Differential gene expression of salt-tolerant alfalfa in response to salinity and inoculation by Ensifer meliloti.

BACKGROUND: Alfalfa (Medicago sativa L.) experiences many negative effects under salinity stress, which may be mediated by recurrent selection. Salt-tolerant alfalfa may display unique adaptations in association with rhizobium under salt stress. RESULTS: To elucidate inoculation effects on salt-tolerant alfalfa under salt stress, this study leveraged a salt-tolerant alfalfa population selected through two cycles of recurrent selection under high salt stress. After experiencing 120-day salt stress, mRNA was extracted from 8 random genotypes either grown in 0 or 8 dS/m salt stress with or without inoculation by Ensifer meliloti. Results showed 320 and 176 differentially expressed genes (DEGs) modulated in response to salinity stress or inoculation x salinity stress, respectively. Notable results in plants under 8 dS/m stress included upregulation of a key gene involved in the Target of Rapamycin (TOR) signaling pathway with a concomitant decrease in expression of the SNrK pathway. Inoculation of salt-stressed plants stimulated increased transcription of a sulfate-uptake gene as well as upregulation of the Lysine-27-trimethyltransferase (EZH2), Histone 3 (H3), and argonaute (AGO, a component of miRISC silencing complexes) genes related to epigenetic and post-transcriptional gene control. CONCLUSIONS: Salt-tolerant alfalfa may benefit from improved activity of TOR and decreased activity of SNrK1 in salt stress, while inoculation by rhizobiumstimulates production of sulfate uptake- and other unique genes.

The symbiovar mediterranense of Sinorhizobium meliloti nodulates Phaseolus vulgaris across Lanzarote (Canary Islands): A revision of this symbiovar supports a proposal to delimit symbiovars boundaries in Sinorhizobium and to define four new symbiovars.

The symbiovar mediterranense of Sinorhizobium meliloti was initially found in Phaseolus vulgaris nodules in Tunisia and in an eastern location of Lanzarote (Canary Islands). Here we show that the symbiovar mediterranense of S. meliloti also nodulates P. vulgaris in two western locations of this Island. The analyses of the symbiotic nodA and nodC genes reveal the complexity of the symbiovar mediterranense which encompasses strains belonging to several phylogenetic lineages and clusters. The comparison of the nodA and nodC phylogenies showed that the nodC was the most resolutive phylogenetic marker for the delineation of Sinorhizobium symbiovars. Considering that the similarity of this gene within several symbiovars, particularly mediterranense, is around 95 %, the cut-off value for their differentiation should be lower. Considering that a nodC gene cut-off similarity value of around 92 % is accepted for the genus Bradyrhizobium and that the symbiovar concept is identical in all rhizobial genera, we propose to apply this value for symbiovars delineation within all these genera. Therefore, using this cut-off value for the nodC gene analysis of Sinorhizobium symbiovars, we propose to merge the symbiovars aegeanense and fredii into the single symbiovar fredii and to define four novel symbiovars with the names asiaense, culleni, sudanense and tunisiaense.

Genotype-by-genotype interkingdom cross-talk between symbiotic nitrogen fixing Sinorhizobium meliloti strains and Trichoderma species.

In the understanding of the molecular interaction between plants and their microbiome, a key point is to identify simplified models of the microbiome including relevant bacterial and fungal partners which could also be effective in plant growth promotion. Here, as proof-of-concept, we aim to identify the possible molecular interactions between symbiotic nitrogen-fixing rhizobia and soil fungi (Trichoderma spp.), hence shed light on synergistic roles rhizospheric fungi could have in the biology of symbiotic nitrogen fixation bacteria. We selected 4 strains of the model rhizobium Sinorhizobium meliloti and 4 Trichoderma species (T. velutinum, T. tomentosum, T. gamsii and T. harzianum). In an experimental scheme of 4 ×4 strains x species combinations, we investigated the rhizobia physiological and transcriptomic responses elicited by fungal spent media, as well as spent media effects on rhizobia-host legume plant (alfalfa, Medicago sativa L.) symbiosis. Fungal spent media had large effects on rhizobia, specific for each fungal species and rhizobial strains combination, indicating a generalized rhizobia genotype x fungal genotype interaction, including synergistic, neutral and antagonistic effects on alfalfa symbiotic phenotypes. Differential expression of a high number of genes was shown in rhizobia strains with up to 25% of total genes differentially expressed upon treatment of cultures with fungal spent media. Percentages over total genes and type of genes differentially expressed changed according to both fungal species and rhizobial strain. To support the hypothesis of a relevant rhizobia genotype x fungal genotype interaction, a nested Likelihood Ratio Test indicated that the model considering the fungus-rhizobium interaction explained 23.4% of differentially expressed genes. Our results provide insights into molecular interactions involving nitrogen-fixing rhizobia and rhizospheric fungi, highlighting the panoply of genes and genotypic interactions (fungus, rhizobium, host plant) which may concur to plant symbiosis.

