Nitrogen-fixing bacteria promote growth and bioactive components accumulation of Astragalus mongholicus by regulating plant metabolism and rhizosphere microbiota.

BACKGROUND: The excessive application of chemical fertilizers in the cultivation of Astragalus mongholicus Bunge results in a reduction in the quality of the medicinal plant and compromises the sustainable productivity of the soil. PGPB inoculant is a hot topic in ecological agriculture research. In the cultivation of Astragalus mongholicus, the screened nitrogen-fixing bacteria can promote plant growth, however, whether it can promote the accumulation of main bioactive components remains unknown. In this study, mixed inoculants containing 5 strains of growth promoting bacteria (Rhizobium T16 , Sinorhizobium T21 , Bacillus J1 , Bacillus G4 and Arthrobacter J2) were used in the field experiment. The metabolic substances in the root tissues of Astragalus mongholicus were identified during the harvest period by non-targeted metabolomics method, and the differential metabolites between groups were identified by statistical analysis. Meanwhile, high-throughput sequencing was performed to analyze the changes of rhizosphere soil and endophytic microbial community structure after mixed microbial treatment. RESULTS: The results of non-targeted metabolism indicated a significant increase in the levels of 26 metabolites after treatment including 13 flavonoids, 3 saponins and 10 other components. The contents of three plant hormones (abscisic acid, salicylic acid and spermidine) also increased after treatment, which presumed to play an important role in regulating plant growth and metabolism. Studies on endosphere and rhizosphere bacterial communities showed that Rhzobiaceae, Micromonosporaceae, and Hypomicrobiaceae in endophytic, and Oxalobactereae in rhizosphere were significantly increased after treatment. These findings suggest their potential importance in plant growth promotion and secondary metabolism regulation. CONCLUSIONS: This finding provides a basis for developing nitrogen-fixing bacteria fertilizer and improving the ecological planting efficiency of Astragalus mongholicus.

Polyvinyl chloride and polybutylene adipate microplastics affect peanut and rhizobium symbiosis by interfering with multiple metabolic pathways.

Microplastics (MPs), widely presented in cultivated soil, have caused serious stresses on crop growth. However, the mechanism by which MPs affect legumes and rhizobia symbiosis is still unclear. Here, peanut seedlings were inoculated with Bradyrhizobium zhanjiangense CCBAU 51778 and were grown in vermiculite with 3 %/5 % (w/w) addition of PVC (polyvinyl chloride)-MPs/PBAT (polybutylene adipate)-MPs. PVC-MPs and PBAT-MPs separately decreased nodule number by 33-100 % and 2.62-80.91 %. Transcriptome analysis showed that PVC-MPs affected more DEGs (differentially expressed genes) than PBAT-MPs, indicating PVC-MPs were more devastating for the symbiosis than PBAT-MPs. Functional annotation revealed that PVC-MPs and PBAT-MPs enriched DEGs related to biosynthesis pathways such as flavonoid, isoflavonoid, and phenylpropanoid, in peanut. And when the dose increased from 3 % to 5 %, PVC-MPs mainly enriched the pathways of starch and sucrose metabolism, alanine, aspartate and glutamate metabolism, diterpenoid biosynthesis, etc.; PBAT-MPs enriched cysteine and methionine metabolism, photosynthesis, MAPK signaling, and other pathways. These significantly enriched pathways functioned in reducing nodule number and promoting peanut tolerance to MPs stresses. This study reveals the effect of PVC-MPs and PBAT-MPs on peanut and rhizobium symbiosis, and provides new perspectives for legume production and environmental safety.

Rhizobia-diatom symbiosis fixes missing nitrogen in the ocean.

