Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches

The legume plant Medicago truncatula establishes a symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti which takes place in root nodules. The formation of nodules employs a complex developmental program involving organogenesis, specific cellular differentiation of the host cells and the endosymbiotic bacteria, called bacteroids, as well as the specific activation of a large number of plant genes. By using a collection of plant and bacterial mutants inducing non-functional, Fix− nodules, we studied the differentiation processes of the symbiotic partners together with the nodule transcriptome, with the aim of unravelling links between cell differentiation and transcriptome activation. Two waves of transcriptional reprogramming involving the repression and the massive induction of hundreds of genes were observed during wild-type nodule formation. The dominant features of this “nodule-specific transcriptome” were the repression of plant defense-related genes, the transient activation of cell cycle and protein synthesis genes at the early stage of nodule development and the activation of the secretory pathway along with a large number of transmembrane and secretory proteins or peptides throughout organogenesis. The fifteen plant and bacterial mutants that were analyzed fell into four major categories. Members of the first category of mutants formed non-functional nodules although they had differentiated nodule cells and bacteroids. This group passed the two transcriptome switch-points similarly to the wild type. The second category, which formed nodules in which the plant cells were differentiated and infected but the bacteroids did not differentiate, passed the first transcriptome switch but not the second one. Nodules in the third category contained infection threads but were devoid of differentiated symbiotic cells and displayed a root-like transcriptome. Nodules in the fourth category were free of bacteria, devoid of differentiated symbiotic cells and also displayed a root-like transcriptome. A correlation thus exists between the differentiation of symbiotic nodule cells and the first wave of nodule specific gene activation and between differentiation of rhizobia to bacteroids and the second transcriptome wave in nodules. The differentiation of symbiotic cells and of bacteroids may therefore constitute signals for the execution of these transcriptome-switches

Reconstruction of ancestral chromosome architecture and gene repertoire reveals principles of genome evolution in a model yeast genus

International audienceReconstructing genome history is complex but necessary to reveal quantitative principles governing genome evolution. Such reconstruction requires recapitulating into a single evolutionary framework the evolution of genome architecture and gene repertoire. Here, we reconstructed the genome history of the genus Lachancea that appeared to cover a continuous evolutionary range from closely related to more diverged yeast species. Our approach integrated the generation of a high-quality genome data set; the development of AnChro, a new algorithm for reconstructing ancestral genome architecture; and a comprehensive analysis of gene repertoire evolution. We found that the ancestral genome of the genus Lachancea contained eight chromosomes and about 5173 protein-coding genes. Moreover, we characterized 24 horizontal gene transfers and 159 putative gene creation events that punctuated species diversification. We retraced all chromosomal rearrangements, including gene losses, gene duplications, chromosomal inversions and translocations at single gene resolution. Gene duplications outnumbered losses and balanced rearrangements with 1503, 929, and 423 events, respectively. Gene content variations between extant species are mainly driven by differential gene losses, while gene duplications remained globally constant in all lineages. Remarkably, we discovered that balanced chromosomal rearrangements could be responsible for up to 14% of all gene losses by disrupting genes at their breakpoints. Finally, we found that nonsynonymous substitutions reached fixation at a coordinated pace with chromosomal inversions, translocations, and duplications, but not deletions. Overall, we provide a granular view of genome evolution within an entire eukaryotic genus, linking gene content, chromosome rearrangements , and protein divergence into a single evolutionary framework

Genomewide and biochemical analyses of DNA-binding activity of Cdc6/Orc1 and Mcm proteins in Pyrococcus sp.

The origin of DNA replication (oriC) of the hyperthermophilic archaeon Pyrococcus abyssi contains multiple ORB and mini-ORB repeats that show sequence similarities to other archaeal ORB (origin recognition box). We report here that the binding of Cdc6/Orc1 to a 5 kb region containing oriC in vivo was highly specific both in exponential and stationary phases, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip). The oriC region is practically the sole binding site for the Cdc6/Orc1, thereby distinguishing oriC in the 1.8 M bp genome. We found that the 5 kb region contains a previously unnoticed cluster of ORB and mini-ORB repeats in the gene encoding the small subunit (dp1) for DNA polymerase II (PolD). ChIP and the gel retardation analyses further revealed that Cdc6/Orc1 specifically binds both of the ORB clusters in oriC and dp1. The organization of the ORB clusters in the dp1 and oriC is conserved during evolution in the order Thermococcales, suggesting a role in the initiation of DNA replication. Our ChIP-chip analysis also revealed that Mcm alters the binding specificity to the oriC region according to the growth phase, consistent with its role as a licensing factor

