H2O2 Is Required for Optimal Establishment of the Medicago sativa/Sinorhizobium meliloti Symbiosis▿

The symbiotic interaction between Medicago sativa and Sinorhizobium meliloti RmkatB++ overexpressing the housekeeping catalase katB is delayed, and this delay is combined with an enlargement of infection threads. This result provides evidence that H2O2 is required for optimal progression of infection threads through the root hairs and plant cell layers

Involvement of Glutaredoxin and Thioredoxin Systems in the Nitrogen-Fixing Symbiosis between Legumes and Rhizobia

Leguminous plants can form a symbiotic relationship with Rhizobium bacteria, during which plants provide bacteria with carbohydrates and an environment appropriate to their metabolism, in return for fixed atmospheric nitrogen. The symbiotic interaction leads to the formation of a new organ, the root nodule, where a coordinated differentiation of plant cells and bacteria occurs. The establishment and functioning of nitrogen-fixing symbiosis involves a redox control important for both the plant-bacteria crosstalk and the regulation of nodule metabolism. In this review, we discuss the involvement of thioredoxin and glutaredoxin systems in the two symbiotic partners during symbiosis. The crucial role of glutathione in redox balance and S-metabolism is presented. We also highlight the specific role of some thioredoxin and glutaredoxin systems in bacterial differentiation. Transcriptomics data concerning genes encoding components and targets of thioredoxin and glutaredoxin systems in connection with the developmental step of the nodule are also considered in the model system Medicago truncatula⁻Sinorhizobium meliloti

Overexpression of BetS, a Sinorhizobium meliloti high-affinity betaine transporter, in bacteroids from Medicago sativa nodules sustains nitrogen fixation during early salt stress adaptation

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Redox-sensitive fluorescent biosensors detect Sinorhizobium meliloti intracellular redox changes under free-living and symbiotic lifestyles

International audienceReactive oxygen species such as hydrogen peroxide (H2O2) are key signaling molecules that control the setup and functioning of Rhizobium-legume symbiosis. This interaction results in the formation of a new organ, the root nodule, in which bacteria enter the host cells and differentiate into nitrogen (N2)-fixing bacteroids. The interaction between Sinorhizobium meliloti and Medicago truncatula is a genetic model to study N2-fixing symbiosis. In previous work, S. meliloti mutants impaired in the antioxidant defense, showed altered symbiotic properties, emphasizing the importance of redox-based regulation in the bacterial partner. However, direct measurements of S. meliloti intracellular redox state have never been performed. Here, we measured dynamic changes of intracellular H2O2 and glutathione redox potential by expressing roGFP2-Orp1 and Grx1-roGFP2 biosensors in S. meliloti. Kinetic analyses of redox changes under free-living conditions showed that these biosensors are suitable to monitor the bacterial redox state in real-time, after H2O2 challenge and in different genetic backgrounds. In planta, flow cytometry and confocal imaging experiments allowed the determination of sensor oxidation state in nodule bacteria. These cellular studies establish the existence of an oxidative shift in the redox status of S. meliloti during bacteroid differentiation. Our findings open up new possibilities for in vivo studies of redox dynamics during N2-fixing symbiosis

Role of the fixGHI region of Azorhizobium caulinodans in free-living and symbiotic nitrogen fixation

International audienceA 19-kb DNA region containing genes sharing homology with Rhizobium meliloti fixNOQP and fixGHI was isolated from a genomic library of Azorhizobium caulinodans. Identity of fixG was confirmed by partial nucleotide sequencing. Mutant strains in the fixGHI region were constructed by deletion or Tn5 insertions. In contrast with the situation in R. meliloti, the mutants still displayed a significant nitrogenase activity in symbiosis

The Sinorhizobium meliloti ABC Transporter Cho Is Highly Specific for Choline and Expressed in Bacteroids from Medicago sativa Nodules

In Sinorhizobium meliloti, choline is the direct precursor of phosphatidylcholine, a major lipid membrane component in the Rhizobiaceae family, and glycine betaine, an important osmoprotectant. Moreover, choline is an efficient energy source which supports growth. Using a PCR strategy, we identified three chromosomal genes (choXWV) which encode components of an ABC transporter: ChoX (binding protein), ChoW (permease), and ChoV (ATPase). Whereas the best homology scores were obtained with components of betaine ProU-like systems, Cho is not involved in betaine transport. Site-directed mutagenesis of choX strongly reduced (60 to 75%) the choline uptake activity, and purification of ChoX, together with analysis of the ligand-binding specificity, showed that ChoX binds choline with a high affinity (K(D), 2.7 μM) and acetylcholine with a low affinity (K(D), 145 μM) but binds none of the betaines. Uptake competition experiments also revealed that ectoine, various betaines, and choline derivatives were not effective competitors for Cho-mediated choline transport. Thus, Cho is a highly specific high-affinity choline transporter. Choline transport activity and ChoX expression were induced by choline but not by salt stress. Western blotting experiments with antibodies raised against ChoX demonstrated the presence of ChoX in bacteroids isolated from nitrogen-fixing nodules obtained from Medicago sativa roots. The choX mutation did not have an effect on growth under standard conditions, and neither Nod nor Fix phenotypes were impaired in the mutant, suggesting that the remaining choline uptake system(s) still present in the mutant strain can compensate for the lack of Cho transporter

Reactive oxygen and nitrogen species and glutathione: key players in the legume - Rhizobium symbiosis

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Poly-β-Hydroxybutyrate Turnover in Azorhizobium caulinodans Is Required for Growth and Affects nifA Expression

Azorhizobium caulinodans is able to fix nitrogen in the free-living state and in symbiosis with the tropical legume Sesbania rostrata. The bacteria accumulate poly-β-hydroxybutyrate (PHB) under both conditions. The structural gene for PHB synthase, phbC, was inactivated by insertion of an interposon. The mutant strains obtained were devoid of PHB, impaired in their growth properties, totally devoid of nitrogenase activity ex planta (Nif(−)), and affected in nucleotide pools and induced Fix(−) nodules devoid of bacteria. The Nif(−) phenotype was the consequence of the lack of nifA transcription. Nitrogenase activity was partially restored to a phbC mutant by constitutive expression of the nifA gene. However, this constitutive nifA expression had no effect on the nucleotide content or on growth of the phbC mutant. It is suggested that PHB is required for maintaining the reducing power of the cell and therefore the bacterial growth. These observations also suggest a new control of nifA expression to adapt nitrogen fixation to the availability of carbon and reducing equivalents