Nodulation Genes Research Articles

Genetic and transcriptomic analysis of the Bradyrhizobium T3SS-triggered nodulation in the legume Aeschynomene evenia.

Some Bradyrhizobium strains nodulate certain Aeschynomene species independently of Nod factors, but thanks to their type III secretion system (T3SS). While different T3 effectors triggering nodulation (ErnA and Sup3) have been identified, the plant signalling pathways they activate remain unknown. Here, we explored the intraspecies variability in T3SS-triggered nodulation within Aeschynomene evenia and investigated transcriptomic responses that occur during this symbiosis. Furthermore, Bradyrhizobium strains having different effector sets were tested on A. evenia mutants altered in various symbiotic signalling genes. We identified the A. evenia accession N21/PI 225551 as appropriate for deciphering the T3SS-dependent process. Comparative transcriptomic analysis of A. evenia N21 roots inoculated with ORS3257 strain and its ∆ernA mutant revealed genes differentially expressed, including some involved in plant defences and auxin signalling. In the other A. evenia accession N76, all tested strains nodulated the AeCRK mutant but not the AeNIN and AeNSP2 mutants, indicating a differential requirement of these genes for T3SS-dependent nodulation. Furthermore, the effects of AePOLLUX, AeCCaMK and AeCYCLOPS mutations differed between the strains. Notably, ORS86 nodulated these three mutant lines and required for this both ErnA and Sup3. Taken together, these results shed light on how the T3SS-dependent nodulation process is achieved in legumes.

Nitrogen Fertilization in Snap Bean Following a Legume in the Sandy Soils of North Florida

Despite being a legume, snap bean (Phaseolus vulgaris L.) lacks nodulation genes, restricting its ability for nitrogen (N) fixation through rhizobia, making N fertilization essential for maximizing yields. However, conventional fertilizer application may lead to N losses due to a lack of synchronization between plant uptake and N availability. A promising option could be controlled-release fertilizers (CRFs), which can be custom-formulated to synchronize nutrient release with plant growth needs, promoting efficient resource use. Field studies were conducted at the North Florida Research and Education Center-Suwannee Valley (NFREC-SV) in Live Oak, FL, during the spring of 2021 and 2022. The experiments also investigated broadcasting and banding application methods of the University of Florida’s recommended N rate (112 kg·ha−1). The experimental design comprised 10 treatments, including five N rates (0, 56, 112, 168, and 224 kg·ha−1 N) and two fertilizer sources (ammonium nitrate and CRF), applied to ‘Caprice’ in a randomized complete block design with four replications. Results showed a significant yield improvement with N fertilization vs. the zero-N control. However, no yield increase was observed beyond 56 kg·ha−1. This finding could be due to the residual N from a preceding peanut crop rotation in both years. No notable variation in quality metrics (pod length and width) occurred above the 56 kg·ha−1 threshold. The results also revealed that the choice between conventional or CRF did not exert any statistically significant differences in snap bean yield. In addition, fertilizer broadcast and banding treatments yielded the same results.

Arbuscular mycorrhizal hyphae facilitate rhizobia dispersal and nodulation in legumes.

In soil ecosystems, rhizobia occupy the rhizosphere of legume roots to form nodules, a process triggered by microbial recognition of specific root-derived signals (i.e., flavonoids). However, soil conditions can limit bacterial motility, restricting signal perception to the area directly influenced by roots. Legumes, like most plants of agricultural interest, associate with arbuscular mycorrhizal fungi, whose hyphae develop extensively in the soil, potentially providing an effective dispersal network for rhizobia. We hypothesized that mycelial networks of arbuscular mycorrhizal fungi play a role in signal transmission and act as a highway, enabling rhizobia to migrate from distant soil to the roots of leguminous plants. Using in vitro and greenhouse microcosm systems, we demonstrated that Rhizophagus irregularis helps Shinorhizobium meliloti to migrate towards the legume Medicago truncatula, triggering nodulation, a mechanism absent without the arbuscular mycorrhizal fungus. Metabolomics analysis revealed eight flavonoids unique to the compartment containing extraradical hyphae of the arbuscular mycorrhizal fungus linked to M. truncatula roots, associated with S. meliloti growth and nod gene expression. Rhizobia plated on the extraradical hyphae connecting two plants (the legume M. truncatula and non-legume Solanum tuberosum) by a common mycelium network, showed preference for the legume, suggesting the chemoattraction by specific signals transported by the fungus connected to the legume. Simultaneously, S. meliloti stimulated the cytoplasmic/protoplasmic flow in the hyphae, likely increasing the release of nutrients and signals. Our results highlight the importance of extraradical hyphae (i.e. the mycorrhizal pathway) of arbuscular mycorrhizal fungi for the migration of rhizobia over long distances to the roots, leading to nodulation.

