The ex planta signal activity of a Medicago ribosomal uL2 protein suggests a moonlighting role in controlling secondary rhizobial infection.

We recently described a regulatory loop, which we termed autoregulation of infection (AOI), by which Sinorhizobium meliloti, a Medicago endosymbiont, downregulates the root susceptibility to secondary infection events via ethylene. AOI is initially triggered by so-far unidentified Medicago nodule signals named signal 1 and signal 1' whose transduction in bacteroids requires the S. meliloti outer-membrane-associated NsrA receptor protein and the cognate inner-membrane-associated adenylate cyclases, CyaK and CyaD1/D2, respectively. Here, we report on advances in signal 1 identification. Signal 1 activity is widespread as we robustly detected it in Medicago nodule extracts as well as in yeast and bacteria cell extracts. Biochemical analyses indicated a peptidic nature for signal 1 and, together with proteomic analyses, a universally conserved Medicago ribosomal protein of the uL2 family was identified as a candidate signal 1. Specifically, MtRPuL2A (MtrunA17Chr7g0247311) displays a strong signal activity that requires S. meliloti NsrA and CyaK, as endogenous signal 1. We have shown that MtRPuL2A is active in signaling only in a non-ribosomal form. A Medicago truncatula mutant in the major symbiotic transcriptional regulator MtNF-YA1 lacked most signal 1 activity, suggesting that signal 1 is under developmental control. Altogether, our results point to the MtRPuL2A ribosomal protein as the candidate for signal 1. Based on the Mtnf-ya1 mutant, we suggest a link between root infectiveness and nodule development. We discuss our findings in the context of ribosomal protein moonlighting.

Endosymbiotic Sinorhizobium meliloti modulate Medicago root susceptibility to secondary infection via ethylene.

A complex network of pathways coordinates nodulation and epidermal root hair infection in the symbiotic interaction between rhizobia and legume plants. Whereas nodule formation was known to be autoregulated, it was so far unclear whether a similar control is exerted on the infection process. We assessed the capacity of Medicago plants nodulated by Sinorhizobium meliloti to modulate root susceptibility to secondary bacterial infection or to purified Nod factors in split-root and volatile assays using bacterial and plant mutant combinations. Ethylene implication in this process emerged from gas production measurements, use of a chemical inhibitor of ethylene biosynthesis and of a Medicago mutant affected in ethylene signal transduction. We identified a feedback mechanism that we named AOI (for Autoregulation Of Infection) by which endosymbiotic bacteria control secondary infection thread formation by their rhizospheric peers. AOI involves activation of a cyclic adenosine 3',5'-monophosphate (cAMP) cascade in endosymbiotic bacteria, which decreases both root infectiveness and root susceptibility to bacterial Nod factors. These latter two effects are mediated by ethylene. AOI is a novel component of the complex regulatory network controlling the interaction between Sinorhizobium meliloti and its host plants that emphasizes the implication of endosymbiotic bacteria in fine-tuning the interaction.

NsrA, a Predicted ß-Barrel Outer Membrane Protein Involved in Plant Signal Perception and the Control of Secondary Infection in Sinorhizobium meliloti.

