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Lyon, France

Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.

Jany J.-L.,Cornell University | Jany J.-L.,CNRS Microbial Ecology | Pawlowska T.E.,Cornell University
American Naturalist | Year: 2010

Arbuscular mycorrhizal fungi (Glomeromycota) are the dominant symbionts of land plants and one of the oldest multicellular lineages that exist without evidence of sexual reproduction. The mechanisms that protect these organisms from extinction due to accumulation of deleterious mutations in the absence of sexual recombination are unclear. Glomeromycota reproduce by spores containing hundreds of nuclei, which represents a departure from the typical eukaryotic developmental pattern, where a multicellular organism is re-created from a uninucleate propagule. To understand whether the multinucleate spore makeup may have contributed to the evolutionary success of Glomeromycota, we examined the dynamics of spore nuclei in Glomus etunicatum using live threedimensional imaging and mathematical models. We show that the spores are populated by an influx of a stream of nuclei from the surrounding mycelium rather than by divisions of a single founder nucleus. We present evidence that mechanisms of selection are likely to operate at the level of individual nuclei. On the basis of mathematical analyses of the effects that these nuclear dynamics have on the population mutation load, we postulate that the developmental patterns of sporogenesis have adaptive significance for moderating the accumulation of deleterious mutations and may have contributed to the evolutionary longevity of Glomeromycota. © 2010 by The University of Chicago.

The influence exerted by the biocontrol oomycete Pythium oligandrum on the bacterial populations proliferating in the rhizosphere of tomato plants grown in a hydroponic system and in the circulating solutions is studied in the present experiment. Quantitative PCR and single-strand conformation polymorphism were used to investigate the genetic structure and dynamics of the bacterial communities colonizing the root systems and the various circulating solutions. Quantitative PCR assays showed that bacteria heavily colonized the rhizosphere of tomato plants with, however, no significant density changes throughout the cultural season (April-September). Single strand conformation polymorphism fingerprints revealed the occurrence of transient perturbations in the rhizospheric indigenous bacterial communities following P. oligandrum introduction in the root system of plants. This effect was, however, transient and did not persist until the end of the cropping season. Interestingly, the genetic structure of the bacterial microflora colonizing either the roots or the nutrient solutions evolved throughout the cropping season. This temporal evolution occurred whatever the presence and persistence of P. oligandrum in the rhizosphere. Evidence is also provided that bacterial microflora that colonize the root system are different from the ones colonizing the circulating solutions. The relationships between these 2 microflora (at the root and solution levels) are discussed.

Cheynier V.,French National Institute for Agricultural Research | Comte G.,CNRS Microbial Ecology | Davies K.M.,The New Zealand Institute for Plant and Food Research Ltd | Martens S.,Research and Innovation Center
Plant Physiology and Biochemistry | Year: 2013

Land-adapted plants appeared between about 480 and 360 million years ago in the mid-Palaeozoic era, originating from charophycean green algae. The successful adaptation to land of these prototypes of amphibious plants - when they emerged from an aquatic environment onto the land - was achieved largely by massive formation of "phenolic UV light screens". In the course of evolution, plants have developed the ability to produce an enormous number of phenolic secondary metabolites, which are not required in the primary processes of growth and development but are of vital importance for their interaction with the environment, for their reproductive strategy and for their defense mechanisms.From a biosynthetic point of view, beside methylation catalyzed by O-methyltransferases, acylation and glycosylation of secondary metabolites, including phenylpropanoids and various derived phenolic compounds, are fundamental chemical modifications. Such modified metabolites have altered polarity, volatility, chemical stability in cells but also in solution, ability for interaction with other compounds (co-pigmentation) and biological activity.The control of the production of plant phenolics involves a matrix of potentially overlapping regulatory signals. These include developmental signals, such as during lignification of new growth or the production of anthocyanins during fruit and flower development, and environmental signals for protection against abiotic and biotic stresses. For some of the key compounds, such as the flavonoids, there is now an excellent understanding of the nature of those signals and how the signal transduction pathway connects through to the activation of the phenolic biosynthetic genes.Within the plant environment, different microorganisms can coexist that can establish various interactions with the host plant and that are often the basis for the synthesis of specific phenolic metabolites in response to these interactions. In the rhizosphere, increasing evidence suggests that root specific chemicals (exudates) might initiate and manipulate biological and physical interactions between roots and soil organisms. These interactions include signal traffic between roots of competing plants, roots and soil microbes, and one-way signals that relate the nature of chemical and physical soil properties to the roots. Plant phenolics can also modulate essential physiological processes such as transcriptional regulation and signal transduction. Some interesting effects of plant phenolics are also the ones associated with the growth hormone auxin. An additional role for flavonoids in functional pollen development has been observed. Finally, anthocyanins represent a class of flavonoids that provide the orange, red and blue/purple colors to many plant tissues. According to the coevolution theory, red is a signal of the status of the tree to insects that migrate to (or move among) the trees in autumn. © 2013 Elsevier Masson SAS.

Pommier T.,CNRS Microbial Ecology | Douzery E.J.P.,Montpellier University | Mouillot D.,Montpellier University | Mouillot D.,James Cook University
Biology Letters | Year: 2012

Although environmental filtering has been observed to influence the biodiversity patterns of marine bacterial communities, it was restricted to the regional scale and to the species level, leaving the main drivers unknown at large biogeographic scales and higher taxonomic levels. Bacterial communities with different species compositions may nevertheless share phylogenetic lineages, and phylogenetic turnover (PT) among those communities may be surprisingly low along any biogeographic or environmental gradient. Here, we investigated the relative influence of environmental filtering and geographical distance on the PT between marine bacterial communities living more than 8000 km apart in contrasted abiotic conditions. PT was high between communities and was more structured by local environmental factors than by geographical distance, suggesting the predominance of a lineage filtering process. Strong phenotype-environment mismatches observed in the ocean may surpass high connectivity between marine microbial communities. This journal is © 2012 The Royal Society.

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