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RNA-seq Analysis Reveal Global Differential Transcritomes of Mesorhizobium huakuii 7653R between Bacteroids and Free-living Cells

ABSTRACT: Mesorhizobium huakuii 7653R is an α-proteobacterium that occurs either in a nitrogen-fixing symbiosis with its host plant, A. sinicus, or free-living in the soil. We performed RNA-Seq on free-living cells grown in rich media and in bacteroids isolated from root nodules to understand how M. huakuii 7653R responds to alterations in its environment and to the physiological changes that occur during bacteroid differentiation. Understand how M. huakuii 7653R responds to alterations in its environment and to the physiological changes that occur during bacteroid differentiation.

ORGANISM(S): Mesorhizobium huakuii 7653R

PROVIDER: GSE48103 | GEO | 2014/06/30

SECONDARY ACCESSION(S): PRJNA208944

REPOSITORIES: GEO

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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:Fatty acids β-oxidation (FAO) is the main pathway of fatty acid degradation in organisms. Acyl-CoA dehydrogenase as the first major enzyme in FAO pathways, catalyzes the dehydrogenation reaction of α and β carbon atoms at the site under the action of FAD cofactor. Bioinformatics analysis showed that acdA gene in Mesorhizobium huakuii 7653R encodes the acyl-CoA dehydrogenase. M. huakuii 7653R acdA gene mutant strain and complementary strain were constructed by homologous recombination. mutant HKacdA exhibited delay in growth in AMS minimal medium with NH4Cl as nitrogen source and glucose as carbon source, and showed more sensitivity to H2O2. The function of M. huakuii acdA gene in symbiotic nitrogen fixation was studied through pot experiment of Astragalus sinicus. Mutant HKacdA was severely impaired in its ability to colonize the rhizosphere of A. sinicus. Compared with the plants inoculated with wild-type 7653R, the leaves of HKacdA-inoculated plants appeared were abnormally sparse and yellow, and exhibited obvious symptoms of nitrogen deficiency. Observation of root nodules and bacteroid morphology revealed that the nodules inoculated with HKacdA contained an abnormally thick cortex and bacteroid-containing cells, and some bacteroids accumulated poly-β-hydroxybutyrate (PHB) granules and appeared dissociation and senescent. The mass spectrometry combined with label-free technique was applied to study the differential expression of proteins in bacteroids. A total of 198 differentially expressed proteins were identifified were identifified including 120 up-regulated and 78 down-regulated proteins. Among the differentially expressed proteins, 38 proteins were related to redox，31 proteins were related to amino acid transport, and 11 proteins were connected with nitrogen fixation. Results showed that acdA gene plays a role in rhizobial bacteroid differentiation and nitrogen fixation by affecting amino acid transport, electron transport in nitrogen fixation and nitrogenase activity in nodule bacteroids.

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RNA-seq Analysis Reveal Global Differential Transcritomes of Mesorhizobium huakuii 7653R between Bacteroids and Free-living Cells

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