Nod Gene Induction Research Articles

O-Methylated Isoflavones Induce nod Genes of Mesorhizobium ciceri and Pratensein Promotes Nodulation in Chickpea.

Legume plants form symbiotic relationships with rhizobia, which allow plants to utilize atmospheric nitrogen as a nutrient. This symbiosis is initiated by secretion of specific signaling metabolites from the roots, which induce the expression of nod genes in rhizobia. These metabolites are called nod gene inducers (NGIs), and various flavonoids have been found to act as NGIs. However, NGIs of chickpea, the second major pulse crop, remain elusive. We conducted untargeted metabolome analysis of chickpea root exudates to explore metabolites with increased secretion under nitrogen deficiency. Principal component (PC) analysis showed a clear difference between nitrogen deficiency and control, with PC1 alone accounting for 37.5% of the variance. The intensity of two features with the highest PC1 loading values significantly increased under nitrogen deficiency; two prominent peaks were identified as O-methylated isoflavones, pratensein and biochanin A. RNA-seq analysis showed that they induce nodABC gene expression in the Mesorhizobium ciceri symbiont, suggesting that pratensein and biochanin A are chickpea NGIs. Pratensein applied concurrently with M. ciceri at sowing promoted chickpea nodulation. These results demonstrate that pratensein and biochanin A are chickpea NGIs, and pratensein can be useful for increasing nodulation efficiency in chickpea production.

Methylated chalcones are required for rhizobial nod gene induction in the Medicago truncatula rhizosphere.

Legume nodulation requires the detection of flavonoids in the rhizosphere by rhizobia to activate their production of Nod factor countersignals. Here we investigated the flavonoids involved in nodulation of Medicago truncatula. We biochemically characterized five flavonoid-O-methyltransferases (OMTs) and a lux-based nod gene reporter was used to investigate the response of Sinorhizobium medicae NodD1 to various flavonoids. We found that chalcone-OMT 1 (ChOMT1) and ChOMT3, but not OMT2, 4, and 5, were able to produce 4,4'-dihydroxy-2'-methoxychalcone (DHMC). The bioreporter responded most strongly to DHMC, while isoflavones important for nodulation of soybean (Glycine max) showed no activity. Mutant analysis revealed that loss of ChOMT1 strongly reduced DHMC levels. Furthermore, chomt1 and omt2 showed strongly reduced bioreporter luminescence in their rhizospheres. In addition, loss of both ChOMT1 and ChOMT3 reduced nodulation, and this phenotype was strengthened by the further loss of OMT2. We conclude that: the loss of ChOMT1 greatly reduces root DHMC levels; ChOMT1 or OMT2 are important for nod gene activation in the rhizosphere; and ChOMT1/3 and OMT2 promote nodulation. Our findings suggest a degree of exclusivity in the flavonoids used for nodulation in M. truncatula compared to soybean, supporting a role for flavonoids in rhizobial host range.

Surface Motility Regulation of Sinorhizobium fredii HH103 by Plant Flavonoids and the NodD1, TtsI, NolR, and MucR1 Symbiotic Bacterial Regulators.

Bacteria can spread on surfaces to colonize new environments and access more resources. Rhizobia, a group of α- and β-Proteobacteria, establish nitrogen-fixing symbioses with legumes that rely on a complex signal interchange between the partners. Flavonoids exuded by plant roots and the bacterial transcriptional activator NodD control the transcription of different rhizobial genes (the so-called nod regulon) and, together with additional bacterial regulatory proteins (such as TtsI, MucR or NolR), influence the production of different rhizobial molecular signals. In Sinorhizobium fredii HH103, flavonoids and NodD have a negative effect on exopolysaccharide production and biofilm production. Since biofilm formation and motility are often inversely regulated, we have analysed whether flavonoids may influence the translocation of S. fredii HH103 on surfaces. We show that the presence of nod gene-inducing flavonoids does not affect swimming but promotes a mode of surface translocation, which involves both flagella-dependent and -independent mechanisms. This surface motility is regulated in a flavonoid-NodD1-TtsI-dependent manner, relies on the assembly of the symbiotic type 3 secretion system (T3SS), and involves the participation of additional modulators of the nod regulon (NolR and MucR1). To our knowledge, this is the first evidence indicating the participation of T3SS in surface motility in a plant-interacting bacterium. Interestingly, flavonoids acting as nod-gene inducers also participate in the inverse regulation of surface motility and biofilm formation, which could contribute to a more efficient plant colonisation.

