Arthrobacter sp. UMCV2, and its compound N,N-dimethylhexadecilamine promote nodulation in Medicago truncatula by Sinorhizobium medicae.

The actinobacterium Arthrobacter sp. UMCV2 promotes plant growth through the emission of N,N-dimethylhexadecilamine (DMHDA). The Medicago-Sinorhizobium nodulation has been employed to study symbiotic nitrogen fixation by rhizobia in nodulating Fabaceae. Herein, we isolated three Sinorhizobium medicae strains that were used to induce nodules in Medicago truncatula. The co-inoculation of M. truncatula with Arthrobacter sp. strain UMCV2 produced a higher number of effective nodules than inoculation with only Sinorhizobium strains. Similarly, the exposure of inoculated M. truncatula to DMHDA produced a greater number of effective nodules compared to non-exposed plants. Thus, we conclude that Arthrobacter sp. UMCV2 promotes nodulation, and propose that this effect is produced, at least partly, via DMHDA emission.

Endogenous gibberellins affect root nodule symbiosis via transcriptional regulation of NODULE INCEPTION in Lotus japonicus.

Legumes and nitrogen-fixing rhizobial bacteria establish root nodule symbiosis, which is orchestrated by several plant hormones. Exogenous addition of biologically active gibberellic acid (GA) is known to inhibit root nodule symbiosis. However, the precise role of GA has not been elucidated because of the trace amounts of these hormones in plants and the multiple functions of GAs. Here, we found that GA signaling acts as a key regulator in a long-distance negative-feedback system of root nodule symbiosis called autoregulation of nodulation (AON). GA biosynthesis is activated during nodule formation in and around the nodule vascular bundles, and bioactive GAs accumulate in the nodule. In addition, GA signaling induces expression of the symbiotic transcription factor NODULE INCEPTION (NIN) via a cis-acting region on the NIN promoter. Mutants with deletions of this cis-acting region have increased susceptibility to rhizobial infection and reduced GA-induced CLE-RS1 and CLE-RS2 expression, suggesting that the inhibitory effect of GAs occurs through AON. This is supported by the GA-insensitive phenotypes of an AON-defective mutant of HYPERNODULATION ABERRANT ROOT FORMATION1 (HAR1) and a reciprocal grafting experiment. Thus, endogenous GAs induce NIN expression via its GA-responsive cis-acting region, and subsequently the GA-induced NIN activates the AON system to regulate nodule formation.

Nitrate-induced CLE35 signaling peptides inhibit nodulation through the SUNN receptor and miR2111 repression.

Legume plants form nitrogen (N)-fixing symbiotic nodules when mineral N is limiting in soils. As N fixation is energetically costly compared to mineral N acquisition, these N sources, and in particular nitrate, inhibit nodule formation and N fixation. Here, in the model legume Medicago truncatula, we characterized a CLAVATA3-like (CLE) signaling peptide, MtCLE35, the expression of which is upregulated locally by high-N environments and relies on the Nodule Inception-Like Protein (NLP) MtNLP1. MtCLE35 inhibits nodule formation by affecting rhizobial infections, depending on the Super Numeric Nodules (MtSUNN) receptor. In addition, high N or the ectopic expression of MtCLE35 represses the expression and accumulation of the miR2111 shoot-to-root systemic effector, thus inhibiting its positive effect on nodulation. Conversely, ectopic expression of miR2111 or downregulation of MtCLE35 by RNA interference increased miR2111 accumulation independently of the N environment, and thus partially bypasses the nodulation inhibitory action of nitrate. Overall, these results demonstrate that the MtNLP1-dependent, N-induced MtCLE35 signaling peptide acts through the MtSUNN receptor and the miR2111 systemic effector to inhibit nodulation.

GmbZIP1 negatively regulates ABA-induced inhibition of nodulation by targeting GmENOD40-1 in soybean.

