Role of Nitric Oxide of Bacterial Origin in the Medicago truncatula-Sinorhizobium meliloti Symbiosis.

Nitric oxide (NO) is a small ubiquitous gaseous molecule that has been found in many host-pathogen interactions. NO has been shown to be part of the defense arsenal of animal cells and more recently of plant cells. To fight this molecular weapon, pathogens have evolved responses consisting of adaptation to NO or degradation of this toxic molecule. More recently, it was shown that NO could also be produced by the pathogen and contributes likewise to the success of the host cell infection. NO is also present during symbiotic interactions. Despite growing knowledge about the role of NO during friendly interactions, data on the specificity of action of NO produced by each partner are scarce, partly due to the multiplicity of NO production systems. In the nitrogen-fixing symbiosis between the soil bacterium Sinorhizobium meliloti and the model legume Medicago truncatula, NO has been detected at all steps of the interaction, where it displays various roles. Both partners contribute to NO production inside the legume root nodules where nitrogen fixation occurs. The study focuses on the role of bacterial NO in this interaction. We used a genetic approach to identify bacterial NO sources in the symbiotic context and to test the phenotype in planta of bacterial mutants affected in NO production. Our results show that only denitrification is a source of bacterial NO in Medicago nodules, giving insight into the role of bacteria-derived NO at different steps of the symbiotic interaction. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A dual legume-rhizobium transcriptome of symbiotic nodule senescence reveals coordinated plant and bacterial responses.

Senescence determines plant organ lifespan depending on aging and environmental cues. During the endosymbiotic interaction with rhizobia, legume plants develop a specific organ, the root nodule, which houses nitrogen (N)-fixing bacteria. Unlike earlier processes of the legume-rhizobium interaction (nodule formation, N fixation), mechanisms controlling nodule senescence remain poorly understood. To identify nodule senescence-associated genes, we performed a dual plant-bacteria RNA sequencing approach on Medicago truncatula-Sinorhizobium meliloti nodules having initiated senescence either naturally (aging) or following an environmental trigger (nitrate treatment or salt stress). The resulting data allowed the identification of hundreds of plant and bacterial genes differentially regulated during nodule senescence, thus providing an unprecedented comprehensive resource of new candidate genes associated with this process. Remarkably, several plant and bacterial genes related to the cell cycle and stress responses were regulated in senescent nodules, including the rhizobial RpoE2-dependent general stress response. Analysis of selected core nodule senescence plant genes allowed showing that MtNAC969 and MtS40, both homologous to leaf senescence-associated genes, negatively regulate the transition between N fixation and senescence. In contrast, overexpression of a gene involved in the biosynthesis of cytokinins, well-known negative regulators of leaf senescence, may promote the transition from N fixation to senescence in nodules.

Stress-inducible NHEJ in bacteria: function in DNA repair and acquisition of heterologous DNA.

DNA double-strand breaks (DSB) in bacteria can be repaired by non-homologous end-joining (NHEJ), a two-component system relying on Ku and LigD. While performing a genetic characterization of NHEJ in Sinorhizobium meliloti, a representative of bacterial species encoding several Ku and LigD orthologues, we found that at least two distinct functional NHEJ repair pathways co-exist: one is dependent on Ku2 and LigD2, while the other depends on Ku3, Ku4 and LigD4. Whereas Ku2 likely acts as canonical bacterial Ku homodimers, genetic evidences suggest that Ku3-Ku4 form eukaryotic-like heterodimers. Strikingly, we found that the efficiency of both NHEJ systems increases under stress conditions, including heat and nutrient starvation. We found that this stimulation results from the transcriptional up-regulation of the ku and/or ligD genes, and that some of these genes are controlled by the general stress response regulator RpoE2. Finally, we provided evidence that NHEJ not only repairs DSBs, but can also capture heterologous DNA fragments into genomic breaks. Our data therefore suggest that NHEJ could participate to horizontal gene transfer from distantly related species, bypassing the need of homology to integrate exogenous DNA. This supports the hypothesis that NHEJ contributes to evolution and adaptation of bacteria under adverse environmental conditions.

A partner-switching system controls activation of mixed-linkage ß-glucan synthesis by c-di-GMP in Sinorhizobium meliloti.