Revealing potential functions of hypothetical proteins induced by genistein in the symbiosis island of Bradyrhizobium japonicum commercial strain SEMIA 5079 (= CPAC 15).

BackgroundBradyrhizobium japonicum strain SEMIA 5079 (= CPAC 15) is a nitrogen-fixing symbiont of soybean broadly used in commercial inoculants in Brazil. Its genome has about 50% of hypothetical (HP) protein-coding genes, many in the symbiosis island, raising questions about their putative role on the biological nitrogen fixation (BNF) process. This study aimed to infer functional roles to 15 HP genes localized in the symbiosis island of SEMIA 5079, and to analyze their expression in the presence of a nod-gene inducer.ResultsA workflow of bioinformatics tools/databases was established and allowed the functional annotation of the HP genes. Most were enzymes, including transferases in the biosynthetic pathways of cobalamin, amino acids and secondary metabolites that may help in saprophytic ability and stress tolerance, and hydrolases, that may be important for competitiveness, plant infection, and stress tolerance. Putative roles for other enzymes and transporters identified are discussed. Some HP proteins were specific to the genus Bradyrhizobium, others to specific host legumes, and the analysis of orthologues helped to predict roles in BNF.ConclusionsAll 15 HP genes were induced by genistein and high induction was confirmed in five of them, suggesting major roles in the BNF process.Supplementary informationThe online version contains supplementary material available at 10.1186/s12866-022-02527-9.

Phenolic Acids Induce Nod Factor Production in Lotus japonicus-Mesorhizobium Symbiosis.

In legume–rhizobia symbiosis, partner recognition and the initiation of symbiosis processes require the mutual exchange of chemical signals. Chemicals, generally (iso)flavonoids, in the root exudates of the host plant induce the expression of nod genes in rhizobia, and, thus, are called nod gene inducers. The expression of nod genes leads to the production of lipochitooligosaccharides (LCOs) called Nod factors. Natural nod gene inducer(s) in Lotus japonicus–Mesorhizobium symbiosis remain unknown. Therefore, we developed an LCO detection method based on ultra-high-performance liquid chromatography–tandem-quadrupole mass spectrometry (UPLC-TQMS) to identify these inducers and used it herein to screen 40 phenolic compounds and aldonic acids for their ability to induce LCOs in Mesorhizobium japonicum MAFF303099. We identified five phenolic acids with LCO-inducing activities, including p-coumaric, caffeic, and ferulic acids. The induced LCOs caused root hair deformation, and nodule numbers in L. japonicus inoculated with M. japonicum were increased by these phenolic acids. The three phenolic acids listed above induced the expression of the nodA, nodB, and ttsI genes in a strain harboring a multicopy plasmid encoding NodD1, but not that encoding NodD2. The presence of p-coumaric and ferulic acids in the root exudates of L. japonicus was confirmed by UPLC-TQMS, and the induction of ttsI::lacZ in the strain harboring the nodD1 plasmid was detected in the rhizosphere of L. japonicus. Based on these results, we propose that phenolic acids are a novel type of nod gene inducer in L. japonicus–Mesorhizobium symbiosis.

Facing the communication between soybean plants and microorganisms (Bradyrhizobium and Delftia) by quantitative shotgun proteomics

The effect of plant-produced flavonoids in rhizobial cells is well-known, and flavonoids are considered the first signals in the rhizobia-plant communication. We previously reported that Delftia strains increase the performance (nodulation and nitrogen fixation) of leguminous plants in co-inoculation experiments, as compared with the single rhizobial inoculation. By using an approach based on quantitative shotgun proteomics, we explored the response of Delftia sp. JD2 cells to the presence of the flavonoid genistein (nod gene-inducer of Bradyrhizobium cells). We found that genistein up-regulates the production of regulatory proteins associated with the epiphytic habit (TetR and diguanylate cyclase), proteins involved in the formation of flagella, iron acquisition, resistance to several drugs, the rhizoplane colonization (biofilm establishment), and the rearrangement of the peptidoglycan structure and membrane proteins. These results indicate that genistein induces chemotaxis, mobility and prepares JD2 strain for an epiphytic lifestyle. We also analyzed the effect of the co-inoculation on the proteome of symbiosome-enriched fractions from soybean nodules. The fractions from co-inoculated plants showed an up-regulation of proteins involved in stress endurance (“stem 31 kDa glycoprotein”, “Kunitz trypsin protease inhibitor”, proteasome subunits) and ferritin (involved in iron storage and homeostasis), suggesting that co-inoculation with JD2 prepares the soybean plants to deal with unfriendly environmental conditions. In summary, the plant-secreted genistein functions as a chemical signal in the communications between soybean plants and Delftia sp. JD2, while the presence of JD2 would prepare the plant to cope with abiotic stresses in co-inoculation experiments.