BACKGROUND: Abscisic acid (ABA) plays an important role in plant growth and adaptation through the ABA signaling pathway. The ABA-responsive element binding (AREB/ABF) family transcriptional factors are central regulators that integrate ABA signaling with various signaling pathways. It has long been known that ABA inhibits rhizobial infection and nodule formation in legumes, but the underlying molecular mechanisms remain elusive. RESULTS: Here, we show that nodulation is very sensitive to ABA and exogenous ABA dramatically inhibits rhizobial infection and nodule formation in soybean. In addition, we proved that GmbZIP1, an AREB/ABF transcription factor, is a major regulator in both nodulation and plant response to ABA in soybean. GmbZIP1 was specifically expressed during nodule formation and development. Overexpression of GmbZIP1 resulted in reduced rhizobial infection and decreased nodule number. Furthermore, GmbZIP1 is responsive to ABA, and ectopic overexpression of GmbZIP1 increased sensitivity of Arabidopsis plants to ABA during seed germination and postgerminative growth, and conferred enhanced drought tolerance of plants. Remarkably, we found that GmbZIP1 directly binds to the promoter of GmENOD40-1, a marker gene for nodule formation, to repress its expression. CONCLUSION: Our results identified GmbZIP1 as a node regulator that integrates ABA signaling with nodulation signaling to negatively regulate nodule formation.

Application of Nitrate, Ammonium, or Urea Changes the Concentrations of Ureides, Urea, Amino Acids and Other Metabolites in Xylem Sap and in the Organs of Soybean Plants (Glycine max (L.) Merr.).

Soybean (Glycine max (L.) Merr.) plants form root nodules and fix atmospheric dinitrogen, while also utilizing the combined nitrogen absorbed from roots. In this study, nodulated soybean plants were supplied with 5 mM N nitrate, ammonium, or urea for 3 days, and the changes in metabolite concentrations in the xylem sap and each organ were analyzed. The ureide concentration in the xylem sap was the highest in the control plants that were supplied with an N-free nutrient solution, but nitrate and asparagine were the principal compounds in the xylem sap with nitrate treatment. The metabolite concentrations in both the xylem sap and each organ were similar between the ammonium and urea treatments. Considerable amounts of urea were present in the xylem sap and all the organs among all the treatments. Positive correlations were observed between the ureides and urea concentrations in the xylem sap as well as in the roots and leaves, although no correlations were observed between the urea and arginine concentrations, suggesting that urea may have originated from ureide degradation in soybean plants, possibly in the roots. This is the first finding of the possibility of ureide degradation to urea in the underground organs of soybean plants.

Physical Properties of Carbon Nanomaterials and Nanoceria Affect Pathways Important to the Nodulation Competitiveness of the Symbiotic N2 -Fixing Bacterium Bradyrhizobium diazoefficiens.

The pathogenicity and antimicrobial properties of engineered nanomaterials (ENMs) are relatively well studied. However, less is known regarding the interactions of ENMs and agriculturally beneficial microorganisms that affect food security. Nanoceria (CeO2 nanoparticles (NPs)), multiwall carbon nanotubes (MWCNTs), graphene nanoplatelets (GNPs), and carbon black (CB) have been previously shown to inhibit symbiotic N2 fixation in soybeans, but direct rhizobial susceptibility is uncertain. Here, Bradyrhizobium diazoefficiens associated with symbiotic N2 fixation in soybeans is assessed, evaluating the role of soybean root exudates (RE) on ENM-bacterial interactions and the effects of CeO2 NPs, MWCNTs, GNPs, and CB on bacterial growth and gene expression. Although bacterial growth is inhibited by 50 mg L-1 CeO2 NPs, MWCNTs, and CB, all ENMs at 0.1 and 10 mg L-1 cause a global transcriptomic response that is mitigated by RE. ENMs may interfere with plant-bacterial signaling, as evidenced by suppressed upregulation of genes induced by RE, and downregulation of genes encoding transport RNA, which facilitates nodulation signaling. MWCNTs and CeO2 NPs inhibit the expression of genes conferring B. diazoefficiens nodulation competitiveness. Surprisingly, the transcriptomic effects on B. diazoefficiens are similar for these two ENMs, indicating that physical, not chemical, ENM properties explain the observed effects.

Impact of seed-applied pesticides on rhizobial survival and legume nodulation.

AIMS: Compatibility of seed-applied pesticides and rhizobial inoculants is an important consideration for farmers when sowing legumes. Some of the seed-applied pesticides may influence rhizobial growth and nodulation, but there is currently little available information on the potential inhibitory effects. Therefore, common seed fungicidal and insecticidal treatments were assessed to determine adverse impacts on rhizobial inoculants both in vitro, on treated seed, and in the field. METHODS AND RESULTS: Initially, the in vitro toxicity of the seed-applied fungicides Thiram 600, P-Pickel T (PPT), their active ingredients (thiram and thiabendazole) and the insecticide Gaucho to rhizobia was measured with filter discs containing varying concentrations of the pesticides. Pea and chickpea seed was then coated with the same pesticides and inoculated with rhizobia in different inoculant substrates to determine bacterial survival and nodulation. Finally, a field trial using the fungicide PPT and commercial inoculants was conducted. Some seed fungicide treatments were found to be inhibitory to rhizobia and reduce nodulation under monoxenic conditions and in the field. SIGNIFICANCE AND IMPACT OF THE STUDY: These data provide more detailed information on the compatibility of specific rhizobial inoculants with common seed-applied pesticides. This research will provide information on the compatibility of rhizobia and seed-applied pesticides, and assist farmers to select sowing practices which reduce the risk of crop nodulation failures.