An ongoing signal exchange fine-tunes the symbiotic interactions between rhizobia and legumes, ensuring the establishment and maintenance of mutualism. In a recently identified regulatory loop, endosymbiotic Sinorhizobium meliloti exerts negative feedback on root infection in response to unknown plant cues. Upon signal perception, three bacterial adenylate cyclases (ACs) of the inner membrane, namely, CyaD1, CyaD2, and CyaK, synthesize the second messenger cAMP, which, together with the cAMP-dependent Clr transcriptional activator, activates the expression of genes involved in root infection control. The pathway that links signal perception at the surface of the cell to cytoplasmic cAMP production by ACs was thus far unknown. Here we first show that CyaK is the cognate AC for the plant signal, called signal 1, that was observed previously in mature nodule and shoot extracts. We also show that inactivation of the gene immediately upstream of cyaK, nsrA (smb20775), which encodes a ß-barrel protein of the outer membrane, abolished signal 1 perception ex planta, whereas nsrA overexpression increased signal 1 responsiveness. Inactivation of the nsrA gene abolished all Clr-dependent gene expression in nodules and led to a marked hyperinfection phenotype on plants, similar to that of a cyaD1 cyaD2 cyaK triple mutant. We suggest that the NsrA protein acts as the (co)receptor for two signal molecules, signal 1 and a hypothetical signal 1', in mature and young nodules that cooperate in controlling secondary infection in S. meliloti-Medicago symbiosis. The predicted topology and domain composition of the NsrA protein hint at a mechanism of transmembrane signaling.IMPORTANCE Symbiotic interactions, especially mutualistic ones, rely on a continuous signal exchange between the symbionts. Here we report advances regarding a recently discovered signal transduction pathway that fine-tunes the symbiotic interaction between S. meliloti and its Medicago host plant. We have identified an outer membrane protein of S. meliloti, called NsrA, that transduces Medicago plant signals to adenylate cyclases in the inner membrane, thereby triggering a cAMP signaling cascade that controls infection. Besides their relevance for the rhizobium-legume symbiosis, these findings shed light on the mechanisms of signal perception and transduction by adenylate cyclases and transmembrane signaling in bacteria.

Transcriptomic Insight in the Control of Legume Root Secondary Infection by the Sinorhizobium meliloti Transcriptional Regulator Clr.

The cAMP-dependent transcriptional regulator Clr of Sinorhizobium meliloti regulates the overall number of infection events on Medicago roots by a so-far unknown mechanism requiring smc02178, a Clr-target gene of unknown function. In order to shed light on the mode of action of Clr on infection and potentially reveal additional biological functions for Clr, we inventoried genomic Clr target genes by transcriptome profiling. We have found that Clr positively controls the synthesis of cAMP-dependent succinoglycan as well as the expression of genes involved in the synthesis of a so-far unknown polysaccharide compound. In addition, Clr activated expression of 24 genes of unknown function in addition to smc02178. Genes negatively controlled by Clr were mainly involved in swimming motility and chemotaxis. Functional characterization of two novel Clr-activated genes of unknown function, smb20495 and smc02177, showed that their expression was activated by the same plant signal as smc02178 ex planta. In planta, however, symbiotic expression of smc02177 proved independent of clr. Both smc02177 and smb20495 genes were strictly required for the control of secondary infection on M. sativa. None of the three smc02177, smc02178 and smb20495 genes were needed for plant signal perception. Altogether this work provides a refined view of the cAMP-dependent Clr regulon of S. meliloti. We specifically discuss the possible roles of smc02177, smc02178, smb20495 genes and other Clr-controlled genes in the control of secondary infection of Medicago roots.

Transient hypermutagenesis accelerates the evolution of legume endosymbionts following horizontal gene transfer.

Horizontal gene transfer (HGT) is an important mode of adaptation and diversification of prokaryotes and eukaryotes and a major event underlying the emergence of bacterial pathogens and mutualists. Yet it remains unclear how complex phenotypic traits such as the ability to fix nitrogen with legumes have successfully spread over large phylogenetic distances. Here we show, using experimental evolution coupled with whole genome sequencing, that co-transfer of imuABC error-prone DNA polymerase genes with key symbiotic genes accelerates the evolution of a soil bacterium into a legume symbiont. Following introduction of the symbiotic plasmid of Cupriavidus taiwanensis, the Mimosa symbiont, into pathogenic Ralstonia solanacearum we challenged transconjugants to become Mimosa symbionts through serial plant-bacteria co-cultures. We demonstrate that a mutagenesis imuABC cassette encoded on the C. taiwanensis symbiotic plasmid triggered a transient hypermutability stage in R. solanacearum transconjugants that occurred before the cells entered the plant. The generated burst in genetic diversity accelerated symbiotic adaptation of the recipient genome under plant selection pressure, presumably by improving the exploration of the fitness landscape. Finally, we show that plasmid imuABC cassettes are over-represented in rhizobial lineages harboring symbiotic plasmids. Our findings shed light on a mechanism that may have facilitated the dissemination of symbiotic competency among α- and ß-proteobacteria in natura and provide evidence for the positive role of environment-induced mutagenesis in the acquisition of a complex lifestyle trait. We speculate that co-transfer of complex phenotypic traits with mutagenesis determinants might frequently enhance the ecological success of HGT.