Nitrogen (N2) fixation in oligotrophic surface waters is the main source of new nitrogen to the ocean1 and has a key role in fuelling the biological carbon pump2. Oceanic N2 fixation has been attributed almost exclusively to cyanobacteria, even though genes encoding nitrogenase, the enzyme that fixes N2 into ammonia, are widespread among marine bacteria and archaea3-5. Little is known about these non-cyanobacterial N2 fixers, and direct proof that they can fix nitrogen in the ocean has so far been lacking. Here we report the discovery of a non-cyanobacterial N2-fixing symbiont, 'Candidatus Tectiglobus diatomicola', which provides its diatom host with fixed nitrogen in return for photosynthetic carbon. The N2-fixing symbiont belongs to the order Rhizobiales and its association with a unicellular diatom expands the known hosts for this order beyond the well-known N2-fixing rhizobia-legume symbioses on land6. Our results show that the rhizobia-diatom symbioses can contribute as much fixed nitrogen as can cyanobacterial N2 fixers in the tropical North Atlantic, and that they might be responsible for N2 fixation in the vast regions of the ocean in which cyanobacteria are too rare to account for the measured rates.

Timely symbiosis: circadian control of legume-rhizobia symbiosis.

Legumes house nitrogen-fixing endosymbiotic rhizobia in specialised polyploid cells within root nodules. This results in a mutualistic relationship whereby the plant host receives fixed nitrogen from the bacteria in exchange for dicarboxylic acids. This plant-microbe interaction requires the regulation of multiple metabolic and physiological processes in both the host and symbiont in order to achieve highly efficient symbiosis. Recent studies have showed that the success of symbiosis is influenced by the circadian clock of the plant host. Medicago and soybean plants with altered clock mechanisms showed compromised nodulation and reduced plant growth. Furthermore, transcriptomic analyses revealed that multiple genes with key roles in recruitment of rhizobia to plant roots, infection and nodule development were under circadian control, suggesting that appropriate timing of expression of these genes may be important for nodulation. There is also evidence for rhythmic gene expression of key nitrogen fixation genes in the rhizobium symbiont, and temporal coordination between nitrogen fixation in the bacterial symbiont and nitrogen assimilation in the plant host may be important for successful symbiosis. Understanding of how circadian regulation impacts on nodule establishment and function will identify key plant-rhizobial connections and regulators that could be targeted to increase the efficiency of this relationship.

Symphony of survival: Insights into cross-talk mechanisms in plants, bacteria, and fungi for strengthening plant immune responses.

Plants coexist with a diverse array of microorganisms, predominantly bacteria and fungi, in both natural and agricultural environments. While some microorganisms positively influence plant development and yield, others can cause harm to the host, leading to significant adverse impacts on the environment and the economy. Plant growth-promoting microorganisms (PGPM), including plant growth-promoting bacteria, arbuscular mycorrhizal fungus (AMF), and rhizobia, have been found to increase plant biomass production by synthesizing hormones, fixing nitrogen, and solubilizing phosphate and potassium. Numerous studies have contributed to unraveling the complex process of plant-microbe interactions in recent decades. In light of the increasing global challenges such as population growth, climate change, and resource scarcity, it has become imperative to explore the potential of plant-bacteria-fungi crosstalk in promoting sustainability. This review aims to bridge existing knowledge gaps, providing a roadmap for future research in this dynamic field by synthesizing current knowledge and identifying emerging trends.

A Single Latent Plant Growth-Promoting Endophyte BH46 Enhances Houttuynia cordata Thunb. Yield and Quality.

Plant growth-promoting endophytes (PGPE) can effectively regulate plant growth and metabolism. The regulation is modulated by metabolic signals, and the resulting metabolites can have considerable effects on the plant yield and quality. Here, tissue culture Houttuynia cordata Thunb., was inoculated with Rhizobium sp. (BH46) to determine the effect of BH46 on H. cordata growth and metabolism, and elucidate associated regulatory mechanisms. The results revealed that BH46 metabolized indole-3-acetic acid and induced 1-aminocyclopropane-1-carboxylate deaminase to decrease ethylene metabolism. Host peroxidase synthesis MPK3/MPK6 genes were significantly downregulated, whereas eight genes associated with auxins, cytokinins, abscisic acid, jasmonic acid, and antioxidant enzymes were significantly upregulated. Eight genes associated with flavonoid biosynthesis were significantly upregulated, with the CPY75B1 gene regulating the production of rutin and quercitrin and the HCT gene directly regulating the production of chlorogenic acid. Therefore, BH46 influences metabolic signals in H. cordata to modulate its growth and metabolism, in turn, enhancing yield and quality of H. cordata.

