The type III effector NopL interacts with GmREM1a and GmNFR5 to promote symbiosis in soybean.

The establishment of symbiotic interactions between leguminous plants and rhizobia requires complex cellular programming activated by Rhizobium Nod factors (NFs) as well as type III effector (T3E)-mediated symbiotic signaling. However, the mechanisms by which different signals jointly affect symbiosis are still unclear. Here we describe the mechanisms mediating the cross-talk between the broad host range rhizobia Sinorhizobium fredii HH103 T3E Nodulation Outer Protein L (NopL) effector and NF signaling in soybean. NopL physically interacts with the Glycine max Remorin 1a (GmREM1a) and the NFs receptor NFR5 (GmNFR5) and promotes GmNFR5 recruitment by GmREM1a. Furthermore, NopL and NF influence the expression of GmRINRK1, a receptor-like kinase (LRR-RLK) ortholog of the Lotus RINRK1, that mediates NF signaling. Taken together, our work indicates that S. fredii NopL can interact with the NF signaling cascade components to promote the symbiotic interaction in soybean.

Periodic cytokinin responses in Lotus japonicus rhizobium infection and nodule development.

Host plants benefit from legume root nodule symbiosis with nitrogen-fixing bacteria under nitrogen-limiting conditions. In this interaction, the hosts must regulate nodule numbers and distribution patterns to control the degree of symbiosis and maintain root growth functions. The host response to symbiotic bacteria occurs discontinuously but repeatedly at the region behind the tip of the growing roots. Here, live-imaging and transcriptome analyses revealed oscillating host gene expression with approximately 6-hour intervals upon bacterial inoculation. Cytokinin response also exhibited a similar oscillation pattern. Cytokinin signaling is crucial to maintaining the periodicity, as observed in cytokinin receptor mutants displaying altered infection foci distribution. This periodic regulation influences the size of the root region responsive to bacteria, as well as the nodulation process progression.

Mapping the molecular landscape of Lotus japonicus nodule organogenesis through spatiotemporal transcriptomics.

Legumes acquire nitrogen-fixing ability by forming root nodules. Transferring this capability to more crops could reduce our reliance on nitrogen fertilizers, thereby decreasing environmental pollution and agricultural production costs. Nodule organogenesis is complex, and a comprehensive transcriptomic atlas is crucial for understanding the underlying molecular events. Here, we utilized spatial transcriptomics to investigate the development of nodules in the model legume, Lotus japonicus. Our investigation has identified the developmental trajectories of two critical regions within the nodule: the infection zone and peripheral tissues. We reveal the underlying biological processes and provide gene sets to achieve symbiosis and material exchange, two essential aspects of nodulation. Among the candidate regulatory genes, we illustrate that LjNLP3, a transcription factor belonging to the NIN-LIKE PROTEIN family, orchestrates the transition of nodules from the differentiation to maturation. In summary, our research advances our understanding of nodule organogenesis and provides valuable data for developing symbiotic nitrogen-fixing crops.

Water deficit response in nodulated soybean roots: a comprehensive transcriptome and translatome network analysis.

BACKGROUND: Soybean establishes a mutualistic interaction with nitrogen-fixing rhizobacteria, acquiring most of its nitrogen requirements through symbiotic nitrogen fixation. This crop is susceptible to water deficit; evidence suggests that its nodulation status-whether it is nodulated or not-can influence how it responds to water deficit. The translational control step of gene expression has proven relevant in plants subjected to water deficit. RESULTS: Here, we analyzed soybean roots' differential responses to water deficit at transcriptional, translational, and mixed (transcriptional + translational) levels. Thus, the transcriptome and translatome of four combined-treated soybean roots were analyzed. We found hormone metabolism-related genes among the differentially expressed genes (DEGs) at the translatome level in nodulated and water-restricted plants. Also, weighted gene co-expression network analysis followed by differential expression analysis identified gene modules associated with nodulation and water deficit conditions. Protein-protein interaction network analysis was performed for subsets of mixed DEGs of the modules associated with the plant responses to nodulation, water deficit, or their combination. CONCLUSIONS: Our research reveals that the stand-out processes and pathways in the before-mentioned plant responses partially differ; terms related to glutathione metabolism and hormone signal transduction (2 C protein phosphatases) were associated with the response to water deficit, terms related to transmembrane transport, response to abscisic acid, pigment metabolic process were associated with the response to nodulation plus water deficit. Still, two processes were common: galactose metabolism and branched-chain amino acid catabolism. A comprehensive analysis of these processes could lead to identifying new sources of tolerance to drought in soybean.

Inter-species interaction of bradyrhizobia affects their colonization and plant growth promotion in Arachis hypogaea.