Rhizosphere flavonoids alleviates the inhibition of soybean nodulation caused by shading under maize-soybean strip intercropping

The flavonoids produced by legume roots are signal molecules that induce nod genes for symbiotic rhizobium. Nevertheless, the promoting effects of flavonoids in root exudates in intercropping system on soybean nodulation are still unknown. A two years of field experiments was carried with maize soybean strip intercropping, i.e., the interspecific row spacing of 30 cm (MS30), 45 cm (MS45), 60 cm (MS60), and sole soybean/maize:SS/MM, and root interaction, i.e., root no barrier (NB) and root polythene-plastic barrier (PB), to evaluate relationships between flavonoids in root exudates and nodulation. We found that root-root interaction between soybean and maize enhances the nodules number and fresh weight in intercropped soybean. This enhancement increase gradually with expansion of interspecific distance. Proportion of nodules with diameter greater than 0.4cm was higher in intercropped soybean with NB than with PB. The expressions of nodules-related genes (GmENOD40, GmNIN2b and GmEXPB2) were up-regulated. Furthermore, compared with monocropping, isoflavones secretion of soybean roots reduced, flavonoids and flavonols secretion of maize and soybean roots increased under intercropping. The secretion of differential metabolites of flavonoids in the rhizosphere of maize and soybean declined with root barrier. The expressions of GmCHS8 and GmIFS1 in soybean roots were up-regulated and GmICHG was down-regulated under root interaction. The most of the flavonoids and flavonol compounds were positively correlated with nodule diameter. The nodules number, the nodules fresh weight and the proportion of nodules with a diameter greater than 0.2 cm increased in different genotypes of soybean treated with maize root exudate, which promoted the improvement of nitrogen fixation capacity. Therefore, maize-soybean strip intercropping combined with reasonable spacing to enhance the positive effect of underground root interaction, and improve the nodulation and nitrogen fixation capacity of intercropping soybean.

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.

CopG1, a Novel Transcriptional Regulator Affecting Symbiosis in Bradyrhizobium sp. SUTN9-2.

The symbiotic interaction between leguminous and Bradyrhizobium sp. SUTN9-2 mainly relies on the nodulation process through Nod factors (NFs), while the type IV secretion system (T4SS) acts as an alternative pathway in this symbiosis. Two copies of T4SS (T4SS1 and T4SS2) are located on the chromosome of SUTN9-2. ΔT4SS1 reduces both nodule number and nitrogenase activity in all SUTN9-2 nodulating legumes. The functions of three selected genes (copG1, traG1, and virD21) within the region of T4SS1 were examined. We generated deleted mutants and tested them in Vigna radiata cv. SUT4. ΔtraG1 and ΔvirD21 exhibited lower invasion efficiency at the early stages of root infection but could be recently restored. In contrast, ΔcopG1 completely hindered nodule organogenesis and nitrogenase activity in all tested legumes. ΔcopG1 showed low expression of the nodulation gene and ttsI but exhibited high expression levels of the T4SS genes, traG1 and trbE1. The secreted proteins from ΔT4SS1 were down-regulated compared to the wild-type. Although ΔcopG1 secreted several proteins after flavonoid induction, T3SS (nopP and nopX) and the C4-dicarboxylate transporter (dct) were not detected. These results confirm the crucial role of the copG1 gene as a novel key regulator in the symbiotic relationship between SUTN9-2 and legumes.