Shaping bacterial symbiosis with legumes by experimental evolution.

Nitrogen-fixing symbionts of legumes have appeared after the emergence of legumes on earth, approximately 70 to 130 million years ago. Since then, symbiotic proficiency has spread to distant genera of α- and ß-proteobacteria, via horizontal transfer of essential symbiotic genes and subsequent recipient genome remodeling under plant selection pressure. To tentatively replay rhizobium evolution in laboratory conditions, we previously transferred the symbiotic plasmid of the Mimosa symbiont Cupriavidus taiwanensis in the plant pathogen Ralstonia solanacearum, and selected spontaneous nodulating variants of the chimeric Ralstonia sp. using Mimosa pudica as a trap. Here, we pursued the evolution experiment by submitting two of the rhizobial drafts to serial ex planta-in planta (M. pudica) passages that may mimic alternating of saprophytic and symbiotic lives of rhizobia. Phenotyping 16 cycle-evolved clones showed strong and parallel evolution of several symbiotic traits (i.e., nodulation competitiveness, intracellular infection, and bacteroid persistence). Simultaneously, plant defense reactions decreased within nodules, suggesting that the expression of symbiotic competence requires the capacity to limit plant immunity. Nitrogen fixation was not acquired in the frame of this evolutionarily short experiment, likely due to the still poor persistence of final clones within nodules compared with the reference rhizobium C. taiwanensis. Our results highlight the potential of experimental evolution in improving symbiotic proficiency and for the elucidation of relationship between symbiotic capacities and elicitation of immune responses.

Biochemical and functional characterization of SpdA, a 2', 3'cyclic nucleotide phosphodiesterase from Sinorhizobium meliloti.

BACKGROUND: 3', 5'cAMP signaling in Sinorhizobium meliloti was recently shown to contribute to the autoregulation of legume infection. In planta, three adenylate cyclases CyaD1, CyaD2 and CyaK, synthesizing 3', 5'cAMP, together with the Crp-like transcriptional regulator Clr and smc02178, a gene of unknown function, are involved in controlling plant infection. RESULTS: Here we report on the characterization of a gene (smc02179, spdA) at the cyaD1 locus that we predicted to encode a class III cytoplasmic phosphodiesterase.First, we have shown that spdA had a similar pattern of expression as smc02178 in planta but did not require clr nor 3', 5'cAMP for expression.Second, biochemical characterization of the purified SpdA protein showed that, contrary to expectation, it had no detectable activity against 3', 5'cAMP and, instead, high activity against the positional isomers 2', 3'cAMP and 2', 3'cGMP.Third, we provide direct experimental evidence that the purified Clr protein was able to bind both 2', 3'cAMP and 3', 5'cAMP in vitro at high concentration. We further showed that Clr is a 3', 5'cAMP-dependent DNA-binding protein and identified a DNA-binding motif to which Clr binds. In contrast, 2', 3'cAMP was unable to promote Clr specific-binding to DNA and activate smc02178 target gene expression ex planta.Fourth, we have shown a negative impact of exogenous 2', 3'cAMP on 3', 5'cAMP-mediated signaling in vivo. A spdA null mutant was also partially affected in 3', 5'cAMP signaling. CONCLUSIONS: SpdA is a nodule-expressed 2', 3' specific phosphodiesterase whose biological function remains elusive. Circumstantial evidence suggests that SpdA may contribute insulating 3', 5'cAMP-based signaling from 2', 3' cyclic nucleotides of metabolic origin.

Queuosine biosynthesis is required for sinorhizobium meliloti-induced cytoskeletal modifications on HeLa Cells and symbiosis with Medicago truncatula.

Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton.

Experimental evolution of nodule intracellular infection in legume symbionts.

