic yeast mutant by two cDNA originating possibly from an ascomycete and a basidiomycete fungal species. Study of the metatranscriptome has the potential to uncover adaptations of whole microbial communities to local environmental conditions. It also gives access to an abundant source of genes of biotechnological interest.

Metatranscriptomics reveals the diversity of genes expressed by eukaryotes in forest soils.

Eukaryotic organisms play essential roles in the biology and fertility of soils. For example the micro and mesofauna contribute to the fragmentation and homogenization of plant organic matter, while its hydrolysis is primarily performed by the fungi. To get a global picture of the activities carried out by soil eukaryotes we sequenced 2×10,000 cDNAs synthesized from polyadenylated mRNA directly extracted from soils sampled in beech (Fagus sylvatica) and spruce (Picea abies) forests. Taxonomic affiliation of both cDNAs and 18S rRNA sequences showed a dominance of sequences from fungi (up to 60%) and metazoans while protists represented less than 12% of the 18S rRNA sequences. Sixty percent of cDNA sequences from beech forest soil and 52% from spruce forest soil had no homologs in the GenBank/EMBL/DDJB protein database. A Gene Ontology term was attributed to 39% and 31.5% of the spruce and beech soil sequences respectively. Altogether 2076 sequences were putative homologs to different enzyme classes participating to 129 KEGG pathways among which several were implicated in the utilisation of soil nutrients such as nitrogen (ammonium, amino acids, oligopeptides), sugars, phosphates and sulfate. Specific annotation of plant cell wall degrading enzymes identified enzymes active on major polymers (cellulose, hemicelluloses, pectin, lignin) and glycoside hydrolases represented 0.5% (beech soil)–0.8% (spruce soil) of the cDNAs. Other sequences coding enzymes active on organic matter (extracellular proteases, lipases, a phytase, P450 monooxygenases) were identified, thus underlining the biotechnological potential of eukaryotic metatranscriptomes. The phylogenetic affiliation of 12 full-length carbohydrate active enzymes showed that most of them were distantly related to sequences from known fungi. For example, a putative GH45 endocellulase was closely associated to molluscan sequences, while a GH7 cellobiohydrolase was closest to crustacean sequences, thus suggesting a potentially significant contribution of non-fungal eukaryotes in the actual hydrolysis of soil organic matter.

Characterization of a multigene family encoding an endopolygalacturonase in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum produces several polygalacturonases which together with other pectinolytic enzymes are involved in the degradation of pectin. A number of different genomic clones were isolated by screening a genomic DNA library in phage EMBL3. Southern-blot and restriction mapping indicate that seven genes constitute two subfamilies of a multigene family encoding endopolygalactutonase. Using pulsed-field gel electrophoresis to separate S. sclerotiorum chromosomes each subfamily was found to hybridize to a different chromosome. A comparison of the nucleotide sequence for the coding region of three members of the gene family reveals surprisingly few base substitutions suggesting that this gene family arose from recent multiple duplication events.

A novel fungal family of oligopeptide transporters identified by functional metatranscriptomics of soil eukaryotes

Functional environmental genomics has the potential to identify novel biological functions that the systematic sequencing of microbial genomes or environmental DNA may fail to uncover. We targeted the functions expressed by soil eukaryotes using a metatranscriptomic approach based on the use of soil-extracted polyadenylated messenger RNA to construct environmental complementary DNA expression libraries. Functional complementation of a yeast mutant defective in di/tripeptide uptake identified a novel family of oligopeptide transporters expressed by fungi. This family has a patchy distribution in the Basidiomycota and Ascomycota and is present in the genome of a Saccharomyces cerevisiae wine strain. High throughput phenotyping of yeast mutants expressing two environmental transporters showed that they both displayed broad substrate specificity and could transport more than 60–80 dipeptides. When expressed in Xenopus oocytes one environmental transporter induced currents upon dipeptide addition, suggesting proton-coupled co-transport of dipeptides. This transporter was also able to transport specifically cysteine. Deletion of the two copies of the corresponding gene family members in the genome of the wine yeast strain severely reduced the number of dipeptides that it could assimilate. These results demonstrate that these genes are functional and can be used by fungi to efficiently scavenge the numerous, low concentration, oligopeptides continuously generated in soils by proteolysis.

Expression of the Sclerotinia sclerotiorum polygalacturonase pg1 gene: possible involvement of CREA in glucose catabolite repression.

Abstract Northern-blot analysis of RNA isolated from Sclerotinia sclerotiorum grown on either glucose or polygalacturonate as the sole carbon source showed that pg1, encoding a neutral polygalacturonase, was not expressed during growth in both media. In contrast, transcripts of this gene were detected during infection of sunflower germlings. Analysis of the promoter sequence revealed a number of cis-acting sequences known to regulate the expression of many fungal promoters. Protein-DNA-binding experiments showed that proteins extracted from mycelia grown on polygalacturonate or glucose interacted with different regions of the promoter. The GST-CREA fusion protein, containing the two zinc fingers of the Aspergillus nidulans repressor CREA involved in carbon catabolite repression, forms several complexes with DNA fragments carrying the consensus 5′-SYGGRG-3′. These results suggest that a CREA homolog may be involved in the regulation of pg1.