Comprehensive genomic and transcriptomic profiling of pulmonary nodules in synchronous multiple primary lung cancer.

e20022 Background: Synchronous Multiple Primary Lung Cancer (sMPLC) exhibits distinct histopathological characteristics among pulmonary nodules. However, a comprehensive understanding of the somatic mutation landscape and transcriptome heterogeneity is lacking. Therefore, our study aims to meticulously investigate genomic distinctions among multiple pulmonary nodules within individual patients. Methods: We performed targeted sequencing on tumor specimens and conducted bulk RNA transcriptome analysis on 53 multiple nodules originating from 26 lung cancer patients. The multiple nodules from the same patient was determined as major nodule and minor nodule. Additionally, the tumor tissues underwent histopathological evaluation through H&E staining, complemented by a comprehensive series of immunohistochemistry (IHC) analyses to detect protein expression. The detected protein markers encompassed PD-L1, Ki67, and others. Results: For the 53 nodule samples from 26 MPLCs patients, EGFR was the mostly mutated genes, and the TP53 mutation frequency demonstrated notably different between major and minor nodules. Furthermore, pathway enrichment analysis based on the differentially expressed genes (DEGs) between major and minor nodules revealed the significantly active cell cycle and p53 pathways in the major nodules. Additionally, both major and minor nodules demonstrated mostly similar immune microenvironment and PD-L1 protein expression, and a significantly higher expression of Ki67. A noteworthy suppression was observed in the immune microenvironment in nodules, revealed by the expression of macrophage, neutrophils, and NK cells. Furthermore, minor nodules exhibited a modestly elevated expression of macrophages compared to major nodules. Additionally, among the significantly up-regulated cell cycle related genes in the major nodules when compared with minor nodules, CCNE1 mRNA expression demonstrated significant correlation with poor prognosis in the lung cancer. Conclusions: This study validated molecular distinctions between samples from major and minor nodules in patients with sMPLC at both genomic and transcriptomic levels. The major nodules exhibited heightened activity in tumor cell proliferation pathways and demonstrated malignancy-related biological characteristics, which correlated with pathological assessment results.

Rhizobium hidalgonense and Rhizobium redzepovicii as faba bean (Vicia faba L.) microsymbionts in Mexican soils.

As a legume crop widely cultured in the world, faba bean (Vicia faba L.) forms root nodules with diverse Rhizobium species in different regions. However, the symbionts associated with this plant in Mexico have not been studied. To investigate the diversity and species/symbiovar affiliations of rhizobia associated with faba bean in Mexico, rhizobia were isolated from this plant grown in two Mexican sites in the present study. Based upon the analysis of recA gene phylogeny, two genotypes were distinguished among a total of 35 isolates, and they were identified as Rhizobium hidalgonense and Rhizobium redzepovicii, respectively, by the whole genomic sequence analysis. Both the species harbored identical nod gene cluster and the same phylogenetic positions of nodC and nifH. So, all of them were identified into the symbiovar viciae. As a minor group, R. hidalgonense was only isolated from slightly acid soil and R. redzepovicii was the dominant group in both the acid and neutral soils. In addition, several genes related to resistance to metals (zinc, copper etc.) and metalloids (arsenic) were detected in genomes of the reference isolates, which might offer them some adaptation benefits. As conclusion, the community composition of faba bean rhizobia in Mexico was different from those reported in other regions. Furthermore, our study identified sv. viciae as the second symbiovar in the species R. redzepovicii. These results added novel evidence about the co-evolution, diversification and biogeographic patterns of rhizobia in association with their host legumes in distinct geographic regions.

Gene cloning, expression and performance validation of nitric oxide dismutase

Nitrous oxide (N2O) is a significant contributor to global warming and possesses an ozone-depleting impact nearly 298 times that of CO2. To reduce N2O emissions, the newly-discovered nod gene which can directly convert NO into N2 and O2 was successfully cloned from the anaerobic denitrification sludge. The recombinant plasmid containing the nod gene was built, and the expression of nod gene in Escherichia coli was determined, leading to the construction of recombinant engineering bacteria. Results showed that the recombinant engineering bacteria E. coli BL21 (DE3)-pET28a-nod could autonomously degrade NO, with a degradation rate of 72 % within 48 h, and could produce 2479.72 ppm of N2 and 75.12 mL of O2. The cumulative O2 production of the sludge sample and recombinant E. coli within 8 h was 1.75 mL and 8.45 mL, respectively. The cumulative O2 production of recombinant E. coli was at least 4.82 times higher than that of the sludge sample. The investigation proposed a new biodegradation pathway for nitrogen pollution.

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.

Calcium Induces the Cleavage of NopA and Regulates the Expression of Nodulation Genes and Secretion of T3SS Effectors in Sinorhizobium fredii NGR234.