Production and characterization of bacterial cellulose by Rhizobium sp. isolated from bean root.

Bacterial cellulose (BC) is a natural polymer renowned for its unique physicochemical and mechanical attributes, including notable water-holding capacity, crystallinity, and a pristine fiber network structure. While BC has broad applications spanning agriculture, industry, and medicine, its industrial utilization is hindered by production costs and yield limitations. In this study, Rhizobium sp. was isolated from bean roots and systematically assessed for BC synthesis under optimal conditions, with a comparative analysis against BC produced by Komagataeibacter hansenii. The study revealed that Rhizobium sp. exhibited optimal BC synthesis when supplied with a 1.5% glucose carbon source and a 0.15% yeast extract nitrogen source. Under static conditions at 30 °C and pH 6.5, the most favorable conditions for growth and BC production (2.5 g/L) were identified. Modifications were introduced using nisin to enhance BC properties, and the resulting BC-nisin composites were comprehensively characterized through various techniques, including FE-SEM, FTIR, porosity, swelling, filtration, and antibacterial activity assessments. The results demonstrated that BC produced by Rhizobium sp. displayed properties comparable to K. hansenii-produced BC. Furthermore, the BC-nisin composites exhibited remarkable inhibitory activity against Escherichia coli and Pseudomonas aeruginosa. This study contributes valuable insights into BC's production, modification, and characterization utilizing Rhizobium sp., highlighting the exceptional properties that render it efficacious across diverse applications.

RinRK1 enhances NF receptors accumulation in nanodomain-like structures at root-hair tip.

Legume-rhizobia root-nodule symbioses involve the recognition of rhizobial Nod factor (NF) signals by NF receptors, triggering both nodule organogenesis and rhizobial infection. RinRK1 is induced by NF signaling and is essential for infection thread (IT) formation in Lotus japonicus. However, the precise mechanism underlying this process remains unknown. Here, we show that RinRK1 interacts with the extracellular domains of NF receptors (NFR1 and NFR5) to promote their accumulation at root hair tips in response to rhizobia or NFs. Furthermore, Flotillin 1 (Flot1), a nanodomain-organizing protein, associates with the kinase domains of NFR1, NFR5 and RinRK1. RinRK1 promotes the interactions between Flot1 and NF receptors and both RinRK1 and Flot1 are necessary for the accumulation of NF receptors at root hair tips upon NF stimulation. Our study shows that RinRK1 and Flot1 play a crucial role in NF receptor complex assembly within localized plasma membrane signaling centers to promote symbiotic infection.

Hopanoid lipids promote soybean-Bradyrhizobium symbiosis.

The symbioses between leguminous plants and nitrogen-fixing bacteria known as rhizobia are well known for promoting plant growth and sustainably increasing soil nitrogen. Recent evidence indicates that hopanoids, a family of steroid-like lipids, promote Bradyrhizobium symbioses with tropical legumes. To characterize hopanoids in Bradyrhizobium symbiosis with soybean, we validated a recently published cumate-inducible hopanoid mutant of Bradyrhizobium diazoefficiens USDA110, Pcu-shc::∆shc. GC-MS analysis showed that this strain does not produce hopanoids without cumate induction, and under this condition, is impaired in growth in rich medium and under osmotic, temperature, and pH stress. In planta, Pcu-shc::∆shc is an inefficient soybean symbiont with significantly lower rates of nitrogen fixation and low survival within the host tissue. RNA-seq revealed that hopanoid loss reduces the expression of flagellar motility and chemotaxis-related genes, further confirmed by swim plate assays, and enhances the expression of genes related to nitrogen metabolism and protein secretion. These results suggest that hopanoids provide a significant fitness advantage to B. diazoefficiens in legume hosts and provide a foundation for future mechanistic studies of hopanoid function in protein secretion and motility.A major problem for global sustainability is feeding our exponentially growing human population while available arable land decreases. Harnessing the power of plant-beneficial microbes is a potential solution, including increasing our reliance on the symbioses of leguminous plants and nitrogen-fixing rhizobia. This study examines the role of hopanoid lipids in the symbiosis between Bradyrhizobium diazoefficiens USDA110, an important commercial inoculant strain, and its economically significant host soybean. Our research extends our knowledge of the functions of bacterial lipids in symbiosis to an agricultural context, which may one day help improve the practical applications of plant-beneficial microbes in agriculture.

