Project description:The three-dimensional (3D) architecture of compacted DNA inside cells plays fundamental roles in gene transcription regulation. The higher order of chromatin 3D structures includes cis-regulatory interactions such as enhancer-promoter looping, mediated by transcription factors (TFs) and affecting the activity of promoter regions. In the model legume Medicago truncatula, which has the ability to establish symbiotic interactions with rhizobial bacteria to form nitrogen-fixing nodules, we have explored the changes occurring during symbiosis using Hi-C approaches. Excitingly, the plant genome is re-organized and shows major gene interaction changes in N2-fixing symbiotic nodules versus non-fixing nodules and roots, correlating with specific transcriptome reprogramming. By integrating Hi-C, ChIP-Seq, RNA-Seq and ATAC-Seq data, we revealed that N2-fixing symbiotic plant cells undergo modifications of chromatin compartmentalization that affect gene transcriptional regulation by forming chromatin interactions with active histone marks. Among the detected novel chromatin loops, we identified one connecting a 15kb distal region with the proximal promoter of the TF NODULE INCEPTION (NIN), a major regulator of the developmental transition required for symbiosome development and N2-fixation. Interestingly, the NIN gene displays a complex spatiotemporal regulation governed by distinct cis-regulatory sequences including this conserved distal -15 kb region. Furthermore, NIN distal promoter region contains several putative cytokinin (CK) response elements (CE) and we showed that NIN transcriptional induction by CKs likely depends on the formation of an enhancer-promoter loop bringing the -15 kb conserved distal CE element close to the -5 kb proximal promoter region of NIN. To gain further insight, we showed that NIN transcriptional induction by CKs depends on the TYPE-B RESPONSE REGULATOR 3 (RRB3) TF, which only binds to the distal NIN promoter region. These results suggest that a TF complex involving type-B RRs deposited on NIN distal region controls the formation of a chromatin loop responsible for NIN spatiotemporal regulation and subsequently coordinates nodulation developmental programs. More generally, the reorganization of the linear genome in this symbiotic plant-bacteria interaction reveals a new regulatory level where cell-cell communication of bacterial symbionts impacts 3D genome organization and transcription reprograming of the host eukaryote.

Project description:Legumes can generate two types of nitrogen-fixing nodules depending on both the host plant and the symbiont. In indeterminate nodules, bacteria undergo terminal differentiation into bacteroids, an irreversible state in which they are able to fix atmospheric nitrogen into ammonia. In Medicago truncatula nodules, the differentiation process is controlled by nodule-specific cysteine-rich peptides (NCRs). The NCRs are secreted by the plant and translocated to the bacterial cytosol, where they can be cleaved by bacterial peptidases, as a defense response to the antimicrobial activity of those peptides. Two peptidases involved in the development of nitrogen-fixing nodules have recently been described: HrrP and SapA. Both proteins showed peptidase activity against NCRs. Expression of hrrP (host range restriction peptidase) in S. meliloti B800 inhibits nitrogen fixation in M. truncatula A20. On the other hand, overexpression of sapA generates plants with lower levels of nitrogen fixation. Rhizobium favelukesii LPU83 was isolated from acid soils of Argentina and can nodulate M. truncatula. Even though this bacterium presents a symbiotic plasmid with the necessary genes to establish an effective symbiosis, it is inefficient in nitrogen fixation. In this work, we sought to identify and analyze the role of homologous genes to hrrP and sapA peptidases in the symbiosis between LPU83 and M. truncatula in order to evaluate their implication in the symbiotic phenotype of the strain. Genes homologous to hrrP and sapA were found in the symbiotic plasmid and chromosome of LPU83. Mutants in these genes were generated and their symbiotic phenotype was evaluated in M. truncatula A20. Our results showed that both single and a double mutant on these genes did not exhibit changes in their symbiotic phenotype compared to controls, with no significant differences in shoot dry weight. Previous research indicated that overexpression of hrrP in S. meliloti negatively affected symbiosis, resulting in ineffective nitrogen fixation in plants with white, small nodules. To study the impact of LPU83 peptidases, the homologous genes were cloned into replicative plasmids in rhizobia under the promoter of the hrrP gene and transferred to S. meliloti. However, overexpression of LPU83 peptidases showed no differences with the strain carrying the empty plasmid, with plants exhibiting green leaves and pink, elongated nodules. Confocal microscopy of these nodules revealed that even though the plants fix nitrogen, there were more dead bacteria inside the nodules infected with overexpressed LPU83 peptidases. Overall, these findings suggest that while the hrrP gene plays a crucial role in nodulation and nitrogen fixation in S. meliloti, the peptidases from LPU83 do not significantly impact on the symbiotic effectiveness. Further research is needed to explore and clarify the special phenotype observed in LPU83.

Project description:During the legume-rhizobium symbiosis, free-living soil bacteria known as rhizobia trigger the formation of root nodules. The rhizobia infect these organs and adopt an intracellular lifestyle within the symbiotic nodule cells where they become nitrogen-fixing bacteroids. Several legume lineages enforce their symbionts into an extreme cellular differentiation, comprising cell enlargement and genome endoreduplication. The antimicrobial peptide transporter BclA is a major determinant of this differentiation process in Bradyrhizobium sp. ORS285, a symbiont of Aeschynomene spp.. In the absence of BclA, Bradyrhizobium sp. ORS285 proceeds until the intracellular infection of nodule cells but the bacteria cannot differentiate into enlarged polyploid bacteroids and fix nitrogen. The nodule bacteria of the bclA mutant constitute thus an intermediate stage between the free-living soil bacteria and the intracellular nitrogen-fixing bacteroids. Metabolomics on whole nodules of Aeschynomene afraspera and Aeschynomene indica infected with the ORS285 wild type or the bclA mutant revealed 47 metabolites that differentially accumulated concomitantly with bacteroid differentiation. Bacterial transcriptome analysis of these nodules discriminated nodule-induced genes that are specific to differentiated and nitrogen-fixing bacteroids and others that are activated in the host microenvironment irrespective of bacterial differentiation and nitrogen fixation. These analyses demonstrated that the intracellular settling of the rhizobia in the symbiotic nodule cells is accompanied with a first transcriptome switch involving several hundreds of upregulated and downregulated genes and a second switch accompanying the bacteroid differentiation, involving less genes but that are expressed to extremely elevated levels. The transcriptomes further highlighted the dynamics of oxygen and redox regulation of gene expression during nodule formation and we discovered that bclA represses the expression of non-ribosomal peptide synthetase gene clusters suggesting a non-symbiotic function of BclA. Together, our data uncover the metabolic and gene expression changes that accompany the transition from intracellular bacteria into differentiated nitrogen-fixing bacteroids.

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The three-dimensional genome architecture drives symbiotic nodulation in legumes [Hi-C]

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