Sinorhizobium meliloti synthesizes a linear mixed-linkage (1 → 3)(1 → 4)-ß-d-glucan (ML ß-glucan, MLG) in response to high levels of cyclic diguanylate (c-di-GMP). Two proteins BgsA and BgsB are required for MLG synthesis, BgsA being the glucan synthase which is activated upon c-di-GMP binding to its C-terminal domain. Here we report that the product of bgrR (SMb20447) is a diguanylate cyclase (DGC) that provides c-di-GMP for the synthesis of MLG by BgsA. bgrR is the first gene of a hexacistronic bgrRSTUWV operon, likely encoding a partner-switching regulatory network where BgrR is the final target. Using different approaches, we have determined that the products of genes bgrU (containing a putative PP2C serine phosphatase domain) and bgrW (with predicted kinase effector domain), modulate the phosphorylation status and the activity of the STAS domain protein BgrV. We propose that unphosphorylated BgrV inhibits BgrR DGC activity, perhaps through direct protein-protein interactions as established for other partner switchers. A bgrRSTUWV operon coexists with MLG structural bgsBA genes in many rhizobial genomes but is also present in some MLG non-producers, suggesting a role of this partner-switching system in other processes besides MLG biosynthesis.

DNA double-strand break repair is involved in desiccation resistance of Sinorhizobium meliloti, but is not essential for its symbiotic interaction with Medicago truncatula.

The soil bacterium Sinorhizobium meliloti, a nitrogen-fixing symbiont of legume plants, is exposed to numerous stress conditions in nature, some of which cause the formation of harmful DNA double-strand breaks (DSBs). In particular, the reactive oxygen species (ROS) and the reactive nitrogen species (RNS) produced during symbiosis, and the desiccation occurring in dry soils, are conditions which induce DSBs. Two major systems of DSB repair are known in S. meliloti: homologous recombination (HR) and non-homologous end-joining (NHEJ). However, their role in the resistance to ROS, RNS and desiccation has never been examined in this bacterial species, and the importance of DSB repair in the symbiotic interaction has not been properly evaluated. Here, we constructed S. meliloti strains deficient in HR (by deleting the recA gene) or in NHEJ (by deleting the four ku genes) or both. Interestingly, we observed that ku and/or recA genes are involved in S. meliloti resistance to ROS and RNS. Nevertheless, an S. meliloti strain deficient in both HR and NHEJ was not altered in its ability to establish and maintain an efficient nitrogen-fixing symbiosis with Medicago truncatula, showing that rhizobial DSB repair is not essential for this process. This result suggests either that DSB formation in S. meliloti is efficiently prevented during symbiosis or that DSBs are not detrimental for symbiosis efficiency. In contrast, we found for the first time that both recA and ku genes are involved in S. meliloti resistance to desiccation, suggesting that DSB repair could be important for rhizobium persistence in the soil.

An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing.

Rhizobium-induced root nodules are specialized organs for symbiotic nitrogen fixation. Indeterminate-type nodules are formed from an apical meristem and exhibit a spatial zonation which corresponds to successive developmental stages. To get a dynamic and integrated view of plant and bacterial gene expression associated with nodule development, we used a sensitive and comprehensive approach based upon oriented high-depth RNA sequencing coupled to laser microdissection of nodule regions. This study, focused on the association between the model legume Medicago truncatula and its symbiont Sinorhizobium meliloti, led to the production of 942 million sequencing read pairs that were unambiguously mapped on plant and bacterial genomes. Bioinformatic and statistical analyses enabled in-depth comparison, at a whole-genome level, of gene expression in specific nodule zones. Previously characterized symbiotic genes displayed the expected spatial pattern of expression, thus validating the robustness of our approach. We illustrate the use of this resource by examining gene expression associated with three essential elements of nodule development, namely meristem activity, cell differentiation and selected signaling processes related to bacterial Nod factors and redox status. We found that transcription factor genes essential for the control of the root apical meristem were also expressed in the nodule meristem, while the plant mRNAs most enriched in nodules compared with roots were mostly associated with zones comprising both plant and bacterial partners. The data, accessible on a dedicated website, represent a rich resource for microbiologists and plant biologists to address a variety of questions of both fundamental and applied interest.

A putative bifunctional histidine kinase/phosphatase of the HWE family exerts positive and negative control on the Sinorhizobium meliloti general stress response.