The type III secretion system (T3SS) is a key factor for the symbiosis between rhizobia and legumes. In this study, we investigated the effect of calcium on the expression and secretion of T3SS effectors (T3Es) in Sinorhizobium fredii NGR234, a broad host range rhizobial strain. We performed RNA-Seq analysis of NGR234 grown in the presence of apigenin, calcium, and apigenin plus calcium and compared it with NGR234 grown in the absence of calcium and apigenin. Calcium treatment resulted in a differential expression of 65 genes, most of which are involved in the transport or metabolism of amino acids and carbohydrates. Calcium had a pronounced effect on the transcription of a gene (NGR_b22780) that encodes a putative transmembrane protein, exhibiting a 17-fold change when compared to NGR234 cells grown in the absence of calcium. Calcium upregulated the expression of several sugar transporters, permeases, aminotransferases, and oxidoreductases. Interestingly, calcium downregulated the expression of nodABC, genes that are required for the synthesis of nod factors. A gene encoding a putative outer membrane protein (OmpW) implicated in antibiotic resistance and membrane integrity was also repressed by calcium. We also observed that calcium reduced the production of nodulation outer proteins (T3Es), especially NopA, the main subunit of the T3SS pilus. Additionally, calcium mediated the cleavage of NopA into two smaller isoforms, which might affect the secretion of other T3Es and the symbiotic establishment. Our findings suggest that calcium regulates the T3SS at a post-transcriptional level and provides new insights into the role of calcium in rhizobia-legume interactions.

Novel Alphaproteobacteria transcribe genes for nitric oxide transformation at high levels in a marine oxygen-deficient zone.

Marine oxygen-deficient zones (ODZs) are portions of the ocean where intense nitrogen loss occurs primarily via denitrification and anammox. Despite many decades of study, the identity of the microbes that catalyze nitrogen loss in ODZs is still being elucidated. Intriguingly, high transcription of genes in the same family as the nitric oxide dismutase (nod) gene from Methylomirabilota has been reported in the anoxic core of ODZs. Here, we show that the most abundantly transcribed nod genes in the Eastern Tropical North Pacific ODZ belong to a new order (UBA11136) of Alphaproteobacteria, rather than Methylomirabilota as previously assumed. Gammaproteobacteria and Planctomycetia also transcribe nod, but at lower relative abundance than UBA11136 in the upper ODZ. The nod-transcribing Alphaproteobacteria likely use formaldehyde and formate as a source of electrons for aerobic respiration, with additional electrons possibly from sulfide oxidation. They also transcribe multiheme cytochrome (here named ptd) genes for a putative porin-cytochrome protein complex of unknown function, potentially involved in extracellular electron transfer. Molecular oxygen for aerobic respiration may originate from nitric oxide dismutation via cryptic oxygen cycling. Our results implicate Alphaproteobacteria order UBA11136 as a significant player in marine nitrogen loss and highlight their potential in one-carbon, nitrogen, and sulfur metabolism in ODZs.IMPORTANCEIn marine oxygen-deficient zones (ODZs), microbes transform bioavailable nitrogen to gaseous nitrogen, with nitric oxide as a key intermediate. The Eastern Tropical North Pacific contains the world's largest ODZ, but the identity of the microbes transforming nitric oxide remains unknown. Here, we show that highly transcribed nitric oxide dismutase (nod) genes belong to Alphaproteobacteria of the novel order UBA11136, which lacks cultivated isolates. These Alphaproteobacteria show evidence for aerobic respiration, using oxygen potentially sourced from nitric oxide dismutase, and possess a novel porin-cytochrome protein complex with unknown function. Gammaproteobacteria and Planctomycetia transcribe nod at lower levels. Our results pinpoint the microbes mediating a key step in marine nitrogen loss and reveal an unexpected predicted metabolism for marine Alphaproteobacteria.

Pseudomonas chlororaphis IRHB3 assemblies beneficial microbes and activates JA-mediated resistance to promote nutrient utilization and inhibit pathogen attack.