Hydrogen Sulfide Promotes Nodulation and Nitrogen Fixation in Soybean-Rhizobia Symbiotic System.

The rhizobium-legume symbiotic system is crucial for nitrogen cycle balance in agriculture. Hydrogen sulfide (H2S), a gaseous signaling molecule, may regulate various physiological processes in plants. However, whether H2S has regulatory effect in this symbiotic system remains unknown. Herein, we investigated the possible role of H2S in the symbiosis between soybean (Glycine max) and rhizobium (Sinorhizobium fredii). Our results demonstrated that an exogenous H2S donor (sodium hydrosulfide [NaHS]) treatment promoted soybean growth, nodulation, and nitrogenase (Nase) activity. Western blotting analysis revealed that the abundance of Nase component nifH was increased by NaHS treatment in nodules. Quantitative real-time polymerase chain reaction data showed that NaHS treatment upregulated the expressions of symbiosis-related genes nodA, nodC, and nodD of S. fredii. In addition, expression of soybean nodulation marker genes, including early nodulin 40 (GmENOD40), ERF required for nodulation (GmERN), nodulation signaling pathway 2b (GmNSP2b), and nodulation inception genes (GmNIN1a, GmNIN2a, and GmNIN2b), were upregulated. Moreover, the expressions of glutamate synthase (GmGOGAT), asparagine synthase (GmAS), nitrite reductase (GmNiR), ammonia transporter (GmSAT1), leghemoglobin (GmLb), and nifH involved in nitrogen metabolism were upregulated in NaHS-treated soybean roots and nodules. Together, our results suggested that H2S may act as a positive signaling molecule in the soybean-rhizobia symbiotic system and enhance the system's nitrogen fixation ability.

Rhizobium tropici CIAT 899 copA gene plays a fundamental role in copper tolerance in both free life and symbiosis with Phaseolus vulgaris.

Rhizobium tropici CIAT 899 is a facultative symbiotic diazotroph able to deal with stressful concentrations of metals. Nevertheless the molecular mechanisms involved in metal tolerance have not been elucidated. Copper (Cu2+) is a metal component essential for the heme-copper respiratory oxidases and enzymes that catalyse redox reactions, however, it is highly toxic when intracellular trace concentrations are surpassed. In this study, we report that R. tropici CIAT 899 is more tolerant to Cu2+ than other Rhizobium and Sinorhizobium species. Through Tn5 random mutagenesis we identify a R. tropici mutant strain with a severe reduction in Cu2+ tolerance. The Tn5 insertion disrupted the gene RTCIAT899_CH17575, encoding a putative heavy metal efflux P1B-1-type ATPase designated as copA. Phaseolus vulgaris plants inoculated with the copA::Tn5 mutant in the presence of toxic Cu2+ concentrations showed a drastic reduction in plant and nodule dry weight, as well as nitrogenase activity. Nodules induced by the copA::Tn5 mutant present an increase in H2O2 concentration, lipoperoxidation and accumulate 40-fold more Cu2+ than nodules formed by the wild-type strain. The copA::Tn5 mutant complemented with the copA gene recovered the wild-type symbiotic phenotypes. Therefore, the copA gene is essential for R. tropici CIAT 899 to survive in copper-rich environments in both free life and symbiosis with P. vulgaris plants.

Fungicide application increased copper-bioavailability and impaired nitrogen fixation through reduced root nodule formation on alfalfa.