The EcfG-type sigma factor RpoE2 is the regulator of the general stress response in Sinorhizobium meliloti. RpoE2 activity is negatively regulated by two NepR-type anti-sigma factors (RsiA1/A2), themselves under the control of two anti-anti-sigma factors (RsiB1/B2) belonging to the PhyR family of response regulators. The current model of RpoE2 activation suggests that in response to stress, RsiB1/B2 are activated by phosphorylation of an aspartate residue in their receiver domain. Once activated, RsiB1/B2 become able to interact with the anti-sigma factors and release RpoE2, which can then associate with the RNA polymerase to transcribe its target genes. The purpose of this work was to identify and characterize proteins involved in controlling the phosphorylation status of RsiB1/B2. Using in vivo approaches, we show that the putative histidine kinase encoded by the rsiC gene (SMc01507), located downstream from rpoE2, is able to both positively and negatively regulate the general stress response. In addition, our data suggest that the negative action of RsiC results from inhibition of RsiB1/B2 phosphorylation. From these observations, we propose that RsiC is a bifunctional histidine kinase/phosphatase responsible for RsiB1/B2 phosphorylation or dephosphorylation in the presence or absence of stress, respectively. Two proteins were previously proposed to control PhyR phosphorylation in Caulobacter crescentus and Sphingomonas sp. strain FR1. However, these proteins contain a Pfam:HisKA_2 domain of dimerization and histidine phosphotransfer, whereas S. meliloti RsiC harbors a Pfam:HWE_HK domain instead. Therefore, this is the first report of an HWE_HK-containing protein controlling the general stress response in Alphaproteobacteria.

Dual control of Sinorhizobium meliloti RpoE2 sigma factor activity by two PhyR-type two-component response regulators.

RpoE2 is an extracytoplasmic function (ECF) sigma factor involved in the general stress response of Sinorhizobium meliloti, the nitrogen-fixing symbiont of the legume plant alfalfa. RpoE2 orthologues are widely found among alphaproteobacteria, where they play various roles in stress resistance and/or host colonization. In this paper, we report a genetic and biochemical investigation of the mechanisms of signal transduction leading to S. meliloti RpoE2 activation in response to stress. We showed that RpoE2 activity is negatively controlled by two paralogous anti-sigma factors, RsiA1 (SMc01505) and RsiA2 (SMc04884), and that RpoE2 activation by stress requires two redundant paralogous PhyR-type response regulators, RsiB1 (SMc01504) and RsiB2 (SMc00794). RsiB1 and RsiB2 do not act at the level of rpoE2 transcription but instead interact with the anti-sigma factors, and we therefore propose that they act as anti-anti-sigma factors to relieve RpoE2 inhibition in response to stress. This model closely resembles a recently proposed model of activation of RpoE2-like sigma factors in Methylobacterium extorquens and Bradyrhizobium japonicum, but the existence of two pairs of anti- and anti-anti-sigma factors in S. meliloti adds an unexpected level of complexity, which may allow the regulatory system to integrate multiple stimuli.

The Nitrate Assimilatory Pathway in Sinorhizobium meliloti: Contribution to NO Production.

The interaction between rhizobia and their legume host plants culminates in the formation of specialized root organs called nodules in which differentiated endosymbiotic bacteria (bacteroids) fix atmospheric nitrogen to the benefit of the plant. Interestingly, nitric oxide (NO) has been detected at various steps of the rhizobium-legume symbiosis where it has been shown to play multifaceted roles. It is recognized that both bacterial and plant partners of the Sinorhizobium meliloti-Medicago truncatula symbiosis are involved in NO synthesis in nodules. S. meliloti can also produce NO from nitrate when living as free cells in the soil. S. meliloti does not possess any NO synthase gene in its genome. Instead, the denitrification pathway is often described as the main driver of NO production with nitrate as substrate. This pathway includes the periplasmic nitrate reductase (Nap) which reduces nitrate into nitrite, and the nitrite reductase (Nir) which reduces nitrite into NO. However, additional genes encoding putative nitrate and nitrite reductases (called narB and nirB, respectively) have been identified in the S. meliloti genome. Here we examined the conditions where these genes are expressed, investigated their involvement in nitrate assimilation and NO synthesis in culture and their potential role in planta. We found that narB and nirB are expressed under aerobic conditions in absence of ammonium in the medium and most likely belong to the nitrate assimilatory pathway. Even though these genes are clearly expressed in the fixation zone of legume root nodule, they do not play a crucial role in symbiosis. Our results support the hypothesis that in S. meliloti, denitrification remains the main enzymatic way to produce NO while the assimilatory pathway involving NarB and NirB participates indirectly to NO synthesis by cooperating with the denitrification pathway.