The rhizosphere microbiome is critical to plant health and resistance. PGPR are well known as plant-beneficial bacteria and generally regulate nutrient utilization as well as plant responses to environmental stimuli. In our previous work, one typical PGPR strain, Pseudomonas chlororaphis IRHB3, isolated from the soybean rhizosphere, had positive impacts on soil-borne disease suppression and growth promotion in the greenhouse, but its biocontrol mechanism and application in the field are not unclear. In the current study, IRHB3 was introduced into field soil, and its effects on the local rhizosphere microbiome, disease resistance, and soybean growth were comprehensively analyzed through high-throughput sequencing and physiological and molecular methods. We found that IRHB3 significantly increased the richness of the bacterial community but not the structure of the soybean rhizosphere. Functional bacteria related to phosphorus solubilization and nitrogen fixation, such as Geobacter, Geomonas, Candidatus Solibacter, Occallatibacter, and Candidatus Koribacter, were recruited in rich abundance by IRHB3 to the soybean rhizosphere as compared to those without IRHB3. In addition, the IRHB3 supplement obviously maintained the homeostasis of the rhizosphere microbiome that was disturbed by F. oxysporum, resulting in a lower disease index of root rot when compared with F. oxysporum. Furthermore, JA-mediated induced resistance was rapidly activated by IRHB3 following PDF1.2 and LOX2 expression, and meanwhile, a set of nodulation genes, GmENOD40b, GmNIN-2b, and GmRIC1, were also considerably induced by IRHB3 to improve nitrogen fixation ability and promote soybean yield, even when plants were infected by F. oxysporum. Thus, IRHB3 tends to synergistically interact with local rhizosphere microbes to promote host growth and induce host resistance in the field.

Bacterial Endophytes from Legumes Native to Arid Environments Are Promising Tools to Improve Mesorhizobium-Chickpea Symbiosis under Salinity.

Symbiotic nitrogen fixation is a major contributor of N in agricultural ecosystems, but the establishment of legume-rhizobium symbiosis is highly affected by soil salinity. Our interest is focused on the use of non-rhizobial endophytes to assist the symbiosis between chickpea and its microsymbiont under salinity to avoid loss of production and fertility. Our aims were (1) to investigate the impact of salinity on both symbiotic partners; including on early events of the Mesorhizobium-chickpea symbiosis, and (2) to evaluate the potential of four non-rhizobial endophytes isolated from legumes native to arid regions (Phyllobacterium salinisoli, P. ifriqiyense, Xanthomonas translucens, and Cupriavidus respiraculi) to promote chickpea growth and nodulation under salinity. Our results show a significant reduction in chickpea seed germination rate and in the microsymbiont Mesorhizobium ciceri LMS-1 growth under different levels of salinity. The composition of phenolic compounds in chickpea root exudates significantly changed when the plants were subjected to salinity, which in turn affected the nod genes expression in LMS-1. Furthermore, the LMS-1 response to root exudate stimuli was suppressed by the presence of salinity (250 mM NaCl). On the contrary, a significant upregulation of exoY and otsA genes, which are involved in exopolysaccharide and trehalose biosynthesis, respectively, was registered in salt-stressed LMS-1 cells. In addition, chickpea co-inoculation with LMS-1 along with the consortium containing two non-rhizobial bacterial endophytes, P. salinisoli and X. translucens, resulted in significant improvement of the chickpea growth and the symbiotic performance of LMS-1 under salinity. These results indicate that this non-rhizobial endophytic consortium may be an appropriate ecological and safe tool to improve chickpea growth and its adaptation to salt-degraded soils.

Soybean-mediated suppression of BjaI/BjaR1 quorum sensing in Bradyrhizobium diazoefficiens impacts symbiotic nitrogen fixation.

