Joëlle Fourment

Global Changes in Gene Expression in Sinorhizobium meliloti 1021 under Microoxic and Symbiotic Conditions

Sinorhizobium meliloti is an alpha-proteobacterium that alternates between a free-living phase in bulk soil or in the rhizosphere of plants and a symbiotic phase within the host plant cells, where the bacteria ultimately differentiate into nitrogen-fixing organelle-like cells, called bacteroids. As a step toward understanding the physiology of S. meliloti in its free-living and symbiotic forms and the transition between the two, gene expression profiles were determined under two sets of biological conditions: growth under oxic versus microoxic conditions, and in free-living versus symbiotic state. Data acquisition was based on both macro- and microarrays. Transcriptome profiles highlighted a profound modification of gene expression during bacteroid differentiation, with 16% of genes being altered. The data are consistent with an overall slow down of bacteroid metabolism during adaptation to symbiotic life and acquisition of nitrogen fixation capability. A large number of genes of unknown function, including potential regulators, that may play a role in symbiosis were identified. Transcriptome profiling in response to oxygen limitation indicated that up to 5% of the genes were oxygen regulated. However, the microoxic and bacteroid transcriptomes only partially overlap, implying that oxygen contributes to a limited extent to the control of symbiotic gene expression.

Phosphorylation‐induced dimerization of the FixJ receiver domain

The ‘two‐component’ transcriptional activator FixJ controls nitrogen fixation in Sinorhizobium meliloti. Phosphorylation of FixJ induces its dimerization, as evidenced by gel permeation chromatography and equilibrium sedimentation analysis. Phosphorylation‐induced dimerization is an intrinsic property of the isolated receiver domain FixJN. Accordingly, chemical phosphorylation of both FixJ and FixJN are second‐order reactions with respect to protein concentration. However, the second‐order phosphorylation constant is 44‐fold higher for FixJN than for FixJ. Therefore, the C‐terminal transcriptional activator domain FixJC inhibits the chemical phosphorylation of the receiver domain FixJN. Conversely, FixJN has been shown previously to inhibit FixJC activity ≈ 40‐fold, reflecting the interaction between FixJN and FixJC. Therefore, we propose that modulation of FixJ activity involves both its dimerization and the disruption of the interface between FixJN and FixJC, resulting in the opening of the protein structure. Alanine scanning mutagenesis of FixJN indicated that the FixJ~P dimerization interface involves Val‐91 and Lys‐95 in helix α4. Dimerization was required for high‐affinity binding to fixK promoter DNA.

Molecular genetic analysis of the Rhizobium meliloti fixK promoter: identification of sequences involved in positive and negative regulation

Transcription of the Rhizobium meliloti fixK gene is induced in symbiotic and microaerobic growth conditions by the FixL/FixJ modulator/effector pair. Transcription of fixK is also negatively autoregulated. By 5’deletion analysis, the involvement in negative regulation of a DNA region between ‐514 and ‐450 with respect to the transcription start was demonstrated. Site‐directed mutagenesis allowed us to show that a sequence homologous to the binding site of the Escherichia coli Fnr protein, centred at position ‐487, participates in this effect. However, deletion or mutagenesis of this Fnr‐like sequence does not completely eliminate FixK‐dependent repression, which suggests that either an additional DNA region is involved in negative regulation or that it is mediated at the level of fixLJ transcription. Deletion analysis also allowed the definition of a DNA region involved in FixJ‐mediated activation of the fixK promoter, between ‐79 and ‐42. Different point mutations in the ‐60, ‐45 and ‐35 regions were shown to affect promoter activity. In some cases, the activity of mutant promoters could be partly or fully restored by increasing the expression of the fixLJ regulatory genes. In an E. coli strain harbouring a plasmid with fixLJ under the control of an inducible (p‐tac) promoter.