Response of Sinorhizobium meliloti to elevated concentrations of cadmium and zinc.

Whole-genome transcriptional profiling was used to identify genes in Sinorhizobium meliloti 1021 that are differentially expressed during exposure to elevated concentrations of cadmium and zinc. Mutant strains with insertions in metal-regulated genes and in genes encoding putative metal efflux pumps were analyzed for their metal sensitivities, revealing a crucial role for the SMc04128-encoded P-type ATPase in the defense of S. meliloti against cadmium and zinc stress.

Next-generation annotation of prokaryotic genomes with EuGene-P: application to Sinorhizobium meliloti 2011.

The availability of next-generation sequences of transcripts from prokaryotic organisms offers the opportunity to design a new generation of automated genome annotation tools not yet available for prokaryotes. In this work, we designed EuGene-P, the first integrative prokaryotic gene finder tool which combines a variety of high-throughput data, including oriented RNA-Seq data, directly into the prediction process. This enables the automated prediction of coding sequences (CDSs), untranslated regions, transcription start sites (TSSs) and non-coding RNA (ncRNA, sense and antisense) genes. EuGene-P was used to comprehensively and accurately annotate the genome of the nitrogen-fixing bacterium Sinorhizobium meliloti strain 2011, leading to the prediction of 6308 CDSs as well as 1876 ncRNAs. Among them, 1280 appeared as antisense to a CDS, which supports recent findings that antisense transcription activity is widespread in bacteria. Moreover, 4077 TSSs upstream of protein-coding or non-coding genes were precisely mapped providing valuable data for the study of promoter regions. By looking for RpoE2-binding sites upstream of annotated TSSs, we were able to extend the S. meliloti RpoE2 regulon by ∼3-fold. Altogether, these observations demonstrate the power of EuGene-P to produce a reliable and high-resolution automatic annotation of prokaryotic genomes.

Sinorhizobium meliloti sigma factors RpoE1 and RpoE4 are activated in stationary phase in response to sulfite.

Rhizobia are soil bacteria able to establish a nitrogen-fixing symbiosis with legume plants. Both in soil and in planta, rhizobia spend non-growing periods resembling the stationary phase of in vitro-cultured bacteria. The primary objective of this work was to better characterize gene regulation in this biologically relevant growth stage in Sinorhizobium meliloti. By a tap-tag/mass spectrometry approach, we identified five sigma factors co-purifying with the RNA polymerase in stationary phase: the general stress response regulator RpoE2, the heat shock sigma factor RpoH2, and three extra-cytoplasmic function sigma factors (RpoE1, RpoE3 and RpoE4) belonging to the poorly characterized ECF26 subgroup. We then showed that RpoE1 and RpoE4 i) are activated upon metabolism of sulfite-generating compounds (thiosulfate and taurine), ii) display overlapping regulatory activities, iii) govern a dedicated sulfite response by controlling expression of the sulfite dehydrogenase SorT, iv) are activated in stationary phase, likely as a result of endogenous sulfite generation during bacterial growth. We showed that SorT is required for optimal growth of S. meliloti in the presence of sulfite, suggesting that the response governed by RpoE1 and RpoE4 may be advantageous for bacteria in stationary phase either by providing a sulfite detoxification function or by contributing to energy production through sulfite respiration. This paper therefore reports the first characterization of ECF26 sigma factors, the first description of sigma factors involved in control of sulphur metabolism, and the first indication that endogenous sulfite may act as a signal for regulation of gene expression upon entry of bacteria in stationary phase.

An extracytoplasmic function sigma factor acts as a general stress response regulator in Sinorhizobium meliloti.

Sinorhizobium meliloti genes transcriptionally up-regulated after heat stress, as well as upon entry into stationary phase, were identified by microarray analyses. Sixty stress response genes were thus found to be up-regulated under both conditions. One of them, rpoE2 (smc01506), encodes a putative extracytoplasmic function (ECF) sigma factor. We showed that this sigma factor controls its own transcription and is activated by various stress conditions, including heat and salt, as well as entry into stationary phase after either carbon or nitrogen starvation. We also present evidence that the product of the gene cotranscribed with rpoE2 negatively regulates RpoE2 activity, and we therefore propose that it plays the function of anti-sigma factor. By combining transcriptomic, bioinformatic, and quantitative reverse transcription-PCR analyses, we identified 44 RpoE2-controlled genes and predicted the number of RpoE2 targets to be higher. Strikingly, more than one-third of the 60 stress response genes identified in this study are RpoE2 targets. Interestingly, two genes encoding proteins with known functions in stress responses, namely, katC and rpoH2, as well as a second ECF-encoding gene, rpoE5, were found to be RpoE2 regulated. Altogether, these data suggest that RpoE2 is a major global regulator of the general stress response in S. meliloti. Despite these observations, and although this sigma factor is well conserved among alphaproteobacteria, no in vitro nor in planta phenotypic difference from the wild-type strain could be detected for rpoE2 mutants. This therefore suggests that other important actors in the general stress response have still to be identified in S. meliloti.