Soil bacteria known as rhizobia are able to establish an endosymbiosis with legumes that takes place in neoformed nodules in which intracellularly hosted bacteria fix nitrogen. Intracellular accommodation that facilitates nutrient exchange between the two partners and protects bacteria from plant defense reactions has been a major evolutionary step towards mutualism. Yet the forces that drove the selection of the late event of intracellular infection during rhizobium evolution are unknown. To address this question, we took advantage of the previous conversion of the plant pathogen Ralstonia solanacearum into a legume-nodulating bacterium that infected nodules only extracellularly. We experimentally evolved this draft rhizobium into intracellular endosymbionts using serial cycles of legume-bacterium cocultures. The three derived lineages rapidly gained intracellular infection capacity, revealing that the legume is a highly selective environment for the evolution of this trait. From genome resequencing, we identified in each lineage a mutation responsible for the extracellular-intracellular transition. All three mutations target virulence regulators, strongly suggesting that several virulence-associated functions interfere with intracellular infection. We provide evidence that the adaptive mutations were selected for their positive effect on nodulation. Moreover, we showed that inactivation of the type three secretion system of R. solanacearum that initially allowed the ancestral draft rhizobium to nodulate, was also required to permit intracellular infection, suggesting a similar checkpoint for bacterial invasion at the early nodulation/root infection and late nodule cell entry levels. We discuss our findings with respect to the spread and maintenance of intracellular infection in rhizobial lineages during evolutionary times.

Plant-activated bacterial receptor adenylate cyclases modulate epidermal infection in the Sinorhizobium meliloti-Medicago symbiosis.

Legumes and soil bacteria called rhizobia have coevolved a facultative nitrogen-fixing symbiosis. Establishment of the symbiosis requires bacterial entry via root hair infection threads and, in parallel, organogenesis of nodules that subsequently are invaded by bacteria. Tight control of nodulation and infection is required to maintain the mutualistic character of the interaction. Available evidence supports a passive bacterial role in nodulation and infection after the microsymbiont has triggered the symbiotic plant developmental program. Here we identify in Sinorhizobium meliloti, the Medicago symbiont, a cAMP-signaling regulatory cascade consisting of three receptor-like adenylate cyclases, a Crp-like regulator, and a target gene of unknown function. The cascade is activated specifically by a plant signal during nodule organogenesis. Cascade inactivation results in a hyperinfection phenotype consisting of abortive epidermal infection events uncoupled from nodulation. These findings show that, in response to a plant signal, rhizobia play an active role in the control of infection. We suggest that rhizobia may modulate the plant's susceptibility to infection. This regulatory loop likely aims at optimizing legume infection.

Peptide signalling in the rhizobium-legume symbiosis.

For two decades, signalling research in the rhizobium-legume symbiosis field has been dominated by oligosaccharide signals (mainly Nod factors and, to a lesser extent, surface polysaccharides made by the microsymbionts) and phytohormones. Recently, plant peptides have emerged as another major class of signalling molecules in the rhizobium-legume symbioses contributing to the control of nodulation, infection and bacteroid differentiation. Here we focus on three examples of symbiotically relevant peptides, namely Enod40, CLE and NCR peptides. The number of genes encoding these peptides, as well as the recent discovery of additional peptide players in the context of symbiosis, suggests that we might be seeing only the tip of the peptide iceberg in the sea of symbiotic regulations.

Cupriavidus taiwanensis bacteroids in Mimosa pudica Indeterminate nodules are not terminally differentiated.

The beta-rhizobium Cupriavidus taiwanensis forms indeterminate nodules on Mimosa pudica. C. taiwanensis bacteroids resemble free-living bacteria in terms of genomic DNA content, cell size, membrane permeability, and viability, in contrast to bacteroids in indeterminate nodules of the galegoid clade. Bacteroid differentiation is thus unrelated to nodule ontogeny.

Experimental evolution of a plant pathogen into a legume symbiont.

Rhizobia are phylogenetically disparate alpha- and beta-proteobacteria that have achieved the environmentally essential function of fixing atmospheric nitrogen in symbiosis with legumes. Ample evidence indicates that horizontal transfer of symbiotic plasmids/islands has played a crucial role in rhizobia evolution. However, adaptive mechanisms that allow the recipient genomes to express symbiotic traits are unknown. Here, we report on the experimental evolution of a pathogenic Ralstonia solanacearum chimera carrying the symbiotic plasmid of the rhizobium Cupriavidus taiwanensis into Mimosa nodulating and infecting symbionts. Two types of adaptive mutations in the hrpG-controlled virulence pathway of R. solanacearum were identified that are crucial for the transition from pathogenicity towards mutualism. Inactivation of the hrcV structural gene of the type III secretion system allowed nodulation and early infection to take place, whereas inactivation of the master virulence regulator hrpG allowed intracellular infection of nodule cells. Our findings predict that natural selection of adaptive changes in the legume environment following horizontal transfer has been a major driving force in rhizobia evolution and diversification and show the potential of experimental evolution to decipher the mechanisms leading to symbiosis.