Exploring the role of symbiotic modifier peptidases in the legume - rhizobium symbiosis.

Legumes can establish a mutual association with soil-derived nitrogen-fixing bacteria called 'rhizobia' forming lateral root organs called root nodules. Rhizobia inside the root nodules get transformed into 'bacteroids' that can fix atmospheric nitrogen to ammonia for host plants in return for nutrients and shelter. A substantial 200 million tons of nitrogen is fixed annually through biological nitrogen fixation. Consequently, the symbiotic mechanism of nitrogen fixation is utilized worldwide for sustainable agriculture and plays a crucial role in the Earth's ecosystem. The development of effective nitrogen-fixing symbiosis between legumes and rhizobia is very specialized and requires coordinated signaling. A plethora of plant-derived nodule-specific cysteine-rich (NCR or NCR-like) peptides get actively involved in this complex and tightly regulated signaling process of symbiosis between some legumes of the IRLC (Inverted Repeat-Lacking Clade) and Dalbergioid clades and nitrogen-fixing rhizobia. Recent progress has been made in identifying two such peptidases that actively prevent bacterial differentiation, leading to symbiotic incompatibility. In this review, we outlined the functions of NCRs and two nitrogen-fixing blocking peptidases: HrrP (host range restriction peptidase) and SapA (symbiosis-associated peptidase A). SapA was identified through an overexpression screen from the Sinorhizobium meliloti 1021 core genome, whereas HrrP is inherited extra-chromosomally. Interestingly, both peptidases affect the symbiotic outcome by degrading the NCR peptides generated from the host plants. These NCR-degrading peptidases can shed light on symbiotic incompatibility, helping to elucidate the reasons behind the inefficiency of nitrogen fixation observed in certain groups of rhizobia with specific legumes.

Autophagy and Symbiosis: Membranes, ER, and Speculations.

The interaction of plants and soil bacteria rhizobia leads to the formation of root nodule symbiosis. The intracellular form of rhizobia, the symbiosomes, are able to perform the nitrogen fixation by converting atmospheric dinitrogen into ammonia, which is available for plants. The symbiosis involves the resource sharing between two partners, but this exchange does not include equivalence, which can lead to resource scarcity and stress responses of one of the partners. In this review, we analyze the possible involvement of the autophagy pathway in the process of the maintenance of the nitrogen-fixing bacteria intracellular colony and the changes in the endomembrane system of the host cell. According to in silico expression analysis, ATG genes of all groups were expressed in the root nodule, and the expression was developmental zone dependent. The analysis of expression of genes involved in the response to carbon or nitrogen deficiency has shown a suboptimal access to sugars and nitrogen in the nodule tissue. The upregulation of several ER stress genes was also detected. Hence, the root nodule cells are under heavy bacterial infection, carbon deprivation, and insufficient nitrogen supply, making nodule cells prone to autophagy. We speculate that the membrane formation around the intracellular rhizobia may be quite similar to the phagophore formation, and the induction of autophagy and ER stress are essential to the success of this process.

Identification and characterization of the TetR family transcriptional regulator NffT in Rhizobium johnstonii.