A tandem array of CBF/DREB1 genes is located in a major freezing tolerance QTL region on Medicago truncatula chromosome 6

BackgroundFreezing provokes severe yield losses to different fall-sown annual legumes. Understanding the molecular bases of freezing tolerance is of great interest for breeding programs. Medicago truncatula Gaertn. is an annual temperate forage legume that has been chosen as a model species for agronomically and economically important legume crops. The present study aimed to identify positional candidate genes for a major freezing tolerance quantitative trait locus that was previously mapped to M. truncatula chromosome 6 (Mt-FTQTL6) using the LR3 population derived from a cross between the freezing-tolerant accession F83005-5 and the freezing-sensitive accession DZA045-5.ResultsThe confidence interval of Mt-FTQTL6 was narrowed down to the region comprised between markers MTIC153 and NT6054 using recombinant F7 and F8 lines. A bacterial-artificial chromosome (BAC) clone contig map was constructed in an attempt to close the residual assembly gap existing therein. Twenty positional candidate genes including twelve C-repeat binding factor (CBF)/dehydration-responsive element binding factor 1 (DREB1) genes were identified from BAC-derived sequences and whole-genome shotgun sequences (WGS). CBF/DREB1 genes are organized in a tandem array within an approximately 296-Kb region. Eleven CBF/DREB1 genes were isolated and sequenced from F83005-5 and DZA045-5 which revealed high polymorphism among these accessions. Unique features characterizing CBF/DREB1 genes from M. truncatula, such as alternative splicing and large tandem duplication, are elucidated for the first time.ConclusionsOverall, twenty genes were identified as potential candidates to explain Mt-FTQTL6 effect. Their future functional characterization will uncover the gene(s) involved in freezing tolerance difference observed between F83005-5 and DZA045-5. Knowledge transfer for breeding improvement of crop legumes is expected. Furthermore, CBF/DREB1 related data will certainly have a large impact on research studies targeting this group of transcriptional activators in M. truncatula and other legume species.

The Impact of Open Pollination on the Structural Evolutionary Dynamics, Meiotic Behavior and Fertility of Resynthesized Allotetraploid Brassica napus L.

Allopolyploidy, which results from the merger and duplication of two divergent genomes, has played a major role in the evolution and diversification of flowering plants. The genomic changes that occur in resynthesized or natural neopolyploids have been extensively studied, but little is known about the effects of the reproductive mode in the initial generations that may precede its successful establishment. To truly reflect the early generations of a nascent polyploid, two resynthesized allotetraploid Brassica napus populations were obtained for the first time by open pollination. In these populations, we detected a much lower level of aneuploidy (third generation) compared with those previously published populations obtained by controlled successive selfing. We specifically studied 33 resynthesized B. napus individuals from our two open pollinated populations, and showed that meiosis was affected in both populations. Their genomes were deeply shuffled after allopolyploidization: up to 8.5 and 3.5% of the C and A subgenomes were deleted in only two generations. The identified deletions occurred mainly at the distal part of the chromosome, and to a significantly greater extent on the C rather than the A subgenome. Using Fluorescent In Situ Hybridization (BAC-FISH), we demonstrated that four of these deletions corresponded to fixed translocations (via homeologous exchanges). We were able to evaluate the size of the structural variations and their impact on the whole genome size, gene content, and allelic diversity. In addition, the evolution of fertility was assessed, to better understand the difficulty encountered by novel polyploid individuals before the putative formation of a novel stable species.