Insights into the Impact of Trans-Zeatin Overproduction-Engineered Sinorhizobium meliloti on Alfalfa (Medicago sativa L.) Tolerance to Drought Stress.

Plant growth-promoting rhizobacteria have been shown to enhance plant tolerance to drought stress through various mechanisms. However, there is limited research on improving drought resistance in alfalfa by genetically modifying PGPR to produce increased levels of cytokinins. Herein, we employed synthetic biology approaches to engineer two novel strains of Sinorhizobium meliloti capable of overproducing trans-Zeatin and investigated their potential in enhancing drought tolerance in alfalfa. Our results demonstrate that alfalfa plants inoculated with these engineered S. meliloti strains exhibited reduced wilting and yellowing while maintaining higher relative water content under drought conditions. The engineered S. meliloti-induced tZ activated the activity of antioxidant enzymes and the accumulation of osmolytes. Additionally, the increased endogenous tZ content in plants alleviated the impact of drought stress on the alfalfa photosynthetic rate. However, under nondrought conditions, inoculation with the engineered S. meliloti strains had no significant effect on alfalfa biomass and nodule formation.

Getting to the point: unipolar growth of Hyphomicrobiales.

The governing principles and suites of genes for lateral elongation or incorporation of new cell wall material along the length of a rod-shaped cell are well described. In contrast, relatively little is known about unipolar elongation or incorporation of peptidoglycan at one end of the rod. Recent work in three related model systems of unipolar growth (Agrobacterium tumefaciens, Brucella abortus, and Sinorhizobium meliloti) has clearly established that unipolar growth in the Hyphomicrobiales order relies on a set of genes distinct from the canonical elongasome. Polar incorporation of envelope components relies on homologous proteins shared by the Hyphomicrobiales, reviewed here. Ongoing and future work will reveal how unipolar growth is integrated into the alphaproteobacterial cell cycle and coordinated with other processes such as chromosome segregation and cell division.

The swimming defect caused by the absence of the transcriptional regulator LdtR in Sinorhizobium meliloti is restored by mutations in the motility genes motA and motS.

The flagellar motor is a powerful macromolecular machine used to propel bacteria through various environments. We determined that flagellar motility of the alpha-proteobacterium Sinorhizobium meliloti is nearly abolished in the absence of the transcriptional regulator LdtR, known to influence peptidoglycan remodeling and stress response. LdtR does not regulate motility gene transcription. Remarkably, the motility defects of the ΔldtR mutant can be restored by secondary mutations in the motility gene motA or a previously uncharacterized gene in the flagellar regulon, which we named motS. MotS is not essential for S. meliloti motility and may serve an accessory role in flagellar motor function. Structural modeling predicts that MotS comprised an N-terminal transmembrane segment, a long-disordered region, and a conserved ß-sandwich domain. The C terminus of MotS is localized in the periplasm. Genetics based substitution of MotA with MotAG12S also restored the ΔldtR motility defect. The MotAG12S variant protein features a local polarity shift at the periphery of the MotAB stator units. We propose that MotS may be required for optimal alignment of stators in wild-type flagellar motors but becomes detrimental in cells with altered peptidoglycan. Similarly, the polarity shift in stator units composed of MotB/MotAG12S might stabilize its interaction with altered peptidoglycan.

Co-inoculation with novel nodule-inhabiting bacteria reduces the benefits of legume-rhizobium symbiosis.