Copper-based fungicides have been used for a long time in viticulture and have accumulated in many vineyard soils. In this study, incrementing Cu(OH)2-based fungicide application from 0.05 to 5 g Cu kg-1 on two agricultural soils (an acidic sandy loam (L, pH 4.95) and an alkaline silt loam (D, pH 7.45)) resulted in 5 times more mobile Cu in the acidic soil. The most sensitive parameters of alfalfa (Medicago sativa) growing in these soils were the root nodule number, decreasing to 34% and 15% of the control at 0.1 g Cu kg-1 in soil L and at 1.5 g Cu kg-1 in soil D, respectively, as well as the nodule biomass, decreasing to 25% and 27% at 0.5 g Cu kg-1 in soil L and at 1.5 g Cu kg-1 in soil D, respectively. However, the enzymatic N2-fixation was not directly affected by Cu in spite of the presence of Cu in the meristem and the zone of effective N2-fixation, as illustrated by chemical imaging. The strongly different responses observed in the two tested soils reflect the higher buffering capacity of the alkaline silt loam and showed that Cu mitigation and remediation strategies should especially target vineyards with acidic, sandy soils.

Heterologous Expression of Rhizobial CelC2 Cellulase Impairs Symbiotic Signaling and Nodulation in Medicago truncatula.

The infection of legume plants by rhizobia is tightly regulated to ensure accurate bacterial penetration, infection, and development of functionally efficient nitrogen-fixing root nodules. Rhizobial Nod factors (NF) have key roles in the elicitation of nodulation signaling. Infection of white clover roots also involves the tightly regulated specific breakdown of the noncrystalline apex of cell walls in growing root hairs, which is mediated by Rhizobium leguminosarum bv. trifolii cellulase CelC2. Here, we have analyzed the impact of this endoglucanase on symbiotic signaling in the model legume Medicago truncatula. Ensifer meliloti constitutively expressing celC gene exhibited delayed nodulation and elicited aberrant ineffective nodules, hampering plant growth in the absence of nitrogen. Cotreatment of roots with NF and CelC2 altered Ca2+ spiking in root hairs and induction of the early nodulin gene ENOD11. Our data suggest that CelC2 alters early signaling between partners in the rhizobia-legume interaction.

Determining the Site of Action of Strigolactones during Nodulation.

Strigolactones (SLs) influence the ability of legumes to associate with nitrogen-fixing bacteria. In this study, we determine the precise stage at which SLs influence nodulation. We show that SLs promote infection thread formation, as a null SL-deficient pea (Pisum sativum) mutant forms significantly fewer infection threads than wild-type plants, and this reduction can be overcome by the application of the synthetic SL GR24. We found no evidence that SLs influence physical events in the plant before or after infection thread formation, since SL-deficient plants displayed a similar ability to induce root hair curling in response to rhizobia or Nod lipochitooligosaccharides (LCOs) and SL-deficient nodules appear to fix nitrogen at a similar rate to those of wild-type plants. In contrast, an SL receptor mutant displayed no decrease in infection thread formation or nodule number, suggesting that SL deficiency may influence the bacterial partner. We found that this influence of SL deficiency was not due to altered flavonoid exudation or the ability of root exudates to stimulate bacterial growth. The influence of SL deficiency on infection thread formation was accompanied by reduced expression of some early nodulation genes. Importantly, SL synthesis is down-regulated by mutations in genes of the Nod LCO signaling pathway, and this requires the downstream transcription factor NSP2 but not NIN This, together with the fact that the expression of certain SL biosynthesis genes can be elevated in response to rhizobia/Nod LCOs, suggests that Nod LCOs may induce SL biosynthesis. SLs appear to influence nodulation independently of ethylene action, as SL-deficient and ethylene-insensitive double mutant plants display essentially additive phenotypes, and we found no evidence that SLs influence ethylene synthesis or vice versa.

Strigolactones promote rhizobia interaction and increase nodulation in soybean (Glycine max).

Strigolactones (SLs) play an important role in controlling root growth, shoot branching, and plant-symbionts interaction. Despite the importance, the components of SL biosynthesis and signaling have not been unequivocally explored in soybean. Here we identified the putative components of SL synthesis enzymes GmMAX1a and GmMAX4a with tissue expression patterns and were apparently regulated by rhizobia infection and changed during nodule development. GmMAX1a and GmMAX4a were further characterized in soybean nodulation with knockdown transgenic hairy roots. GmMAX1a and GmMAX4a knockdown lines exhibit decreased nodule number and expression levels of several nodulation genes required for nodule development. Hormone analysis showed that GmMAX1a and GmMAX4a knockdown hairy roots had increased physiological level of ABA and JA but significantly decreased auxin content. This study not only revealed the conservation of SL biosynthesis but also showed close interactions between SL and other hormone signaling in controlling plant development and legume-rhizobia interaction.

