Project description:Legumes perform symbiotic nitrogen fixation through rhizobial bacteroids housed in specialised root nodules. The biochemical process is energy‐intensive and consumes a huge carbon source to generate sufficient reducing power. To maintain the symbiosis, malate is supplied by legume nodules to bacteroids as their major carbon and energy source in return for ammonium ions and nitrogenous compounds. To sustain the carbon supply to bacteroids, nodule cells undergo drastic reorganisation of carbon metabolism. Here, a comprehensive quantitative comparison of the mitochondrial proteomes between root nodules and uninoculated roots was performed using data‐independent acquisition proteomics, revealing the modulations in nodule mitochondrial proteins and pathways in response to carbon reallocation. Corroborated our findings with that from the literature, we believe nodules preferably allocate cytosolic phosphoenolpyruvates towards malate synthesis in lieu of pyruvate synthesis, and nodule mitochondria prefer malate over pyruvate as the primary source of NADH for ATP production. Moreover, the differential regulation of respiratory chain‐associated proteins suggests that nodule mitochondria could enhance the efficiencies of complexes I and IV for ATP synthesis. This study highlighted a quantitative proteomic view of the mitochondrial adaptation in soybean nodules.
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.
Project description:To circumvent the paucity of nitrogen sources in the soil Legume plants evolved a symbiotic interaction with nitrogen-fixing soil bacteria called rhizobia. During symbiosis, legumes form root organs called nodules, where bacteria are housed intracellularly and become active nitrogen fixers known as bacteroids. Depending on their host plant, bacteroids can adopt different morphotypes, being either unmodified (U), elongated (E) or spherical (S). E- and S-typr bacteroids undergo a terminal differentiation leading to irreversible morphological changes and DNA endoreduplication. Previous studies suggest that differentiated bacteroids display an increased symbiotic efficiency (E>U & S>U). In this study, we used a combination of Aeschynomene species inducing E- and S-type bacteroids in symbiosis with Bradyrhizobium sp. ORS285 to show that S- performed better than E-type bacteroids. Thus, we performed a transcriptomic analysis on E- and S-type bacteroids to identify the bacterial functions involved in each bacteroid type.
Project description:This experiment constitutes an expression profiling approach to identify genes differentially regulated during the symbiotic interaction between the model legume Medicago truncatula and the nitrogen-fixing bacterium Sinorhizobium meliloti. Macro- and microarrays containing 6144 probes were generated on the basis of three cDNA libraries dedicated to the study of root symbiotic interactions. The experiment performed on wild-type and symbiotic mutant material led to the identification of genes either up- or down-regulated at different stages of the nodulation process.
Project description:The bacterium, Sinorhizobium meliloti, interacts symbiotically with leguminous plants such as Medicago truncatula to form nitrogen-fixing root nodules. During symbiosis, plant and bacterial cells differentiate in a coordinated manner, resulting in specialized plant cells that contain nitrogen-fixing bacteroids. Medicago nodules are organized in structurally distinct tissue zones, representing different stages of bacterial and plant differentiation. We used laser-capture microdissection (LCM) to analyze bacterial and plant gene expression in four root nodule regions. In parallel, we analyzed gene expression in nodules formed by wild type bacteria on six plant mutants with nitrogen fixation deficiencies (dnf). We found that bacteroid metabolism is drastically remodeled during bacteroid differentiation. Many processes required for bacterial growth are down-regulated in the nitrogen fixation zone. The overall transcriptional changes are similar to those occurring during nutrient limitation by the stringent response. We also observed differential expression of bacterial genes involved in nitrogen fixation, cell envelope homeostasis, cell division, stress response and polyamine biosynthesis at distinct stages of nodule development. In M. truncatula we observed the differential regulation of several host processes that may trigger bacteroid differentiation and control bacterial infection. We analyzed plant and bacterial gene expression simultaneously, which allowed us to correlate processes in both organisms.
Project description:A transcriptomic analysis of bacteroids isolated from soybean plants inoculated with B. japonicum USDA 110, relative to cells cultured in HM-arabinose medium was performed and the results combined with two other transcriptomic analyses to form a reiterated pool of transcripts that define genes essential for symbiotic nitrogen fixation.
Project description:Rhizobium and allied bacteria form symbiotic nitrogen-fixing nodules on legume roots. Plant hormones appear to play a role in nodule formation. We treated Medicago truncatula roots with auxin transport inhibitors (ATIs) N-(1-naphthyl)phthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA) to induce the formation of pseudonodules. We compared the transcriptional responses of M. truncatula roots treated with ATIs to roots inoculated with Sinorhizobium meliloti. The transcriptional response of M. truncatula roots 1 and 7 days after ATI treatment were opposite to roots treated with S. meliloti.
Project description:A transcriptomic analysis of bacteroids isolated from soybean plants inoculated with B. japonicum USDA 110, relative to cells cultured in HM-arabinose medium was performed and the results combined with two other transcriptomic analyses to form a reiterated pool of transcripts that define genes essential for symbiotic nitrogen fixation. Four independent biological replicates for bacteroids and free-living cells. Total of 8 arrays including dye swap.
Project description:We used an Affymetrix oligonucleotide microarray consisting of 9,935 tentative consensus (TC)sequences, which are based on cDNA libraries (Mitra et al., 2004 [PMID:15220482]). We examined gene expression of wild-type M. truncatula plants after inoculation with wild-type S. meliloti at 1 d, 4 d, 7d, 14 d, and 21 d. We chose these time points because they span the range of development of the Rhizobium-legume symbiosis, from initiation of the interaction through nitrogen fixation. The gene expression data for the 1-day time point (Mitra et al., 2004 [PMID 15220482]) and the other time points was previously published (Starker el al., 2006 [PMID 16407449]).
Project description:Legume plants can form root organs called nodules where they house intracellular symbiotic rhizobium bacteria. Within nodule cells, rhizobia differentiate into bacteroids, which fix nitrogen for the benefit of the plant. Depending on the combination of host plants and rhizobial strains, the output of rhizobium-legume interactions is varying from non-fixing associations to symbioses that are highly beneficial for the plant. Bradyrhizobium diazoefficiens USDA110 was isolated as a soybean symbiont but it can also establish a functional symbiotic interaction with Aeschynomene afraspera. In contrast to soybean, A. afraspera triggers terminal bacteroid differentiation, a process involving bacterial cell elongation, polyploidy and membrane permeability leading to loss of bacterial viability while plants increase their symbiotic benefit. A combination of plant metabolomics, bacterial proteomics and transcriptomics along with cytological analyses was used to study the physiology of USDA110 bacteroids in these two host plants. We show that USDA110 establish a poorly efficient symbiosis with A. afraspera, despite the full activation of the bacterial symbiotic program. We found molecular signatures of high level of stress in A. afraspera bacteroids whereas those of terminal bacteroid differentiation were only partially activated. Finally, we show that in A. afraspera, USDA110 bacteroids undergo an atypical terminal differentiation hallmarked by the disconnection of the canonical features of this process. This study pinpoints how a rhizobium strain can adapt its physiology to a new host and cope with terminal differentiation when it did not co-evolve with such a host.