Proteome responses of Rhizobium tropici CIAT 899 upon apigenin and salt stress induction

Rhizobium tropici establishes symbiosis with several leguminous species, inducing nitrogen fixing nodules. Communication between rhizobia and legumes starts when the flavonoids secreted by plant roots interact with the R. tropici NodD protein to recognize plant signaling molecules present in the rhizosphere and begin the nodulation process. Some studies in R. tropici CIAT 899 have demonstrated that the induction of nod genes by saline stress influences the formation of nodules in plant roots. The aim of this study was to analyze the proteome profile of R. tropici CIAT 899 to investigate the molecular response of the bacterium under apigenin (flavonoid) and salt stress induction. The bacterium was grown in TY medium, considered as the control condition (CC), and in the presence of apigenin (AC) and NaCl (SSC). Total proteins were extracted and analyzed by two-dimensional electrophoresis (2-DE) and the differential spots were identified by MALDI-TOF MS/MS. A total of 105 differentially abundant protein spots were observed, including 2 present only in CC, 2 exclusive to AC and 6 to SSC. A total of 73% of these protein spots were identified by mass spectrometry. Proteins involved in transport and cellular processes were mostly increased in AC:CC and AC:SSC comparison, while in SSC:CC, these proteins were mostly decreased. Under salt stress, the bacterium reduced the uptake of molecules and energy consumption, reducing the abundance of ABC transporter proteins. These proteins are crucial for bacterial adaptation in environmental conditions and can be important for the nodulation process.

Improved nodulation and seed yield of soybean (Glycine max) with a new isoflavone-based inoculant of Bradyrhizobium elkanii

Flavonoids produced by roots are secondary metabolites involved in the induction of nod genes in rhizobial cells and nodule organogenesis. We describe the development and quality control of a new formulation for soybean (Glycine max (L.) Merr.) based on Bradyrhizobium elkanii cells grown in a culture medium containing isoflavones. As opposed to B. japonicum, the performance of B. elkanii pre-incubated with isoflavones is unknown. Its evaluation implied in vitro, greenhouse, and field research trials. Greenhouse and in vitro treatments comprised: seeds inoculated with a traditional inoculant based on B. elkanii; seeds inoculated with a new formulation based on isoflavones-cultivated B. elkanii cells, the exogenous application of isoflavones into the growth substrate, and the non-inoculated control. Field treatments included non-inoculated controls with and without N-fertilizer and seeds inoculation with the traditional or the new formulation. No changes in quality parameters (bacterial survival on both seeds and liquid formulation) were observed, as compared with the traditionally produced bradyrhizobial cells. Also, improved plant response (nodulation, plant growth, and seed yield) was observed when seeds were inoculated with the new formulation. The exogenous application of isoflavones directly into the substrate had deleterious effects on soybean growth and nodulation; however, the increased values of agronomical parameters in field conditions show that the addition of isoflavones during the industrial fermentation of B. elkanii is a better option over the common bradyrhizobial inoculant.

Qualitative changes in proteins contained in outer membrane vesicles produced by Rhizobium etli grown in the presence of the nod gene inducer naringenin.

In this work, we compared the proteomic profiles of outer membrane vesicles (OMVs) isolated from Rhizobium etli CE3 grown in minimal medium (MM) with and without exogenous naringenin. One-hundred and seven proteins were present only in OMVs from naringenin-containing cultures (N-OMVs), 57 proteins were unique to OMVs from control cultures lacking naringenin (C-OMVs) and 303 proteins were present in OMVs from both culture conditions (S-OMVs). Although we found no absolute predominance of specific types of proteins in the N-, C- or S-OMV classes, there were categories of proteins that were significantly less or more common in the different OMV categories. Proteins for energy production, translation and membrane and cell wall biogenesis were overrepresented in C-OMVs relative to N-OMVs. Proteins for carbohydrate metabolism and transport and those classified as either general function prediction only, function unknown, or without functional prediction were more common in N-OMVs than C-OMVs. This indicates that naringenin increased the proportion of these proteins in the OMVs, although NodD binding sites were only slightly more common in the promoters of genes for proteins found in the N-OMVs. In addition, OMVs from naringenin-containing cultures contained nodulation factor.