The acyl-homoserine lactones (AHLs)-mediated LuxI/LuxR quorum sensing (QS) system orchestrates diverse bacterial behaviors in response to changes in population density. The role of the BjaI/BjaR1 QS system in Bradyrhizobium diazoefficiens USDA 110, which shares homology with LuxI/LuxR, remains elusive during symbiotic interaction with soybean. Here this genetic system in wild-type (WT) bacteria residing inside nodules exhibited significantly reduced activity compared to free-living cells, potentially attributed to soybean-mediated suppression. The deletion mutant strain ΔbjaR1 showed significantly enhanced nodulation induction and nitrogen fixation ability. Nevertheless, its ultimate symbiotic outcome (plant dry weight) in soybeans was compromised. Furthermore, comparative analysis of the transcriptome, proteome, and promoter activity revealed that the inactivation of BjaR1 systematically activated and inhibited genomic modules associated with nodulation and nitrogen metabolism. The former appeared to be linked to a significant decrease in the expression of NodD2, a key cell-density-dependent repressor of nodulation genes, while the latter conferred bacterial growth and nitrogen fixation insensitivity to environmental nitrogen. In addition, BjaR1 exerted a positive influence on the transcription of multiple genes involved in a so-called central intermediate metabolism within the nodule. In conclusion, our findings highlight the crucial role of the BjaI/BjaR1 QS circuit in positively regulating bacterial nitrogen metabolism and emphasize the significance of the soybean-mediated suppression of this genetic system for promoting efficient symbiotic nitrogen fixation by B. diazoefficiens.IMPORTANCEThe present study demonstrates, for the first time, that the BjaI/BjaR1 QS system of Bradyrhizobium diazoefficiens has a significant impact on its nodulation and nitrogen fixation capability in soybean by positively regulating NodD2 expression and bacterial nitrogen metabolism. Moreover, it provides novel insights into the importance of suppressing the activity of this QS circuit by the soybean host plant in establishing an efficient mutual relationship between the two symbiotic partners. This research expands our understanding of legumes' role in modulating symbiotic nitrogen fixation through rhizobial QS-mediated metabolic functioning, thereby deepening our comprehension of symbiotic coevolution theory. In addition, these findings may hold great promise for developing quorum quenching technology in agriculture.

Nodulation Experiment by Cross-Inoculation of Nitrogen-Fixing Bacteria Isolated from Root Nodules of Several Leguminous Plants.

Root-nodule nitrogen-fixing bacteria are known for being specific to particular legumes. This study isolated the endophytic root-nodule bacteria from the nodules of legumes and examined them to determine whether they could be used to promote the formation of nodules in other legumes. Forty-six isolates were collected from five leguminous plants and screened for housekeeping (16S rRNA), nitrogen fixation (nifH), and nodulation (nodC) genes. Based on the 16S rRNA gene sequencing and phylogenetic analysis, the bacterial isolates WC15, WC16, WC24, and GM5 were identified as Rhizobium, Sphingomonas, Methylobacterium, and Bradyrhizobium, respectively. The four isolates were found to have the nifH gene, and the study confirmed that one isolate (GM5) had both the nifH and nodC genes. The Salkowski method was used to measure the isolated bacteria for their capacity to produce phytohormone indole acetic acid (IAA). Additional experiments were performed to examine the effect of the isolated bacteria on root morphology and nodulation. Among the four tested isolates, both WC24 and GM5 induced nodulation in Glycine max. The gene expression studies revealed that GM5 had a higher expression of the nifH gene. The existence and expression of the nitrogen-fixing genes implied that the tested strain had the ability to fix the atmospheric nitrogen. These findings demonstrated that a nitrogen-fixing bacterium, Methylobacterium (WC24), isolated from a Trifolium repens, induced the formation of root nodules in non-host leguminous plants (Glycine max). This suggested the potential application of these rhizobia as biofertilizer. Further studies are required to verify the N2-fixing efficiency of the isolates.

A complex regulatory network governs the expression of symbiotic genes in Sinorhizobium fredii HH103.

The establishment of the rhizobium-legume nitrogen-fixing symbiosis relies on the interchange of molecular signals between the two symbionts. We have previously studied by RNA-seq the effect of the symbiotic regulators NodD1, SyrM, and TtsI on the expression of the symbiotic genes (the nod regulon) of Sinorhizobium fredii HH103 upon treatment with the isoflavone genistein. In this work we have further investigated this regulatory network by incorporating new RNA-seq data of HH103 mutants in two other regulatory genes, nodD2 and nolR. Both genes code for global regulators with a predominant repressor effect on the nod regulon, although NodD2 acts as an activator of a small number of HH103 symbiotic genes. By combining RNA-seq data, qPCR experiments, and b-galactosidase assays of HH103 mutants harbouring a lacZ gene inserted into a regulatory gene, we have analysed the regulatory relations between the nodD1, nodD2, nolR, syrM, and ttsI genes, confirming previous data and discovering previously unknown relations. Previously we showed that HH103 mutants in the nodD2, nolR, syrM, or ttsI genes gain effective nodulation with Lotus japonicus, a model legume, although with different symbiotic performances. Here we show that the combinations of mutations in these genes led, in most cases, to a decrease in symbiotic effectiveness, although all of them retained the ability to induce the formation of nitrogen-fixing nodules. In fact, the nodD2, nolR, and syrM single and double mutants share a set of Nod factors, either overproduced by them or not generated by the wild-type strain, that might be responsible for gaining effective nodulation with L. japonicus.