The ecologically and economically vital symbiosis between nitrogen-fixing rhizobia and leguminous plants is often thought of as a bi-partite interaction, yet studies increasingly show the prevalence of non-rhizobial endophytes (NREs) that occupy nodules alongside rhizobia. Yet, what impact these NREs have on plant or rhizobium fitness remains unclear. Here, we investigated four NRE strains found to naturally co-occupy nodules of the legume Medicago truncatula alongside Sinorhizobium meliloti in native soils. Our objectives were to (1) examine the direct and indirect effects of NREs on M. truncatula and S. meliloti fitness, and (2) determine whether NREs can re-colonize root and nodule tissues upon reinoculation. We identified one NRE strain (522) as a novel Paenibacillus species, another strain (717A) as a novel Bacillus species, and the other two (702A and 733B) as novel Pseudomonas species. Additionally, we found that two NREs (Bacillus 717A and Pseudomonas 733B) reduced the fitness benefits obtained from symbiosis for both partners, while the other two (522, 702A) had little effect. Lastly, we found that NREs were able to co-infect host tissues alongside S. meliloti. This study demonstrates that variation of NREs present in natural populations must be considered to better understand legume-rhizobium dynamics in soil communities.

Phosphatidylcholine-deficient suppressor mutant of Sinorhizobium meliloti, altered in fatty acid synthesis, partially recovers nodulation ability in symbiosis with alfalfa (Medicago sativa).

Rhizobial phosphatidylcholine (PC) is thought to be a critical phospholipid for the symbiotic relationship between rhizobia and legume host plants. A PC-deficient mutant of Sinorhizobium meliloti overproduces succinoglycan, is unable to swim, and lacks the ability to form nodules on alfalfa (Medicago sativa) host roots. Suppressor mutants had been obtained which did not overproduce succinoglycan and regained the ability to swim. Previously, we showed that point mutations leading to altered ExoS proteins can reverse the succinoglycan and swimming phenotypes of a PC-deficient mutant. Here, we report that other point mutations leading to altered ExoS, ChvI, FabA, or RpoH1 proteins also revert the succinoglycan and swimming phenotypes of PC-deficient mutants. Notably, the suppressor mutants also restore the ability to form nodule organs on alfalfa roots. However, nodules generated by these suppressor mutants express only low levels of an early nodulin, do not induce leghemoglobin transcript accumulation, thus remain white, and are unable to fix nitrogen. Among these suppressor mutants, we detected a reduced function mutant of the 3-hydoxydecanoyl-acyl carrier protein dehydratase FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids. This alteration of fatty acid composition probably affects lipid packing thereby partially compensating for the previous loss of PC and contributing to the restoration of membrane homeostasis.

VapC10 toxin of the legume symbiont Sinorhizobium meliloti targets tRNASer and controls intracellular lifestyle.

The soil bacterium Sinorhizobium meliloti can establish a nitrogen-fixing symbiosis with the model legume Medicago truncatula. The rhizobia induce the formation of a specialized root organ called nodule, where they differentiate into bacteroids and reduce atmospheric nitrogen into ammonia. Little is known on the mechanisms involved in nodule senescence onset and in bacteroid survival inside the infected plant cells. Although toxin-antitoxin (TA) systems have been shown to promote intracellular survival within host cells in human pathogenic bacteria, their role in symbiotic bacteria was rarely investigated. S. meliloti encodes several TA systems, mainly of the VapBC family. Here we present the functional characterization, through a multidisciplinary approach, of the VapBC10 TA system of S. meliloti. Following a mapping by overexpression of an RNase in Escherichia coli (MORE) RNA-seq analysis, we demonstrated that the VapC10 toxin is an RNase that cleaves the anticodon loop of two tRNASer. Thereafter, a bioinformatics approach was used to predict VapC10 targets in bacteroids. This analysis suggests that toxin activation triggers a specific proteome reprogramming that could limit nitrogen fixation capability and viability of bacteroids. Accordingly, a vapC10 mutant induces a delayed senescence in nodules, associated to an enhanced bacteroid survival. VapBC10 TA system could contribute to S. meliloti adaptation to symbiotic lifestyle, in response to plant nitrogen status.

Analysis of sRNAs and Their mRNA Targets in Sinorhizobium meliloti: Focus on Half-Life Determination.