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1.

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.

In vitro rhizobia response and symbiosis process under aluminum stress.

Aluminum (Al) toxicity is a major problem affecting soil fertility, microbial diversity, and nutrient uptake of plants. Rhizobia response and legume interaction under Al conditions are still unknown; it is important to understand how to develop and improve legume cultivation under Al stress. In this study, rhizobia response was recorded under different Al concentrations. Al effect on rhizobial cells was characterized by combination with different two pH conditions. Symbiosis process was compared between α- and ß-rhizobia inoculated onto soybean varieties. Rhizobial cell numbers was decreased as Al concentration increased. However, induced Al tolerance considerably depended on rhizobia types and their origins. Accordingly, organic acid results were in correlation with growth rate and cell density which suggested that citric acid might be a positive selective force for Al tolerance and plant interaction on rhizobia. Al toxicity delayed and interrupted the plant-rhizobia interaction and the effect was more pronounced under acidic conditions. Burkholderia fungorum VTr35 significantly improved plant growth under acid-Al stress in combination with all soybean varieties. Moreover, plant genotype was an important factor to establish an effective nodulation and nitrogen fixation under Al stress. Additionally, tolerant rhizobia could be applied as an inoculant on stressful agroecosystems. Furthermore, metabolic pathways have still been unknown under Al stress.

Effects of bamboo biochar on soybean root nodulation in multi-elements contaminated soils.

Improvements in plant physiological performance by means of biochar application in soils contaminated by multi-elements are determinants of agroecosystem functioning. This study analyzed the effects of bamboo-derived biochar on root nodulation and plant growth in a moderately acidic Andosol (pH = 5.56) contaminated with multi-elements during a 70-day investigation of soybean growth. Bamboo biochar that had been pyrolyzed at a temperature below 500°C was applied to soils at three different and moderately high rates (5%, 10%, and 15%, w/w). Biochar amendment beyond 5% stimulated root nodulation as well as soybean growth. The nodule weight per root system was significantly enhanced by 186% and 243% over the control at the 10% and 15% addition rates, respectively. The primary explanation for these stimulatory effects was attributed to an increase in the K and Mo supplies for plant uptake that was induced by the biochar application, whereas the increased availability of P contributed to a lesser extent. Leaf CO2 assimilation rate was slightly enhanced at the highest application rate, but this enhancement was not associated with an increase in biomass. The incorporation of biochar into the soil reduced extractable-NH4NO3 Cd, Cu, Mn, Ni, and Zn, but not Pb, regardless of the application dose. This change was accompanied by a significant (P < 0.05) suppression of the uptake od trace elements in soybean shoots at the optimum application rate (10%); the degree of reduction followed this order: Pb>Mn>Cd>Zn>Cu>Ni. The increase in soil pH and the diffusion/adsorption of trace elements onto the biochar may have contributed to the lowering of the concentration of trace elements in the soil as well as in soybean shoots.

The Sinorhizobium (Ensifer) fredii HH103 Nodulation Outer Protein NopI Is a Determinant for Efficient Nodulation of Soybean and Cowpea Plants.

The type III secretion system (T3SS) is a specialized secretion apparatus that is commonly used by many plant and animal pathogenic bacteria to deliver proteins, termed effectors, to the interior of the host cells. These effectors suppress host defenses and interfere with signal transduction pathways to promote infection. Some rhizobial strains possess a functional T3SS, which is involved in the suppression of host defense responses, host range determination, and symbiotic efficiency. The analysis of the genome of the broad-host-range rhizobial strain Sinorhizobium fredii HH103 identified eight genes that code for putative T3SS effectors. Three of these effectors, NopL, NopP, and NopI, are Rhizobium specific. In this work, we demonstrate that NopI, whose amino acid sequence shows a certain similarity with NopP, is secreted through the S. fredii HH103 T3SS in response to flavonoids. We also determined that NopL can be considered an effector since it is directly secreted to the interior of the host cell as demonstrated by adenylate cyclase assays. Finally, the symbiotic phenotype of single, double, and triple nopI, nopL, and nopP mutants in soybean and cowpea was assayed, showing that NopI plays an important role in determining the number of nodules formed in both legumes and that the absence of both NopL and NopP is highly detrimental for symbiosis.IMPORTANCE The paper is focused on three Rhizobium-specific T3SS effectors of Sinorhizobium fredii HH103, NopL, NopP, and NopI. We demonstrate that S. fredii HH103 is able to secrete through the T3SS in response to flavonoids the nodulation outer protein NopI. Additionally, we determined that NopL can be considered an effector since it is secreted to the interior of the host cell as demonstrated by adenylate cyclase assays. Finally, nodulation assays of soybean and cowpea indicated that NopI is important for the determination of the number of nodules formed and that the absence of both NopL and NopP negatively affected nodulation.

