Metabolomics and Dual RNA-Sequencing on Root Nodules Revealed New Cellular Functions Controlled by Paraburkholderia phymatum NifA
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ABSTRACT:
PROVIDER: PRJNA735727 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14749825.fastq.gz | Fastqsanger.gz | |||
| SRR14749826.fastq.gz | Fastqsanger.gz | |||
| SRR14749827.fastq.gz | Fastqsanger.gz | |||
| SRR14749828.fastq.gz | Fastqsanger.gz |
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Project description:Paraburkholderia phymatum STM815 is a nitrogen-fixing endosymbiont that forms root nodules on the agriculturally important Phaseolus vulgaris and other host plants. We previously showed that the nodules induced by a STM815 mutant of the gene encoding the master regulator of nitrogen fixation NifA showed no nitrogenase activity (Fix-) and increased in number compared to P. vul-garis plants infected with the wild-type strain. To further investigate the role of NifA during symbiosis, nodules from P. phymatum wild-type and nifA mutants were collected and analyzed by metabolomics and dual RNA-Sequencing, allowing us to investigate both host and symbiont transcriptome. Using this approach, several metabolites changes could be assigned to bacterial or plant responses. While the amount of the C4-dicarboxylic acid succinate and of several amino acids was lower in Fix- nodules, the level of indole-acetamide (IAM) and brassinosteroids increased in Fix- nodules. Transcriptome analysis identified P. phymatum genes involved in transport of C4-dicarboxylic acids, carbon metabolism, auxin metabolism and stress response to be differen-tially expressed in absence of NifA. Furthermore, P. vulgaris genes involved in autoregulation of nodulation (AON) are repressed in nodules in absence of NifA potentially explaining the hyper-nodulation phenotype of the nifA mutant. These results and additional validation experiments suggest that P. phymatum STM815 NifA is not only important to control expression of nitrogenase and related enzymes but is also involved in regulating its own auxin production and stress re-sponse. Finally, our data indicate that P. vulgaris does sanction the nifA nodules by depleting the local carbon allocation rather than by mounting a strong systemic immune response to the Fix- rhizobia.
2021-07-28 | GSE176287 | GEO
Project description:RpoN (σ54) is the key sigma factor for the regulation of transcription of nitrogen fixation genes in diazotrophic bacteria, which include alpha- and beta-rhizobia. Our previous studies showed that a rpoN mutant of the beta-rhizobial strain Paraburkholderia phymatum formed root nodules on Phaseolus vulgaris that were unable to reduce atmospheric nitrogen into ammonia. In an effort to further characterize the RpoN regulon of P. phymatum, transcriptomics was combined with a powerful metabolomics approach. The metabolome of P. vulgaris root nodules infected by the P. phymatum rpoN Fix- mutant revealed statistically significant metabolic changes compared to wild-type Fix+ nodules, including reduced amounts of chorismate and elevated levels of flavonoids. A transcriptome analysis on Fix+ and Fix- nodules – combined with a search for RpoN binding sequences in promoter regions of regulated genes – confirmed the expected control of σ54 on nitrogen fixation genes in nodules. The transcriptomic data also identified additional target genes, whose differential expression was able to explain the observed metabolite changes in a numerous cases. Moreover, the genes encoding the two-component regulatory system NtrBC were downregulated in root nodules induced by the rpoN mutant and contained a putative RpoN binding motif in their promoter region, suggesting direct regulation. The construction and characterization of an ntrB mutant strain revealed impaired nitrogen assimilation in free-living conditions, as well as a noticeable symbiotic phenotype by forming less but heavier nodules on P. vulgaris roots.
2018-06-06 | GSE111993 | GEO
Project description:Paraburkholderia phymatum belongs to the β-subclass of proteobacteria. It has recently been shown to be able to nodulate and fix nitrogen in symbiosis with several mimosoid and papillionoid legumes. In contrast to symbiosis of legumes with α-proteobacteria, very little is known about the molecular determinants underlying the successful establishment of this mutualistic relationship with β-proteobacteria. In this study, we analyzed RNA-seq data of free-living P. phymatum growing under nitrogen replete and limited conditions, the latter partially mimicking the situation in nitrogen deprived soils. Among the genes up-regulated under nitrogen limitation, we found genes involved in exopolysaccharide production and motility, two traits relevant for plant root infection. Next, RNA-seq data of P. phymatum grown under free-living conditions and from symbiotic root nodules of Phaseolus vulgaris (common bean) were generated and compared. Among the genes highly up-regulated during symbiosis, we identified an operon encoding a potential cytochrome o ubiquinol oxidase (Bphy_3646-49). Bean root nodules induced by a cyoB mutant strain showed reduced nitrogenase and nitrogen fixation abilities suggesting an important role of the cytochrome for respiration inside the nodule. Analysis of mutant strains for RNA polymerase transcription factor rpoN (σ54) and its activator NifA indicated that – similar to the situation in α-rhizobia – P. phymatum RpoN and NifA are key regulators during symbiosis with P. vulgaris.