Symbiotic nitrogen fixation (SNF) by rhizobia is not only the main natural bionitrogen-source for organisms but also a green process leveraged to increase the fertility of soil for agricultural production. However, an insufficient understanding of the regulatory mechanism of SNF hinders its practical application. During SNF, nifA-fixA signaling is essential for the biosynthesis of nitrogenases and electron transfer chain proteins. In the present study, the TetR regulator NffT, whose mutation increased fixA expression, was discovered through a fixA-promoter-ß-glucuronidase fusion assay performed with Rhizobium johnstonii. Real-time quantitative PCR analysis showed that nffT deletion increased the expression of symbiotic genes including nifA and fixA in nifA-fixA signaling, and fixL, fixK, fnrN, and fixN9 in fixL-fixN signaling. nffT overexpression resulted in disordered nodules and reduced nitrogen-fixing efficiency. Electrophoretic mobility shift assays revealed that NffT directly regulated the transcription of RL0091-93, which encode an ATP-binding ABC transporter predicted to be involved in carbohydrate transport. Purified His-tagged NffT bound to a 68 bp DNA sequence located -32 to -99 bp upstream of RL0091-93 and NffT deletion significantly increased the expression of RL0091-93. nffT-promoter-ß-glucuronidase fusion assay indicated that nffT expression was regulated by the cobNTS genes and cobalamin. Mutations in cobNTS significantly decreased the expression of nffT, and cobalamin restored its expression. These results revealed that NffT affects nodule development and nitrogen-fixing reaction by participating in a complex regulatory network of symbiotic and carbohydrate metabolic genes and, thus, plays a pivotal regulatory role during symbiosis of R. johnstonii-Pisum sativum.IMPORTANCESymbiotic nitrogen fixation (SNF) by rhizobia is a green way to maintain soil fertility without causing environmental pollution or consuming chemical energy. A detailed understanding of the regulatory mechanism of this complex process is essential for promoting sustainable agriculture. In this study, we discovered the TetR-type regulator NffT, which suppressed the expression of fixA in Rhizobium johnstonii. Furthermore, NffT was confirmed to play pleiotropic roles in R. johnstonii-Pisum sativum symbiosis; specifically, it inhibited rhizobial growth, nodule differentiation, and nitrogen-fixing reactions. We revealed that NffT indirectly affected R. johnstonii-P. sativum symbiosis by participating in a complex regulatory network of symbiotic and carbohydrate metabolic genes. Furthermore, cobalamin, a chemical molecule, was reported for the first time to be involved in TetR-type protein transcription during symbiosis. Thus, NffT identification connects SNF regulation with genetic, metabolic, and chemical signals and provides new insights into the complex regulation of SNF, laying an experimental basis for the targeted construction of rhizobial strains with highly efficient nitrogen-fixing capacity.

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.

An Integrated Affinity Chromatography-Based Approach to Unravel the sRNA Interactome in Nitrogen-Fixing Rhizobia.

The activity mechanism and function of bacterial base-pairing small non-coding RNA regulators (sRNAs) are largely shaped by their main interacting cellular partners, i.e., proteins and mRNAs. We describe here an MS2 affinity chromatography-based procedure adapted to unravel the sRNA interactome in nitrogen-fixing legume endosymbiotic bacteria. The method consists of tagging of the bait sRNA at its 5'-end with the MS2 aptamer followed by pulse overexpression and immobilization of the chimeric transcript from cell lysates by an MS2-MBP fusion protein conjugated to an amylose resin. The sRNA-binding proteins and target mRNAs are further profiled by mass spectrometry and RNAseq, respectively.

CRISPR/Cas9-Mediated Knock-Out of the MtCLE35 Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate Conditions.