FANCM Limits Meiotic Crossovers in Brassica Crops

Meiotic crossovers (COs) are essential for proper chromosome segregation and the reshuffling of alleles during meiosis. In WT plants, the number of COs is usually small, which limits the genetic variation that can be captured by plant breeding programs. Part of this limitation is imposed by proteins like FANCM, the inactivation of which results in a 3-fold increase in COs in Arabidopsis thaliana. Whether the same holds true in crops needed to be established. In this study, we identified EMS induced mutations in FANCM in two species of economic relevance within the genus Brassica. We showed that CO frequencies were increased in fancm mutants in both diploid and tetraploid Brassicas, Brassica rapa and Brassica napus respectively. In B. rapa, we observed a 3-fold increase in the number of COs, equal to the increase observed previously in Arabidopsis. In B. napus we observed a lesser but consistent increase (1.3-fold) in both euploid (AACC) and allohaploid (AC) plants. Complementation tests in A. thaliana suggest that the smaller increase in crossover frequency observed in B. napus reflects residual activity of the mutant C copy of FANCM. Altogether our results indicate that the anti-CO activity of FANCM is conserved across the Brassica, opening new avenues to make a wider range of genetic diversity accessible to crop improvement.

Long Read Sequencing Technology to Solve Complex Genomic RegionsAssembly in Plants

Background: Numerous completed or on-going whole genome sequencing projects have highlighted the fact that obtaining a high quality genome sequence is necessary to address comparative genomics questions such as structural variations among genotypes and gain or loss of specific function. Despite the spectacular progress that has been made in sequencing technologies, obtaining accurate and reliable data is still a challenge, both at the whole genome scale and when targeting specific genomic regions. These problems are even more noticeable for complex plant genomes. Most plant genomes are known to be particularly challenging due to their size, high density of repetitive elements and various levels of ploidy. To overcome these problems, we have developed a strategy to reduce genome complexity by using the large insert BAC libraries combined with next generation sequencing technologies. Results: We compared two different technologies (Roche-454 and Pacific Biosciences PacBio RS II) to sequence pools of BAC clones in order to obtain the best quality sequence. We targeted nine BAC clones from different species (maize, wheat, strawberry, barley, sugarcane and sunflower) known to be complex in terms of sequence assembly. We sequenced the pools of the nine BAC clones with both technologies. We compared assembly results and highlighted differences due to the sequencing technologies used. Conclusions: We demonstrated that the long reads obtained with the PacBio RS II technology serve to obtain a better and more reliable assembly, notably by preventing errors due to duplicated or repetitive sequences in the same region.

Genetic variation in a complex polyploid: unveiling the dynamic allelic features of sugarcane

Background Sugarcane (Saccharum spp.) is highly polyploid and aneuploid. Modern cultivars are derived from hybridization between S. officinarum and S. spontaneum. This combination results in a genome exhibiting variable ploidy among different loci, a huge genome size (approximately 10 Gb) and a high content of repetitive regions. Gene expression mechanisms are poorly understood in these cultivars. An approach using genomic, transcriptomic and genetic mapping can improve our knowledge of the behavior of genetics in sugarcane. Results The hypothetical HP600 and centromere protein C (CENP-C) genes from sugarcane were used to elucidate the allelic expression and genomic and genetic behavior of this complex polyploid. The genomically side-by-side genes HP600 and CENP-C were found in two different homeologous chromosome groups with ploidies of eight and ten. The first region (Region01) was a Sorghum bicolor ortholog with all haplotypes of HP600 and CENP- C expressed, but HP600 exhibited an unbalanced haplotype expression. The second region (Region02) was a scrambled sugarcane sequence formed from different noncollinear genes containing duplications of HP600 and CENP-C (paralogs). This duplication occurred before the Saccharum genus formation and after the separation of sorghum and sugarcane, resulting in a nonexpressed HP600 pseudogene and a recombined fusion version of CENP-C and orthologous gene Sobic.003G299500 with at least two chimerical gene haplotypes expressed. The genetic map construction supported the difficulty of mapping markers located in duplicated regions of complex polyploid genomes. Conclusion All these findings describe a low synteny region in sugarcane, formed by events occurring in all members of the Saccharum genus. Additionally, evidence of duplicated and truncate gene expression and the behavior of genetic markers in a duplicated region was found. Thus, we describe the complexity involved in sugarcane genetics and genomics and allelic dynamics, which can be useful for understanding the complex polyploid genome.