2018-02-23 | GSE107381 | GEO
Project description:Paraburkholderia phymatum is a beta-proteobacterium, which lives in the soil and is able to enter nitrogen-fixing symbiosis with different legumes. The biological nitrogen fixation (BNF) process is of great ecological and agronomic importance. We previously showed that the expression of the key P. phymatum BNF enzyme – the nitrogenase –is regulated by the sigma factor σ54 (or RpoN) inside root nodules. This study focused on identifying the σ54 regulon of P. phymatum grown in nitrogen limited conditions using RNA-Sequencing. Among the genes significantly down-regulated in absence of σ54 we found those coding for a C4-dicarboxylate transport system (Bphy_0225-27), a flagellar biosynthesis cluster (Bphy_2926-64) and one of the two type 6 secretion system (T6SS-b) present in P. phymatum genome (Bphy_5978-97). Indeed, the σ54 mutant was unable to grow on C4 dicarboxylates (fumarate, malate and succinate) as the sole carbon source and was less motile compared to the wild-type strain. Both defects were complemented by adding rpoN in trans. Additionally, using reporter fusions we confirmed that T6SS-b expression is regulated by σ54. Finally, a σ54 mutant was less competitive than its parental strain against P. diazotrophica, suggesting a role of σ54 in controlling interbacterial competition.
2020-08-12 | GSE156048 | GEO
Project description:Paraburkholderia phymatum STM815 is a nitrogen-fixing endosymbiont that nodulate the agriculturally important Phaseolus vulgaris and several other host plants. We previously showed that the nodules induced by a STM815 mutant of the gene encoding the master regulator of nitrogen fixation NifA showed no nitrogenase activity (Fix-) and increased in number compared to P. vulgaris plants infected with the wild-type strain. To further investigate the role of NifA during symbiosis, nodules from P. phymatum wild-type and nifA mutants were collected and analyzed by metabolomics and dual RNA-Sequencing, allowing us to investigate both host and symbiont transcriptome. Using this approach, several metabolites' changes could be assigned to bacterial or plant responses. While the amount of the C4-dicarboxylic acid succinate and of several amino acids was lower in Fix- nodules, the level of indole-acetamide (IAM) and brassinosteroids increased. Transcriptome analysis identified P. phymatum genes involved in transport of C4-dicarboxylic acids, carbon metabolism, auxin metabolism and stress response to be differentially expressed in absence of NifA. Furthermore, P. vulgaris genes involved in autoregulation of nodulation (AON) are repressed in nodules in absence of NifA potentially explaining the hypernodulation phenotype of the nifA mutant. These results and additional validation experiments suggest that P. phymatum STM815 NifA is not only important to control expression of nitrogenase and related enzymes but is also involved in regulating its own auxin production and stress response. Finally, our data indicate that P. vulgaris does sanction the nifA nodules by depleting the local carbon allocation rather than by mounting a strong systemic immune response to the Fix- rhizobia.
| S-EPMC8305402 | biostudies-literature
Project description:Metabolomics and transcriptomics identify multiple downstream targets of Paraburkholderia phymatum σ54 during symbiosis with Phaseolus vulgaris
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Project description:Metabolomics and transcriptomics of Bradyrhizobium diazoefficiens-induced root nodules Bradyrhizobium diazoefficiens is a nitrogen-fixing endosymbiont, which can grow inside root-nodule cells of the agriculturally important soybean and other host plants. Our previous studies described B. diazoefficiens host-specific global expression changes occurring during legume infection at the transcript and protein level. In order to further characterize nodule metabolism, we here determine by flow injection -time of flight mass spectrometry analysis the metabolome of i) nodules and roots from four different B. diazoefficiens host plants, ii) soybean nodules harvested at different time points during nodule development, and iii) soybean nodules infected by two strains mutated in key genes for nitrogen fixation, respectively. Ribose (soybean), tartaric acid (mungbean), hydroxybutanoyloxybutanoate (siratro) and catechol (cowpea) were among the metabolites found to be specifically elevated in one of the respective host plants. While the level of C4-dicarboxylic acids decreased during soybean nodule development, we observed an accumulation of trehalose-phosphate at 21 days post infection (dpi). Moreover, nodules from non-nitrogen-fixing bacteroids (nifA and nifH mutants) showed specific metabolic alterations; these were also supported by transcriptomics data that was generated for the two mutant strains and were helpful to separate for some examples the respective bacterial and plant contributions to the metabolic profile. The alterations included signs of nitrogen limitation in both mutants, and an increased level of a phytoalexin in nodules induced by the nifA mutant, suggesting that the tissue of these nodules exhibits defense and stress reactions.
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Project description:Paraburkholderia phymatum σ54 controls utilization of dicarboxylates, motility and interbacterial competition
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