Legume plants have the ability to establish a symbiotic relationship with soil bacteria known as rhizobia. The legume-rhizobium symbiosis results in the formation of symbiotic root nodules, where rhizobia fix atmospheric nitrogen. A host plant controls the number of symbiotic nodules to meet its nitrogen demands. CLE (CLAVATA3/EMBRYO SURROUNDING REGION) peptides produced in the root in response to rhizobial inoculation and/or nitrate have been shown to control the number of symbiotic nodules. Previously, the MtCLE35 gene was found to be upregulated by rhizobia and nitrate treatment in Medicago truncatula, which systemically inhibited nodulation when overexpressed. In this study, we obtained several knock-out lines in which the MtCLE35 gene was mutated using the CRISPR/Cas9-mediated system. M. truncatula lines with the MtCLE35 gene knocked out produced increased numbers of nodules in the presence of nitrate in comparison to wild-type plants. Moreover, in the presence of nitrate, the expression levels of two other nodulation-related MtCLE genes, MtCLE12 and MtCLE13, were reduced in rhizobia-inoculated roots, whereas no significant difference in MtCLE35 gene expression was observed between nitrate-treated and rhizobia-inoculated control roots. Together, these findings suggest the key role of MtCLE35 in the number of nodule numbers under high-nitrate conditions, under which the expression levels of other nodulation-related MtCLE genes are reduced.

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 the regulatory mechanisms of salicylate degradation gene cluster cehGHIR4 in Rhizobium sp. strain X9.

In a previous study, the novel gene cluster cehGHI was found to be involved in salicylate degradation through the CoA-mediated pathway in Rhizobium sp. strain X9 (Mol Microbiol 116:783-793, 2021). In this study, an IclR family transcriptional regulator CehR4 was identified. In contrast to other regulators involved in salicylate degradation, cehR4 forms one operon with the gentisyl-CoA thioesterase gene cehI, while cehG and cehH (encoding salicylyl-CoA ligase and salicylyl-CoA hydroxylase, respectively) form another operon. cehGH and cehIR4 are divergently transcribed, and their promoters overlap. The results of the electrophoretic mobility shift assay and DNase I footprinting showed that CehR4 binds to the 42-bp motif between genes cehH and cehI, thus regulating transcription of cehGH and cehIR4. The repeat sequences IR1 (5'-TTTATATAAA-3') and IR2 (5'-AATATAGAAA-3') in the motif are key sites for CehR4 binding. The arrangement of cehGH and cehIR4 and the conserved binding motif of CehR4 were also found in other bacterial genera. The results disclose the regulatory mechanism of salicylate degradation through the CoA pathway and expand knowledge about the systems controlled by IclR family transcriptional regulators.IMPORTANCEThe long-term residue of aromatic compounds in the environment has brought great threat to the environment and human health. Microbial degradation plays an important role in the elimination of aromatic compounds in the environment. Salicylate is a common intermediate metabolite in the degradation of various aromatic compounds. Recently, Rhizobium sp. strain X9, capable of degrading the pesticide carbaryl, was isolated from carbaryl-contaminated soil. Salicylate is the intermediate metabolite that appeared during the degradation of carbaryl, and a novel salicylate degradation pathway and the involved gene cluster cehGHIR4 have been identified. This study identified and characterized the IclR transcription regulator CehR4 that represses transcription of cehGHIR4 gene cluster. Additionally, the genetic arrangements of cehGH and cehIR4 and the binding sites of CehR4 were also found in other bacterial genera. This study provides insights into the biodegradation of salicylate and provides an application in the bioremediation of aromatic compound-contaminated environments.

What determines symbiotic nitrogen fixation efficiency in rhizobium: recent insights into Rhizobium leguminosarum.

Symbiotic nitrogen fixation (SNF) by rhizobium, a Gram-negative soil bacterium, is an essential component in the nitrogen cycle and is a sustainable green way to maintain soil fertility without chemical energy consumption. SNF, which results from the processes of nodulation, rhizobial infection, bacteroid differentiation and nitrogen-fixing reaction, requires the expression of various genes from both symbionts with adaptation to the changing environment. To achieve successful nitrogen fixation, rhizobia and their hosts cooperate closely for precise regulation of symbiotic genes, metabolic processes and internal environment homeostasis. Many researches have progressed to reveal the ample information about regulatory aspects of SNF during recent decades, but the major bottlenecks regarding improvement of nitrogen-fixing efficiency has proven to be complex. In this mini-review, we summarize recent advances that have contributed to understanding the rhizobial regulatory aspects that determine SNF efficiency, focusing on the coordinated regulatory mechanism of symbiotic genes, oxygen, carbon metabolism, amino acid metabolism, combined nitrogen, non-coding RNAs and internal environment homeostasis. Unraveling regulatory determinants of SNF in the nitrogen-fixing protagonist rhizobium is expected to promote an improvement of nitrogen-fixing efficiency in crop production.

