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Antioxidant mechanism of glutaredoxins and their role in symbiotic nitrogen fixation in Rhizobium leguminosarum bv. viciae 3841

ABSTRACT: Glutaredoxins (Grx) are redoxin family proteins that reduce disulfifides and mixed disulfifides between glutathione and proteins. Rhizobium leguminosarum 3841 contains three genes coding for glutaredoxins: RL4289 (grxA) codes for a dithiolic glutaredoxin, RL2615 (grxB) codes for a monothiol glutaredoxin, while RL4261 (grxC) codes for a glutaredoxin-like NrdH protein. In this study, we have generated mutants which have interrupted one, two, or three glutaredoxin genes. These mutants had no different growth phenotypes from the wild type RL3841. Mutation of grxC did not affect R. leguminosarum antioxidant or symbiotic capacities, while grxA-derived or grxB-derived mutants decreased the antioxidant and fixation nitrogen capacity. grxA-derived mutants was severely impaired in there rhizosphere colonization, and formed maller nodules with defects of bacteroid differentiation. whereas nodules induced by grxB-derived mutants contained abnormally thick cortexs and prematurely senescent bacteroids. Surprisingly the grx triple mutant presented an enhanced defect in antioxidant and symbiotic capacities of R. leguminosarum. LC-MS/MS analysis quantitative proteomics techniques were employed to compare differential grx triple mutant root bacteroids in response to the wild type infection. 137 differentially expressed proteins were identified including 56 up-regulated and 81 down-regulated proteins. By sorting the identified proteins according to metabolic function, twenty-eight proteins were involved in transporter activity, twenty proteins were related to stress response and virulence, and sixteen proteins were related to amino acid metabolism. Overall, R. leguminosarum glutaredoxins play an important role in root nodule symbiosis by involving the functions of anti-oxidant defenses, Fe-S cluster assembly, and control of bacteroid differentiation and senescence.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Rhizobium Leguminosarum Bv. Viciae

SUBMITTER: Guojun Cheng

LAB HEAD: Guojun Cheng

PROVIDER: PXD017835 | Pride | 2020-12-02

REPOSITORIES: Pride

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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.

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Antioxidant mechanism of glutaredoxins and their role in symbiotic nitrogen fixation in Rhizobium leguminosarum bv. viciae 3841

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