Regulation by Galacturonic Acid of Pectinolytic Enzyme Production by Sclerotinia sclerotiorum

Abstract. Production of polygalacturonases and pectinases from Sclerotinia sclerotiorum was induced in vitro by galacturonic acid. The inductive effect of galacturonic acid was abolished by the presence of glucose, leading to a basal enzyme production. Zymograms of extracellular enzymes showed that galacturonic acid induced the synthesis of six polygalacturonase and one pectin-methylesterase isoforms. Immunoblotting revealed that an exo-polygalacturonase and an exo-polymethylgalacturonase were secreted in all conditions. They are not glucose repressed and not regulated by galacturonic acid. These constitutive enzymes provide the pathogen with the inherent ability to release galacturonic acid from plant cell walls and to trigger inducible enzyme synthesis.

Molecular evidence for widespread occurrence of Foraminifera in soils.

Environmental SSU rDNA-based surveys are contributing to the dramatic revision of eukaryotic high-level diversity and phylogeny as the number of sequence data increases. This ongoing revolution gives the opportunity to test for the presence of some eukaryotic taxa in environments where they have not been found using classical microscopic observations. Here, we test whether the foraminifera, a group of single-celled eukaryotes, considered generally as typical for the marine ecosystems are present in soil. We performed foraminiferal-specific nested PCR on 20 soil DNA samples collected in contrasted environments. Unexpectedly, we found that the majority of the samples contain foraminiferal SSU rDNA sequences. In total, we obtained 49 sequences from 17 localities. Phylogenetic analysis clusters them in four groups branching among the radiation of early foraminiferal lineages. Three of these groups also include sequences originated from previous freshwater surveys, suggesting that there were up to four independent colonization events of terrestrial and/or freshwater ecosystems by ancestral foraminifera. As shown by our data, foraminifera are a widespread and diverse component of soil microbial communities. Yet, identification of terrestrial foraminiferal species and understanding of their ecological role represent an exciting challenge for future research.

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.

Performance of the COX1 gene as a marker for the study of metabolically active Pezizomycotina and Agaricomycetes fungal communities from the analysis of soil RNA.

In temperate forest soils, filamentous ectomycorrhizal and saprotrophic fungi affiliated to the Agaricomycetes and Pezizomycotina contribute to key biological processes. The diversity of soil fungal communities is usually estimated by studying molecular markers such as nuclear ribosomal gene regions amplified from soil-extracted DNA. However, this approach only reveals the presence of the corresponding genomic DNA in the soil sample and may not reflect the diversity of the metabolically active species. To circumvent this problem, we investigated the performance of the mitochondrial cytochrome c oxidase 1 (COX1)-encoding gene as a fungal molecular marker for environmental RNA-based studies. We designed PCR primers to specifically amplify Agaricomycetes and Pezizomycotina COX1 partial sequences and amplified them from both soil DNA and reverse-transcribed soil RNA. As a control, we also amplified the nuclear internal transcribed spacer ribosomal region from soil DNA. Fungal COX1 sequences were readily amplified from soil-extracted nucleic acids and were not significantly contaminated by nontarget sequences. We show that the relative abundance of fungal taxonomic groups differed between the different sequence data sets, with for example ascomycete COX1 sequences being more abundant among sequences amplified from soil DNA than from soil cDNAs.

Soil metatranscriptomics for mining eukaryotic heavy metal resistance genes.

Heavy metals are pollutants which affect all organisms. Since a small number of eukaryotes have been investigated with respect to metal resistance, we hypothesize that many genes that control this phenomenon remain to be identified. This was tested by screening soil eukaryotic metatranscriptomes which encompass RNA from organisms belonging to the main eukaryotic phyla. Soil-extracted polyadenylated mRNAs were converted into cDNAs and 35 of them were selected for their ability to rescue the metal (Cd or Zn) sensitive phenotype of yeast mutants. Few of the genes belonged to families known to confer metal resistance when overexpressed in yeast. Several of them were homologous to genes that had not been studied in the context of metal resistance. For instance, the BOLA ones, which conferred cross metal (Zn, Co, Cd, Mn) resistance may act by interfering with Fe homeostasis. Other genes, such as those encoding 110- to 130-amino-acid-long, cysteine-rich polypeptides, had no homologues in databases. This study confirms that functional metatranscriptomics represents a powerful approach to address basic biological processes in eukaryotes. The selected genes can be used to probe new pathways involved in metal homeostasis and to manipulate the resistance level of selected organisms.