CRK12: A Key Player in Regulating the Phaseolus vulgaris-Rhizobium tropici Symbiotic Interaction.

Cysteine-rich receptor-like kinases (CRKs) are a type of receptor-like kinases (RLKs) that are important for pathogen resistance, extracellular reactive oxygen species (ROS) signaling, and programmed cell death in plants. In a previous study, we identified 46 CRK family members in the Phaseolus vulgaris genome and found that CRK12 was highly upregulated under root nodule symbiotic conditions. To better understand the role of CRK12 in the Phaseolus-Rhizobia symbiotic interaction, we functionally characterized this gene by overexpressing (CRK12-OE) and silencing (CRK12-RNAi) it in a P. vulgaris hairy root system. We found that the constitutive expression of CRK12 led to an increase in root hair length and the expression of root hair regulatory genes, while silencing the gene had the opposite effect. During symbiosis, CRK12-RNAi resulted in a significant reduction in nodule numbers, while CRK12-OE roots showed a dramatic increase in rhizobial infection threads and the number of nodules. Nodule cross sections revealed that silenced nodules had very few infected cells, while CRK12-OE nodules had enlarged infected cells, whose numbers had increased compared to controls. As expected, CRK12-RNAi negatively affected nitrogen fixation, while CRK12-OE nodules fixed 1.5 times more nitrogen than controls. Expression levels of genes involved in symbiosis and ROS signaling, as well as nitrogen export genes, supported the nodule phenotypes. Moreover, nodule senescence was prolonged in CRK12-overexpressing roots. Subcellular localization assays showed that the PvCRK12 protein localized to the plasma membrane, and the spatiotemporal expression patterns of the CRK12-promoter::GUS-GFP analysis revealed a symbiosis-specific expression of CRK12 during the early stages of rhizobial infection and in the development of nodules. Our findings suggest that CRK12, a membrane RLK, is a novel regulator of Phaseolus vulgaris-Rhizobium tropici symbiosis.

MsSPL9 Modulates Nodulation under Nitrate Sufficiency Condition in Medicago sativa.

Nodulation in Leguminous spp. is induced by common environmental cues, such as low nitrogen availability conditions, in the presence of the specific Rhizobium spp. in the rhizosphere. Medicago sativa (alfalfa) is an important nitrogen-fixing forage crop that is widely cultivated around the world and relied upon as a staple source of forage in livestock feed. Although alfalfa's relationship with these bacteria is one of the most efficient between rhizobia and legume plants, breeding for nitrogen-related traits in this crop has received little attention. In this report, we investigate the role of Squamosa-Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in nodulation in alfalfa. Transgenic alfalfa plants with SPL9-silenced (SPL9-RNAi) and overexpressed (35S::SPL9) were compared to wild-type (WT) alfalfa for phenotypic changes in nodulation in the presence and absence of nitrogen. Phenotypic analyses showed that silencing of MsSPL9 in alfalfa caused an increase in the number of nodules. Moreover, the characterization of phenotypic and molecular parameters revealed that MsSPL9 regulates nodulation under a high concentration of nitrate (10 mM KNO3) by regulating the transcription levels of the nitrate-responsive genes Nitrate Reductase1 (NR1), NR2, Nitrate transporter 2.5 (NRT2.5), and a shoot-controlled autoregulation of nodulation (AON) gene, Super numeric nodules (SUNN). While MsSPL9-overexpressing transgenic plants have dramatically increased transcript levels of SUNN, NR1, NR2, and NRT2.5, reducing MsSPL9 caused downregulation of these genes and displayed a nitrogen-starved phenotype, as downregulation of the MsSPL9 transcript levels caused a nitrate-tolerant nodulation phenotype. Taken together, our results suggest that MsSPL9 regulates nodulation in alfalfa in response to nitrate.