Exploration of natural nod gene inducers for Mesorhizobium loti in seed and root exudates of Lotus corniculatus

Exploration of natural nod gene inducers for Mesorhizobium loti in seed and root exudates of Lotus corniculatus

Intercropping influences component and content change of flavonoids in root exudates and nodulation of Faba bean

ABSTRACTFlavonoids produced by legume roots are signal molecules acting as nod gene inducers for the symbiotic rhizobium partner. Nevertheless, the changes of flavonoids in root exudates in intercropping system are still unknown. Based on pot experiment of faba bean and wheat intercropping, here we showed that faba bean and wheat intercropping increased the nodules number and dry weight, dry weight per nodule of faba bean compared with those found in monocropping, and the increase of faba bean nodulation was likely caused by the enhancement with flavonol, isoflavone, chalcone and hesperetin in its root exudates. It also promoted exudation of five types of flavonoids by wheat compared with monocropping. Our findings suggest that the flavonoids in root exudates have a positive effect on the nodulation and nitrogen fixation of faba bean in faba bean and wheat intercropping.

The Effect of Root Exudate 7,4'-Dihydroxyflavone and Naringenin on Soil Bacterial Community Structure.

Our goal was to investigate how root exudate flavonoids influence the soil bacterial community structure and to identify members of the community that change their relative abundance in response to flavonoid exudation. Using a model system that approximates flavonoid exudation of Medicago sativa roots, we treated a soil with 7,4′-dihydroxyflavone and naringenin in two separate experiments using three different rates: medium (equivalent to the exudation rate of 7,4′-dihydroxyflavone from M. sativa seedlings), high (10× the medium rate), and low (0.1× the medium rate). Controls received no flavonoid. Soil samples were subjected to ATP assays and 16S rRNA gene amplicon sequencing. The flavonoid treatments caused no significant change in the soil ATP content. With the high 7,4′-dihydroxyflavone treatment rate, operational taxonomic units (OTUs) classified as Acidobacteria subdivision 4 increased in relative abundance compared with the control samples, whereas OTUs classified as Gaiellales, Nocardioidaceae, and Thermomonosporaceae were more prevalent in the control. The naringenin treatments did not cause significant changes in the soil bacterial community structure. Our results suggest that the root exudate flavonoid 7,4′-dihydroxyflavone can interact with a diverse range of soil bacteria and may have other functions in the rhizosphere in addition to nod gene induction in legume—rhizobia symbiosis.

Enhanced nodulation of peanut when co-inoculated with fungal endophyte Phomopsis liquidambari and bradyrhizobium

In peanut continuous cropping soil, the application of fungal endophyte Phomopsis liquidambari B3 showed peanut pod yield promotion and root nodule number increase. P. liquidambari improved soil environment by degrading allelochemicals and thus promoted peanut pod yield. Furthermore, peanut yield promotion is in part due to the root nodule increase since nodular nitrogen fixation provides the largest source of nitrogen for peanut. However, it is unknown whether this nodule number increase is induced by fungal endophyte. We therefore conducted several pot experiments using vermiculite to investigate the effects of P. liquidambari on peanut-bradyrhizobium nodulation. Our results showed that P. liquidambari co-inoculated with bradyrhizobium increased root nodule number and shoot accumulated nitrogen by 28.25% and 29.71%, respectively. Nodulation dynamics analysis showed that P. liquidambari accelerated nodule initiation and subsequent nodule development. Meanwhile, P. liquidambari was able to colonize the peanut root as an endophyte. The dynamics of P. liquidambari and bradyrhizobial root colonization analysis showed that P. liquidambari inoculation significantly increased the rate of bradyrhizobial colonization. Furthermore, P. liquidambari inoculation significantly increased flavonoids synthesis-related enzymes activities, two common types of flavonoid (luteolin and quercetin-peanut rhizobial nod gene inducer) secretion and lateral root (peanut rhizobial infection site) formation, indicating that P. liquidambari altered the peanut nodulation-related physiological and metabolic activities. These obtained results confirmed the direct contribution of P. liquidambari in enhancing peanut-bradyrhizobium interaction, nodulation and yield.