Establishing nitrogen-fixing symbiosis with legumes: how many rhizobium recipes?

Rhizobia are phylogenetically disparate alpha- and beta-proteobacteria that have achieved the environmentally essential function of fixing atmospheric nitrogen (N(2)) in symbiosis with legumes. All rhizobia elicit the formation of root - or occasionally stem - nodules, plant organs dedicated to the fixation and assimilation of nitrogen. Bacterial colonization of these nodules culminates in a remarkable case of sustained intracellular infection in plants. Rhizobial phylogenetic diversity raised the question of whether these soil bacteria shared a common core of symbiotic genes. In this article, we review the cumulative evidence from recent genomic and genetic analyses pointing toward an unexpected variety of mechanisms that lead to symbiosis with legumes.

A portal for rhizobial genomes: RhizoGATE integrates a Sinorhizobium meliloti genome annotation update with postgenome data.

Sinorhizobium meliloti is a symbiotic soil bacterium of the alphaproteobacterial subdivision. Like other rhizobia, S. meliloti induces nitrogen-fixing root nodules on leguminous plants. This is an ecologically and economically important interaction, because plants engaged in symbiosis with rhizobia can grow without exogenous nitrogen fertilizers. The S. meliloti-Medicago truncatula (barrel medic) association is an important symbiosis model. The S. meliloti genome was published in 2001, and the M. truncatula genome currently is being sequenced. Many new resources and data have been made available since the original S. meliloti genome annotation and an update was needed. In June 2008, we submitted our annotation update to the EMBL and NCBI databases. Here we describe this new annotation and a new web-based portal RhizoGATE. About 1000 annotation updates were made; these included assigning functions to 313 putative proteins, assigning EC numbers to 431 proteins, and identifying 86 new putative genes. RhizoGATE incorporates the new annotion with the S. meliloti GenDB project, a platform that allows annotation updates in real time. Locations of transposon insertions, plasmid integrations, and array probe sequences are available in the GenDB project. RhizoGATE employs the EMMA platform for management and analysis of transcriptome data and the IGetDB data warehouse to integrate a variety of heterogeneous external data sources.

Auxotrophy accounts for nodulation defect of most Sinorhizobium meliloti mutants in the branched-chain amino acid biosynthesis pathway.

Some Sinorhizobium meliloti mutants in genes involved in isoleucine, valine, and leucine biosynthesis were previously described as being unable to induce nodule formation on host plants. Here, we present a reappraisal of the interconnection between the branched-chain amino acid biosynthesis pathway and the nodulation process in S. meliloti. We characterized the symbiotic phenotype of seven mutants that are auxotrophic for isoleucine, valine, or leucine in two closely related S. meliloti strains, 1021 and 2011. We showed that all mutants were similarly impaired for nodulation and infection of the Medicago sativa host plant. In most cases, the nodulation phenotype was fully restored by the addition of the missing amino acids to the plant growth medium. This strongly suggests that auxotrophy is the cause of the nodulation defect of these mutants. However, we confirmed previous findings that ilvC and ilvD2 mutants in the S. meliloti 1021 genetic background could not be restored to nodulation by supplementation with exogenous amino acids even though their Nod factor production appeared to be normal.

Genome sequence of the beta-rhizobium Cupriavidus taiwanensis and comparative genomics of rhizobia.