Regulation of gene expression at the level of RNA and/or by regulatory RNA is an integral part of the regulatory circuits in all living cells. In bacteria, transcription and translation can be coupled, enabling regulation by transcriptional attenuation, a mechanism based on mutually exclusive structures in nascent mRNA. Transcriptional attenuation gives rise to small RNAs that are well suited to act in trans by either base pairing or ligand binding. Examples of 5'-UTR-derived sRNAs in the alpha-proteobacterium Sinorhizobium meliloti are the sRNA rnTrpL of the tryptophan attenuator and SAM-II riboswitch sRNAs. Analyses addressing RNA-based gene regulation often include measurements of steady-state levels and of half-lives of specific sRNAs and mRNAs. Using such measurements, recently we have shown that the tryptophan attenuator responds to translation inhibition by tetracycline and that SAM-II riboswitches stabilize RNA. Here we discuss our experience in using alternative RNA purification methods for analysis of sRNA and mRNA of S. meliloti. Additionally, we show that other translational inhibitors (besides tetracycline) also cause attenuation giving rise to the rnTrpL sRNA. Furthermore, we discuss the importance of considering RNA stability changes under different conditions and describe in detail a robust and fast method for mRNA half-life determination. The latter includes rifampicin treatment, RNA isolation using commercially available columns, and mRNA analysis by reverse transcription followed by quantitative PCR (RT-qPCR). The latter can be performed as a one-step procedure or in a strand-specific manner using the same commercial kit and a spike-in transcript as a reference.

A Workflow for the Functional Characterization of Noncoding RNAs in Legume Symbiotic Bacteria.

Computational comparative genomics and, later, high-throughput transcriptome profiling (RNAseq) have uncovered a plethora of small noncoding RNA species (sRNAs) with potential regulatory roles in bacteria. A large fraction of sRNAs are differentially regulated in response to different biotic and abiotic stimuli and have the ability to fine-tune posttranscriptional reprogramming of gene expression through protein-assisted antisense interactions with trans-encoded target mRNAs. However, this level of gene regulation is still understudied in most non-model bacteria. Here, we compile experimental methods to detect expression, determine 5'/3'-ends, assess transcriptional regulation, generate mutants, and validate candidate target mRNAs of trans-acting sRNAs (trans-sRNAs) identified in the nitrogen-fixing α-rhizobium Sinorhizobium meliloti. The workflow, molecular tools, and methods are suited to investigate the function of newly identified base-pairing trans-sRNAs in phylogenetically related α-rhizobia.

Photoacoustic Calorimetry Studies of O2-Sensing FixL and (R200, I209) Variants from Sinorhizobium meliloti Reveal Conformational Changes Coupled to Ligand Photodissociation from the Heme-PAS Domain.

FixL is an oxygen-sensing heme-PAS protein that regulates nitrogen fixation in the root nodules of plants. In this paper, we present the first photothermal studies of the full-length wild-type FixL protein from Sinorhizobium meliloti and the first thermodynamic profile of a full-length heme-PAS protein. Photoacoustic calorimetry studies reveal a quadriphasic relaxation for SmFixL*WT and the five variant proteins (SmFixL*R200H, SmFixL*R200Q, SmFixL*R200E, SmFixL*R200A, and SmFixL*I209M) with four intermediates from <20 ns to ∼1.5 µs associated with the photodissociation of CO from the heme. The altered thermodynamic profiles of the full-length SmFixL* variant proteins confirm that the conserved heme domain residues R200 and I209 are important for signal transduction. In contrast, the truncated heme domain, SmFixLH128-264, shows only a single, fast monophasic relaxation at <50 ns associated with the fast disruption of a salt bridge and release of CO to the solvent, suggesting that the full-length protein is necessary to observe the conformational changes that propagate the signal from the heme domain to the kinase domain.

Selenite resistance and biotransformation to SeNPs in Sinorhizobium meliloti 1021 and the synthetic promotion on alfalfa growth.