Epigenetic Natural Variation in Arabidopsis thaliana

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F2 families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means

Les politiques des sciences. Séminaire alternatif

Michel Agier, Mathieu Arnoux, Alban Bensa, Alain Blum, Simona Cerutti, Francis Chateauraynaud, Robert Descimon, Nicolas Dodier, Jean-Claude Galey, Nancy L. Green, Christian Jouhaud, Christian Topalov, directeurs d’étudesIsabelle Backouche, Juliette Cadiot, Fanny Cosandey, Sophie Desrosiers, André Gunthert, Liora Israël, Cyril Lemieux, Mary Picone, Sylvain Piron, maîtres de conférencesIrène Bellier, directrice de recherche au CNRSMichel Barthélémy, Elie Haddad, Cédric Lomba, Birgit Müller, Sop..

The Mutational Profile of the Yeast Genome Is Shaped by Replication

International audienceDespite the scrutiny that has been directed for years at the yeast genome, relatively little is known about the impact of replication on the substitution dynamics in Saccharomyces cerevisiae. Here, we show that the mutation rate increases with the replication timing by more than 30% between the earliest and the latest replicating regions. In addition, we found a mutational asymmetry associated with the polarity of replication resulting in higher rates of substitutions toward C and A than toward G and T in leading strands (reciprocally more substitutions toward G and T in lagging strands). Such mutational asymmetries applied over long evolutionary periods should generate compositional skews between the two DNA strands. Thus, we show that the leading replicating strands present an excess of C over G and of A over T in the genome of S. cerevisiae (reciprocally an excess of G + T over C + A in lagging strands). We also show that the nucleotide frequencies at mutational equilibrium predict a compositional skew at equilibrium very close to the observed skew between leading and lagging strands, suggesting that compositional equilibrium has been nearly attained in the present day genome of S. cerevisiae. Surprisingly, the direction of this skew is inverted compared with the one in the human genome

A Versatile Procedure to Generate Genome-Wide Spatiotemporal Program of Replication in Yeast Species

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Author response: An evolutionary model identifies the main evolutionary biases for the evolution of genome-replication profiles

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The Spatiotemporal Program of Replication in the Genome of Lachancea kluyveri

International audienceWe generated a genome-wide replication profile in the genome of Lachancea kluyveri and assessed the relationship between replication and base composition. This species diverged from Saccharomyces cerevisiae before the ancestral whole genome duplication. The genome comprises eight chromosomes among which achromosomal arm of 1Mb has a G + C-content much higher than the rest of the genome. We identified 252 active replication origins in L. kluyveri and found considerable divergence in origin location with S. cerevisiae and with Lachancea waltii. Although some global features of S. cerevisiae replication are conserved: Centromeres replicate early, whereas telomeres replicate late, we found that replication origins both in L. kluyveri and L. waltii do not behave as evolutionary fragile sites. In L. kluyveri, replication timing along chromosomes alternates between regions of early and late activating origins, except for the 1Mb GC-rich chromosomal arm. This chromosomal arm contains an origin consensus motif different from other chromosomes and is replicated early during S-phase. We showed that precocious replication results from the specific absence of late firing origins in this chromosomal arm. In addition, we found a correlation between GC-content and distance from replication origins as well as a lack of replication-associated compositional skew between leading and lagging strands specifically in this GC-rich chromosomal arm. These findings suggest that the unusual base composition in the genome of L. kluyveri could be linked to replication

Visualization of extracted genomic DNA size distributions.

(a) PFGE using 0.5 μg of DNA prepared with (+) or without (-) SRE size-selection, embedded in 30 μl of 0.5% low-melting agarose plugs, migrated in a 1% SeaKem GTG agarose (Lonza) gel. The ladder is a mix of PFG mid-range (N0342S, NEB) and GeneRuler 1 kb Plus (SM1331, ThermoFischer). Electrophoresis conditions: 0.5X TBE (Tris Borate EDTA) buffer, 6 V.cm-1, 120° angle, for 11h, switching time ramp from 1 to 60 seconds. Gel stained in ethidium bromide and imaged with UV. (b) Standard gel electrophoresis (0.3% agarose) of the indicated samples. GeneRuler 1 kb Plus (SM1331, ThermoFischer) is used as the ladder. See S3 Fig for the uncropped images.</p