AP2-ERF transcription factors mediate Nod factor dependent Mt ENOD11 activation in root hairs via a novel cis-regulatory motif.

Rhizobium Nod factors (NFs) are specific lipochitooligosaccharides that activate host legume signaling pathways essential for initiating the nitrogen-fixing symbiotic association. This study describes the characterization of cis-regulatory elements and trans-interacting factors that regulate NF-dependent and epidermis-specific gene transcription in Medicago truncatula. Detailed analysis of the Mt ENOD11 promoter using deletion, mutation, and gain-of-function constructs has led to the identification of an NF-responsive regulatory unit (the NF box) sufficient to direct NF-elicited expression in root hairs. NF box-mediated expression requires a major GCC-like motif, which is also essential for the binding of root hair-specific nuclear factors. Yeast one-hybrid screening has identified three closely related AP2/ERF transcription factors (ERN1 to ERN3) that are able to bind specifically to the NF box. ERN1 is identical to an ERF-like factor identified recently. Expression analysis has revealed that ERN1 and ERN2 genes are upregulated in root hairs following NF treatment and that this activation requires a functional NFP gene. Transient expression assays in Nicotiana benthamiana have further shown that nucleus-targeted ERN1 and ERN2 factors activate NF box-containing reporters, whereas ERN3 represses ERN1/ERN2-dependent transcription activation. A model is proposed for the fine-tuning of NF-elicited gene transcription in root hairs involving the interplay between repressor and activator ERN factors.

Transcript enrichment of Nod factor-elicited early nodulin genes in purified root hair fractions of the model legume Medicago truncatula.

This article describes an efficient procedure to study Nod factor-induced gene expression in root hairs of the model legume Medicago truncatula. By developing an improved method of fracturing frozen root hairs, it has been possible to obtain a highly purified root hair fraction from M. truncatula seedlings yielding sufficient RNA for real-time quantitative RT-PCR expression analysis. After Nod factor treatment it was possible to detect up to 100-fold increases of MtENOD11 and pMtENOD11-gus transcript levels in root hair RNA. This corresponds to 5-7-fold higher induction levels than for entire root tissue preparations. Furthermore, the use of these enriched RNA samples has revealed for the first time a very significant induction (30-fold) of the MtENOD40 gene in root hairs in response to Nod factors. It is concluded that the rapid and convenient procedure described here will be particularly useful for detecting tissue-specific low-level gene expression in root hairs responding to Rhizobium Nod factors or other exogenous signals.

ERECTA, an LRR receptor-like kinase protein controlling development pleiotropically affects resistance to bacterial wilt.

Bacterial wilt, one of the most devastating bacterial diseases of plants worldwide, is caused by Ralstonia solanacearum and affects many important crop species. We show that several strains isolated from solanaceous crops in Europe are pathogenic in different accessions of Arabidopsis thaliana. One of these strains, 14.25, causes wilting symptoms in A. thaliana accession Landsberg erecta (Ler) and no apparent symptoms in accession Columbia (Col-0). Disease development and bacterial multiplication in the susceptible Ler accession depend on functional hypersensitive response and pathogenicity (hrp) genes, key elements for bacterial pathogenicity. Genetic analysis using Ler x Col-0 recombinant inbred lines showed that resistance is governed by at least three loci: QRS1 (Quantitative Resistance to R. solanacearum) and QRS2 on chromosome 2, and QRS3 on chromosome 5. These loci explain about 90% of the resistance carried by the Col-0 accession. The ERECTA gene, which encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) and affects development of aerial organs, is dimorphic in our population and lies close to QRS1. Susceptible Ler plants transformed with a wild-type ERECTA gene, and the LER line showed increased disease resistance to R. solanacearum as indicated by reduced wilt symptoms and impaired bacterial growth, suggesting unexpected cross-talk between resistance and developmental pathways.