We report the first complete genome sequence of a beta-proteobacterial nitrogen-fixing symbiont of legumes, Cupriavidus taiwanensis LMG19424. The genome consists of two chromosomes of size 3.42 Mb and 2.50 Mb, and a large symbiotic plasmid of 0.56 Mb. The C. taiwanensis genome displays an unexpected high similarity with the genome of the saprophytic bacterium C. eutrophus H16, despite being 0.94 Mb smaller. Both organisms harbor two chromosomes with large regions of synteny interspersed by specific regions. In contrast, the two species host highly divergent plasmids, with the consequence that C. taiwanensis is symbiotically proficient and less metabolically versatile. Altogether, specific regions in C. taiwanensis compared with C. eutrophus cover 1.02 Mb and are enriched in genes associated with symbiosis or virulence in other bacteria. C. taiwanensis reveals characteristics of a minimal rhizobium, including the most compact (35-kb) symbiotic island (nod and nif) identified so far in any rhizobium. The atypical phylogenetic position of C. taiwanensis allowed insightful comparative genomics of all available rhizobium genomes. We did not find any gene that was both common and specific to all rhizobia, thus suggesting that a unique shared genetic strategy does not support symbiosis of rhizobia with legumes. Instead, phylodistribution analysis of more than 200 Sinorhizobium meliloti known symbiotic genes indicated large and complex variations of their occurrence in rhizobia and non-rhizobia. This led us to devise an in silico method to extract genes preferentially associated with rhizobia. We discuss how the novel genes we have identified may contribute to symbiotic adaptation.

FixJ: a major regulator of the oxygen limitation response and late symbiotic functions of Sinorhizobium meliloti.

Sinorhizobium meliloti exists either in a free-living state in the soil or in symbiosis within legume nodules, where the bacteria differentiate into nitrogen-fixing bacteroids. Expression of genes involved in nitrogen fixation and associated respiration is governed by two intermediate regulators, NifA and FixK, respectively, which are controlled by a two-component regulatory system FixLJ in response to low-oxygen conditions. In order to identify the FixLJ regulon, gene expression profiles were determined in microaerobic free-living cells as well as during the symbiotic life of the bacterium for the wild type and a fixJ null-mutant strain. We identified 122 genes activated by FixJ in either state, including 87 novel targets. FixJ controls 74% of the genes induced in microaerobiosis (2% oxygen) and the majority of genes expressed in mature bacteroids. Ninety-seven percent of FixJ-activated genes are located on the symbiotic plasmid pSymA. Transcriptome profiles of a nifA and a fixK mutant showed that NifA activates a limited number of genes, all specific to the symbiotic state, whereas FixK controls more than 90 genes, involved in free-living and/or symbiotic life. This study also revealed that FixJ has no other direct targets besides those already known. FixJ is involved in the regulation of functions such as denitrification or amino acid/polyamine metabolism and transport. Mutations in selected novel FixJ targets did not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa roots. From these results, we propose an updated model of the FixJ regulon.

Sinorhizobium meliloti differentiation during symbiosis with alfalfa: a transcriptomic dissection.

Sinorhizobium meliloti is a soil bacterium able to induce the formation of nodules on the root of specific legumes, including alfalfa (Medicago sativa). Bacteria colonize nodules through infection threads, invade the plant intracellularly, and ultimately differentiate into bacteroids capable of reducing atmospheric nitrogen to ammonia, which is directly assimilated by the plant. As a first step to describe global changes in gene expression of S. meliloti during the symbiotic process, we used whole genome microarrays to establish the transcriptome profile of bacteria from nodules induced by a bacterial mutant blocked at the infection stage and from wild-type nodules harvested at various timepoints after inoculation. Comparison of these profiles to those of cultured bacteria grown either to log or stationary phase as well as examination of a number of genes with known symbiotic transcription patterns allowed us to correlate global gene-expression patterns to three known steps of symbiotic bacteria bacteroid differentiation, i.e., invading bacteria inside infection threads, young differentiating bacteroids, and fully differentiated, nitrogen-fixing bacteroids. Finally, analysis of individual gene transcription profiles revealed a number of new potential symbiotic genes.

Transcriptome-based identification of the Sinorhizobium meliloti NodD1 regulon.

The NodD1 regulon of Sinorhizobium meliloti was determined through the analysis of the S. meliloti transcriptome in response to the plant flavone luteolin and the overexpression of nodD1. Nine new genes regulated by both NodD1 and luteolin were identified, demonstrating that NodD1 controls few functions behind nodulation in S. meliloti.