Toxic selenite, commonly found in soil and water, can be transformed by microorganisms into selenium nanoparticles (SeNPs) as part of a detoxification process. In this study, a comprehensive investigation was conducted on the resistance and biotransformation of selenite in Sinorhizobium meliloti 1021 and the synergistic impact of SeNPs and the strain on alfalfa growth promotion was explored. Strain 1021 reduced 46% of 5 mM selenite into SeNPs within 72 h. The SeNPs, composed of proteins, lipids and polysaccharides, were primarily located outside rhizobial cells and had a tendency to aggregate. Under selenite stress, many genes participated in multidrug efflux, sulfur metabolism and redox processes were significantly upregulated. Of them, four genes, namely gmc, yedE, dsh3 and mfs, were firstly identified in strain 1021 that played crucial roles in selenite biotransformation and resistance. Biotoxic evaluations showed that selenite had toxic effects on roots and seedlings of alfalfa, while SeNPs exhibited antioxidant properties, promoted growth, and enhanced plant's tolerance to salt stress. Overall, our research provides novel insights into selenite biotransformation and resistance mechanisms in rhizobium and highlights the potential of SeNPs-rhizobium complex as biofertilizer to promote legume growth and salt tolerance.

Targeted mutagenesis of Medicago truncatula Nodule-specific Cysteine-Rich (NCR) genes using the Agrobacterium rhizogenes-mediated CRISPR/Cas9 system.

The host-produced nodule specific cysteine-rich (NCR) peptides control the terminal differentiation of endosymbiotic rhizobia in the nodules of IRLC legumes. Although the Medicago truncatula genome encodes about 700 NCR peptides, only few of them have been proven to be crucial for nitrogen-fixing symbiosis. In this study, we applied the CRISPR/Cas9 gene editing technology to generate knockout mutants of NCR genes for which no genetic or functional data were previously available. We have developed a workflow to analyse the mutation and the symbiotic phenotype of individual nodules formed on Agrobacterium rhizogenes-mediated transgenic hairy roots. The selected NCR genes were successfully edited by the CRISPR/Cas9 system and nodules formed on knockout hairy roots showed wild type phenotype indicating that peptides NCR068, NCR089, NCR128 and NCR161 are not essential for symbiosis between M. truncatula Jemalong and Sinorhizobium medicae WSM419. We regenerated stable mutants edited for the NCR068 from hairy roots obtained by A. rhizogenes-mediated transformation. The analysis of the symbiotic phenotype of stable ncr068 mutants showed that peptide NCR068 is not required for symbiosis with S. meliloti strains 2011 and FSM-MA either. Our study reports that gene editing can help to elicit the role of certain NCRs in symbiotic nitrogen fixation.

Dual RNA-Seq Analysis Pinpoints a Balanced Regulation between Symbiosis and Immunity in Medicago truncatula-Sinorhizobium meliloti Symbiotic Nodules.

Legume-rhizobial symbiosis initiates the formation of root nodules, within which rhizobia reside and differentiate into bacteroids to convert nitrogen into ammonium, facilitating plant growth. This process raises a fundamental question: how is plant immunity modulated within nodules when exposed to a substantial number of foreign bacteria? In Medicago truncatula, a mutation in the NAD1 (Nodules with Activated Defense 1) gene exclusively results in the formation of necrotic nodules combined with activated immunity, underscoring the critical role of NAD1 in suppressing immunity within nodules. In this study, we employed a dual RNA-seq transcriptomic technology to comprehensively analyze gene expression from both hosts and symbionts in the nad1-1 mutant nodules at different developmental stages (6 dpi and 10 dpi). We identified 89 differentially expressed genes (DEGs) related to symbiotic nitrogen fixation and 89 DEGs from M. truncatula associated with immunity in the nad1-1 nodules. Concurrently, we identified 27 rhizobial DEGs in the fix and nif genes of Sinorhizobium meliloti. Furthermore, we identified 56 DEGs from S. meliloti that are related to stress responses to ROS and NO. Our analyses of nitrogen fixation-defective plant nad1-1 mutants with overactivated defenses suggest that the host employs plant immunity to regulate the substantial bacterial colonization in nodules. These findings shed light on the role of NAD1 in inhibiting the plant's immune response to maintain numerous rhizobial endosymbiosis in nodules.

A protease and a lipoprotein jointly modulate the conserved ExoR-ExoS-ChvI signaling pathway critical in Sinorhizobium meliloti for symbiosis with legume hosts.