Does nitrogen affect the interaction between a native hemiparasite and its native or introduced leguminous hosts?

Associations between plants and nitrogen (N)-fixing rhizobia intensify with decreasing N supply and come at a carbon cost to the host. However, what additional impact parasitic plants have on their leguminous hosts' carbon budget in terms of effects on host physiology and growth is unknown. Under glasshouse conditions, Ulex europaeus and Acacia paradoxa either uninfected or infected with the hemiparasite Cassytha pubescens were supplied (high nitrogen (HN)) or not (low nitrogen (LN)) with extra N. The photosynthetic performance and growth of the association were measured. Cassytha pubescens significantly reduced the maximum electron transport rates and total biomass of U. europaeus but not those of A. paradoxa, regardless of N. Infection significantly decreased the root biomass of A. paradoxa only at LN, while the significant negative effect of infection on roots of U. europaeus was less severe at LN. Infection had a significant negative impact on host nodule biomass. Ulex europaeus supported significantly greater parasite biomass (also per unit host biomass) than A. paradoxa, regardless of N. We concluded that rhizobia do not influence the effect of a native parasite on overall growth of leguminous hosts. Our results suggest that C. pubescens will have a strong impact on U. europaeus but not A. paradoxa, regardless of N in the field.

Interkingdom Responses to Bacterial Quorum Sensing Signals Regulate Frequency and Rate of Nodulation in Legume-Rhizobia Symbiosis.

Density-dependent phenotypic switching in bacteria, the phenomenon of quorum sensing (QS), is instrumental in many pathogenic and mutualistic behaviors. In many Gram-negative bacteria, QS is regulated by N-acylated-l-homoserine lactones (AHLs). Synthetic analogues of these AHLs hold significant promise for regulating QS at the host-symbiont interface. Regulation depends on refined temporal and spatial models of quorums under native conditions. Critical to this is an understanding of how the presence of these signals may affect a prospective host. We screened a library of AHL analogues for their ability to regulate the legume-rhizobia mutualistic symbiosis (nodulation) between Medicago truncatula and Sinorhizobium meliloti. Using an established QS-reporter line of S. meliloti and nodulation assays with wild-type bacteria, we identified compounds capable of increasing either the rate of nodule formation or total nodule number. Most importantly, we identified compounds with activity exclusive to either host or pathogen, underscoring the potential to generate QS modulators selective to bacteria with limited effects on a prospective host.

MicroRNA167-Directed Regulation of the Auxin Response Factors GmARF8a and GmARF8b Is Required for Soybean Nodulation and Lateral Root Development.

Legume root nodules convert atmospheric nitrogen gas into ammonium through symbiosis with a prokaryotic microsymbiont broadly called rhizobia. Auxin signaling is required for determinant nodule development; however, the molecular mechanism of auxin-mediated nodule formation remains largely unknown. Here, we show in soybean (Glycine max) that the microRNA miR167 acts as a positive regulator of lateral root organs, namely nodules and lateral roots. miR167c expression was up-regulated in the vasculature, pericycle, and cortex of soybean roots following inoculation with Bradyrhizobium japonicum strain USDA110 (the microsymbiont). It was found to positively regulate nodule numbers directly by repressing the target genes GmARF8a and GmARF8b (homologous genes of Arabidopsis [Arabidopsis thaliana] AtARF8 that encode auxin response factors). Moreover, the expression of miR167 and its targets was up- and down-regulated by auxin, respectively. The miR167-GmARF8 module also positively regulated nodulation efficiency under low microsymbiont density, a condition often associated with environmental stress. The regulatory role of miR167 on nodule initiation was dependent on the Nod factor receptor GmNFR1α, and it acts upstream of the nodulation-associated genes nodule inception, nodulation signaling pathway1, early nodulin40-1, NF-YA1 (previously known as HAEM activator protein2-1), and NF-YA2. miR167 also promoted lateral root numbers. Collectively, our findings establish a key role for the miR167-GmARF8 module in auxin-mediated nodule and lateral root formation in soybean.