Identification of a core set of rhizobial infection genes using data from single cell-types.

Genome-wide expression studies on nodulation have varied in their scale from entire root systems to dissected nodules or root sections containing nodule primordia (NP). More recently efforts have focused on developing methods for isolation of root hairs from infected plants and the application of laser-capture microdissection technology to nodules. Here we analyze two published data sets to identify a core set of infection genes that are expressed in the nodule and in root hairs during infection. Among the genes identified were those encoding phenylpropanoid biosynthesis enzymes including Chalcone-O-Methyltransferase which is required for the production of the potent Nod gene inducer 4′,4-dihydroxy-2-methoxychalcone. A promoter-GUS analysis in transgenic hairy roots for two genes encoding Chalcone-O-Methyltransferase isoforms revealed their expression in rhizobially infected root hairs and the nodule infection zone but not in the nitrogen fixation zone. We also describe a group of Rhizobially Induced Peroxidases whose expression overlaps with the production of superoxide in rhizobially infected root hairs and in nodules and roots. Finally, we identify a cohort of co-regulated transcription factors as candidate regulators of these processes.

The root hair "infectome" of Medicago truncatula uncovers changes in cell cycle genes and reveals a requirement for Auxin signaling in rhizobial infection.

Nitrogen-fixing rhizobia colonize legume roots via plant-made intracellular infection threads. Genetics has identified some genes involved but has not provided sufficient detail to understand requirements for infection thread development. Therefore, we transcriptionally profiled Medicago truncatula root hairs prior to and during the initial stages of infection. This revealed changes in the responses to plant hormones, most notably auxin, strigolactone, gibberellic acid, and brassinosteroids. Several auxin responsive genes, including the ortholog of Arabidopsis thaliana Auxin Response Factor 16, were induced at infection sites and in nodule primordia, and mutation of ARF16a reduced rhizobial infection. Associated with the induction of auxin signaling genes, there was increased expression of cell cycle genes including an A-type cyclin and a subunit of the anaphase promoting complex. There was also induction of several chalcone O-methyltransferases involved in the synthesis of an inducer of Sinorhizobium meliloti nod genes, as well as a gene associated with Nod factor degradation, suggesting both positive and negative feedback loops that control Nod factor levels during rhizobial infection. We conclude that the onset of infection is associated with reactivation of the cell cycle as well as increased expression of genes required for hormone and flavonoid biosynthesis and that the regulation of auxin signaling is necessary for initiation of rhizobial infection threads.

A Sinorhizobium meliloti-specific N-acyl homoserine lactone quorum-sensing signal increases nodule numbers in Medicago truncatula independent of autoregulation.

N-acyl homoserine lactones (AHLs) act as quorum sensing signals that regulate cell-density dependent behaviors in many gram-negative bacteria, in particular those important for plant-microbe interactions. AHLs can also be recognized by plants, and this may influence their interactions with bacteria. Here we tested whether the exposure to AHLs affects the nodule-forming symbiosis between legume hosts and rhizobia. We treated roots of the model legume, Medicago truncatula, with a range of AHLs either from its specific symbiont, Sinorhizobium meliloti, or from the potential pathogens, Pseudomonas aeruginosa and Agrobacterium vitis. We found increased numbers of nodules formed on root systems treated with the S. meliloti-specific AHL, 3-oxo-C14-homoserine lactone, at a concentration of 1 μM, while the other AHLs did not result in significant changes to nodule numbers. We did not find any evidence for altered nodule invasion by the rhizobia. Quantification of flavonoids that could act as nod gene inducers in S. meliloti did not show any correlation with increased nodule numbers. The effects of AHLs were specific for an increase in nodule numbers, but not lateral root numbers or root length. Increased nodule numbers following 3-oxo-C14-homoserine lactone treatment were under control of autoregulation of nodulation and were still observed in the autoregulation mutant, sunn4 (super numeric nodules4). However, increases in nodule numbers by 3-oxo-C14-homoserine lactone were not found in the ethylene-insensitive sickle mutant. A comparison between M. truncatula with M. sativa (alfalfa) and Trifolium repens (white clover) showed that the observed effects of AHLs on nodule numbers were specific to M. truncatula, despite M. sativa nodulating with the same symbiont. We conclude that plant perception of the S. meliloti-specific 3-oxo-C14-homoserine lactone influences nodule numbers in M. truncatula via an ethylene-dependent, but autoregulation-independent mechanism.