Single-cell analysis identifies genes facilitating rhizobium infection in Lotus japonicus

Legume-rhizobium signaling during establishment of symbiotic nitrogen fixation restricts rhizobium colonization to specific cells. A limited number of root hair cells allow infection threads to form, and only a fraction of the epidermal infection threads progress to cortical layers to establish functional nodules. Here we use single-cell analysis to define the epidermal and cortical cell populations that respond to and facilitate rhizobium infection. We then identify high-confidence nodulation gene candidates based on their specific expression in these populations, pinpointing genes stably associated with infection across genotypes and time points. We show that one of these, which we name SYMRKL1, encodes a protein with an ectodomain predicted to be nearly identical to that of SYMRK and is required for normal infection thread formation. Our work disentangles cellular processes and transcriptional modules that were previously confounded due to lack of cellular resolution, providing a more detailed understanding of symbiotic interactions.

Unveiling novel pathways and key contributors in the nitrogen cycle: Validation of enrichment and taxonomic characterization of oxygenic denitrifying microorganisms in environmental samples

Microorganisms play a crucial role in both the nitrogen cycle and greenhouse gas emissions. A recent discovery has unveiled a new denitrification pathway called oxygenic denitrification, entailing the enzymatic reduction of nitrite to nitric oxide (NO) by a putative nitric oxide dismutase (nod) enzyme. In this study, the presence of the nod gene was detected and subsequently enriched in anaerobic-activated sludge, farmland soil, and paddy soil samples. After 150 days, the enriched samples exhibited significant denitrification, and concomitant oxygen production. The removal efficiency of nitrite ranged from 64.6 % to 79.0 %, while the oxygen production rate was between 15.4 μL/min and 18.6 μL/min when exposed to a sole nitrogen source of 80 mg/L sodium nitrite. Additionally, batch experiments and kinetic analyses revealed the intricate pathways and underlying mechanisms governing the oxygenic denitrification reaction by using CARBOXY-PTIO, 18O-labelled water, and acetylene to unravel the intricacies of the reaction. The quantitative polymerase chain reaction (qPCR) results indicated a significant surge in the abundance of nod genes, escalating from 7.59 to 10.12-fold. Moreover, analysis of 16S ribosomal DNA (rDNA) amplicons revealed Proteobacteria as the dominant phylum and Thauera as the main genus, with the presumed affiliation. In this study, a new nitrogen conversion pathway, oxygenic denitrification, was discovered in environmental samples. This process provides the possibility for the control of nitrous oxide in the treatment of nitrogenous wastewater.

Molecular Mimicry of NodA Genes by an N-acetylmuramoyl-L-alanine Amidases Gene in a Nodule Forming Staphylococcus cohnii [RESEARCH NOTE

A rare, nodule-forming, Gram-positive bacterium (Sub4) was isolated from the forage and cover crop Pueraria phaseoloides, which aligned at 99.26% sequence identity to a partial 16S rDNA sequence of Staphylococcus cohnii sp. This was the first record of a strain/species of Staphylococcus capable of independent, unassisted nodule formation in a legume host. When a nodA gene was sought by PCR using a pair of gene-specific primers synthesized using a related (Firmicutes) Paenibacillus sp. nodA gene, the reaction yielded a PCR product of similar size but a distinct identity. The resulting ~400bp PCR product coded for a likely N-acetylmuramoyl-L-alanine amidase and not an acyltransferase. It may be inferred that the N-acetylmuramoyl-L-alanine amidase gene was amplified due to sequence similarities/mimicry between 2 cosmetically unrelated loci. There was also an analogy in protein backbones between homology models of the Sub4 N-acetylmuramoyl-L-alanine amidase and a Paenibacillus nodA gene product, which also suggests that the 2 protein families may have an evolutionary relationship. When the phylogeny of the sequenced locus was inferred, a unique clade with high bootstrap support was formed by the sequence under study with an Azorhizobium nodA gene. Azorhizobia were distinct in relation to their nodule formation on stems and their ability to fix nitrogen in the free-living and the symbiotic forms. N-acetylmuramoyl-L-alanine amidases may have roles to play in remodeling peptidoglycan for LysM receptor interactions, likely without nod factors.