Sinorhizobium meliloti is a model alpha-proteobacterium for investigating microbe-host interactions, in particular nitrogen-fixing rhizobium-legume symbioses. Successful infection requires complex coordination between compatible host and endosymbiont, including bacterial production of succinoglycan, also known as exopolysaccharide-I (EPS-I). In S. meliloti EPS-I production is controlled by the conserved ExoS-ChvI two-component system. Periplasmic ExoR associates with the ExoS histidine kinase and negatively regulates ChvI-dependent expression of exo genes, necessary for EPS-I synthesis. We show that two extracytoplasmic proteins, LppA (a lipoprotein) and JspA (a lipoprotein and a metalloprotease), jointly influence EPS-I synthesis by modulating the ExoR-ExoS-ChvI pathway and expression of genes in the ChvI regulon. Deletions of jspA and lppA led to lower EPS-I production and competitive disadvantage during host colonization, for both S. meliloti with Medicago sativa and S. medicae with M. truncatula. Overexpression of jspA reduced steady-state levels of ExoR, suggesting that the JspA protease participates in ExoR degradation. This reduction in ExoR levels is dependent on LppA and can be replicated with ExoR, JspA, and LppA expressed exogenously in Caulobacter crescentus and Escherichia coli. Akin to signaling pathways that sense extracytoplasmic stress in other bacteria, JspA and LppA may monitor periplasmic conditions during interaction with the plant host to adjust accordingly expression of genes that contribute to efficient symbiosis. The molecular mechanisms underlying host colonization in our model system may have parallels in related alpha-proteobacteria.

MucR from Sinorhizobium meliloti: New Insights into Its DNA Targets and Its Ability to Oligomerize.

Proteins of the MucR/Ros family play a crucial role in bacterial infection or symbiosis with eukaryotic hosts. MucR from Sinorhizobium meliloti plays a regulatory role in establishing symbiosis with the host plant, both dependent and independent of Quorum Sensing. Here, we report the first characterization of MucR isolated from Sinorhizobium meliloti by mass spectrometry and demonstrate that this protein forms higher-order oligomers in its native condition of expression by SEC-MALS. We show that MucR purified from Sinorhizobium meliloti can bind DNA and recognize the region upstream of the ndvA gene in EMSA, revealing that this gene is a direct target of MucR. Although MucR DNA binding activity was already described, a detailed characterization of Sinorhizobium meliloti DNA targets has never been reported. We, thus, analyze sequences recognized by MucR in the rem gene promoter, showing that this protein recognizes AT-rich sequences and does not require a consensus sequence to bind DNA. Furthermore, we investigate the dependence of MucR DNA binding on the length of DNA targets. Taken together, our studies establish MucR from Sinorhizobium meliloti as a member of a new family of Histone-like Nucleoid Structuring (H-NS) proteins, thus explaining the multifaceted role of this protein in many species of alpha-proteobacteria.

A signaling complex of adenylate cyclase CyaC of Sinorhizobium meliloti with cAMP and the transcriptional regulators Clr and CycR.

BACKGROUND: Adenylate cyclases (ACs) generate the second messenger cyclic AMP (cAMP), which is found in all domains of life and is involved in the regulation of various cell physiological and metabolic processes. In the plant symbiotic bacterium Sinorhizobium meliloti, synthesis of cAMP by the membrane-bound AC CyaC responds to the redox state of the respiratory chain and the respiratory quinones. However, nothing is known about the signaling cascade that is initiated by cAMP produced by CyaC. RESULTS: Here, the CRP-like transcriptional regulator Clr and the TetR-like regulator CycR (TR01819 protein) were identified to interact with CyaC using the bacterial two-hybrid system (BACTH), co-sedimentation assays, and surface plasmon resonance spectroscopy. Interaction of CycR with Clr, and of CyaC with Clr requires the presence of cAMP and of ATP, respectively, whereas that of CyaC with CycR was independent of the nucleotides. CONCLUSION: The data implicate a ternary CyaC×CycR×cAMP-Clr complex, functioning as a specific signaling cascade which is formed after activation of CyaC and synthesis of cAMP. cAMP-Clr is thought to work in complex with CycR to regulate a subset of genes of the cAMP-Clr regulon in S. meliloti.