Legume nodulation

Legume nodulation

High NaCl Concentrations Induce the nod Genes of Rhizobium tropici CIAT899 in the Absence of Flavonoid Inducers

The nodulation (nod) genes of Rhizobium tropici CIAT899 can be induced by very low concentrations (micromolar to nanomolar range) of several flavonoid molecules secreted by the roots of leguminous plants under a number of different conditions. Some of these conditions have been investigated and appear to have a great influence on the concentration and the number of different Nod factors, which can induce root nodule primordia and pseudonodules in several leguminous plant roots. In one such condition, we added up to 300 mM NaCl to the induction medium of R. tropici CIAT899 containing the nod gene inducer apigenin. At the higher concentrations of NaCl, larger amounts and more different Nod factors were produced than in the absence of extra NaCl. To our surprise, under control conditions (300 mM NaCl without apigenin), some Nod-factor-like spots were also observed on the thin-layer plates used to detect incorporation of radiolabeled glucosamine into newly synthesized Nod factors. This phenomenon was further investigated with thin-layer plates, fusions of nod genes to the lacZ gene, high-performance liquid chromatography, mass spectrometry, and the formation of pseudonodules on bean roots. Here, we report that, in the absence of flavonoid inducers, high concentrations of NaCl induced nod genes and the production of Nod factors.

Involvement of a Novel Genistein-Inducible Multidrug Efflux Pump of <i>Bradyrhizobium japonicum</i> Early in the Interaction with <i>Glycine max</i> (L.) Merr

The early molecular dialogue between soybean and the bacterium Bradyrhizobium japonicum is crucial for triggering their symbiotic interaction. Here we found a single large genomic locus that is widely separated from the symbiosis island and was conspicuously induced within minutes after the addition of genistein. This locus (named BjG30) contains genes for the multidrug efflux pump, TetR family transcriptional regulator, and polyhydroxybutyrate (PHB) metabolism. The induction of BjG30 by genistein was competitively inhibited by daidzein, although both genistein and daidzein are soybean-derived inducers of nodulation (nod) genes. Such a differential expression pattern is also observed in some legume-derived flavonoids, which structurally differ in the hydroxy/deoxy group at the 5-position. In addition, not only did the induction start far in advance of nodW and nodD1 after the addition of genistein, but the levels showed distinct concentration dependence, indicating that the induction pattern of BjG30 is completely different from that of nod genes. The deletion of genes encoding either the multidrug efflux pump or PHB metabolism, especially the former, resulted in defective nodulation performance and nitrogen-fixing capability. Taken together, these results indicate that BjG30, and especially its multidrug efflux pump, may play a key role in the early stage of symbiosis by balancing the dual functions of genistein as both a nod gene inducer and toxicant.

Proteomics reveals differential expression of proteins related to a variety of metabolic pathways by genistein-induced Bradyrhizobium japonicum strains

The rhizobia-legume symbiosis requires a coordinated molecular interaction between the symbionts, initiated by seed and root exudation of several compounds, mainly flavonoids, that trigger the expression of nodulation genes in the bacteria. Since the role of flavonoids seems to be broader than the induction of nodulation genes, we aimed at characterizing genistein-induced proteins of Bradyrhizobium japonicum CPAC 15 (=SEMIA 5079), used in commercial soybean inoculants in Brazil, and of two genetically related strains grown in vitro. Whole-cell proteins were extracted both from induced (1 μM genistein) and from non-induced cultures of the three strains, and separated by two-dimensional electrophoresis. Spot profiles were compared between the two conditions and selected spots were excised and identified by mass spectrometry. Forty-seven proteins were significantly induced by genistein, including several hypothetical proteins, the cytoplasmic flagellar component FliG, periplasmic ABC transporters, a protein related to biosynthesis of exopolysaccharides (ExoN), and proteins involved in redox-state maintenance. Noteworthy was the induction of the PhyR-σ(EcfG) regulon, recently demonstrated to be involved in the symbiotic efficiency of, and general stress response in B. japonicum. Our results confirm that the role of flavonoids, such as genistein, can go far beyond the expression of nodulation-related proteins in B. japonicum.