Bradyrhizobia are the principal symbiotic partner of the leguminous plant and take active part in biological nitrogen-fixation. The present investigation explores the underlying competition among different strains during colonization in host roots. Six distinct GFP and RFP-tagged Bradyrhizobium strains were engineered to track them inside the peanut roots either independently or in combination. The Bradyrhizobium strains require different time-spans ranging from 4 to 21 days post-infection (dpi) for successful colonization which further varies in presence of another strain. While most of the individual strains enhanced the shoot and root dry weight, number of nodules, and nitrogen fixation capabilities of the host plants, no significant enhancement of plant growth and nodulation efficiency was observed when they were allowed to colonize in combinations. However, if among the combinations one strains is SEMIA 6144, the co-infection results in higher growth and nodulation efficiency of the hosts. From the competition experiments it has been found that Bradyrhizobium japonicum SEMIA 6144 was found to be the most dominant strain for effective nodulation in peanut. The extent of biofilm and exopolysaccharide (EPS) production by these isolates, individually or in combinations, were envisaged to correlate whether these parameters have any impact on the symbiotic association. But the extent of colonization, growth-promotion and nitrogen-fixation ability drastically lowered when a strain present together with other Bradyrhizobium strain. Therefore, it is imperative to understand the interaction between two co-inoculating Bradyrhizobium species for nodulation followed by plant growth promotion to develop suitable consortia for enhancing BNF in peanut and possibly for other legumes.

Comparative transcriptomic analysis of the nodulation-competent zone and inference of transcription regulatory network in silicon applied Glycine max [L.]-Merr. Roots.

KEY MESSAGE: The study unveils Si's regulatory influence by regulating DEGs, TFs, and TRs. Further bHLH subfamily and auxin transporter pathway elucidates the mechanisms enhancing root development and nodulation. Soybean is a globally important crop serving as a primary source of vegetable protein for millions of individuals. The roots of these plants harbour essential nitrogen fixing structures called nodules. This study investigates the multifaceted impact of silicon (Si) application on soybean, with a focus on root development, and nodulation employing comprehensive transcriptomic analyses and gene regulatory network. RNA sequence analysis was utilised to examine the change in gene expression and identify the noteworthy differentially expressed genes (DEGs) linked to the enhancement of soybean root nodulation and root development. A set of 316 genes involved in diverse biological and molecular pathways are identified, with emphasis on transcription factors (TFs) and transcriptional regulators (TRs). The study uncovers TF and TR genes, categorized into 68 distinct families, highlighting the intricate regulatory landscape influenced by Si in soybeans. Upregulated most important bHLH subfamily and the involvement of the auxin transporter pathway underscore the molecular mechanisms contributing to enhanced root development and nodulation. The study bridges insights from other research, reinforcing Si's impact on stress-response pathways and phenylpropanoid biosynthesis crucial for nodulation. The study reveals significant alterations in gene expression patterns associated with cellular component functions, root development, and nodulation in response to Si.

Molecular Mechanism of GmSNE3 Ubiquitin Ligase-Mediated Inhibition of Soybean Nodulation by Halosulfuron Methyl.

In this study, the role of E3 ubiquitin ligase GmSNE3 in halosulfuron methyl (HSM) inhibiting soybean nodulation was investigated. GmSNE3 was strongly induced by HSM stress, and the overexpression of GmSNE3 significantly reduced the number of soybean nodules. Further investigation found that GmSNE3 could interact with a nodulation signaling pathway 1 protein (GmNSP1a) and GmSNE3 could mediate the degradation of GmNSP1a. Importantly, GmSNE3-mediated degradation of GmNSP1a could be promoted by HSM stress. Moreover, HSM stress and the overexpression of GmSNE3 resulted in a substantial decrease in the expression of the downstream target genes of GmNSP1a. These results revealed that HSM promotes the ubiquitin-mediated degradation of GmNSP1a by inducing GmSNE3, thereby inhibiting the regulatory effect of GmNSP1a on its downstream target genes and ultimately leading to a reduction in nodulation. Our findings will promote a better understanding of the toxic mechanism of herbicides on the symbiotic nodulation between legumes and rhizobia.

Dynamic modulation of nodulation factor receptor levels by phosphorylation-mediated functional switch of a RING-type E3 ligase during legume nodulation.

The precise control of receptor levels is crucial for initiating cellular signaling transduction in response to specific ligands; however, such mechanisms regulating nodulation factor (NF) receptor (NFR)-mediated perception of NFs to establish symbiosis remain unclear. In this study, we unveil the pivotal role of the NFR-interacting RING-type E3 ligase 1 (NIRE1) in regulating NFR1/NFR5 homeostasis to optimize rhizobial infection and nodule development in Lotus japonicus. We demonstrated that NIRE1 has a dual function in this regulatory process. It associates with both NFR1 and NFR5, facilitating their degradation through K48-linked polyubiquitination before rhizobial inoculation. However, following rhizobial inoculation, NFR1 phosphorylates NIRE1 at a conserved residue, Tyr-109, inducing a functional switch in NIRE1, which enables NIRE1 to mediate K63-linked polyubiquitination, thereby stabilizing NFR1/NFR5 in infected root cells. The introduction of phospho-dead NIRE1Y109F leads to delayed nodule development, underscoring the significance of phosphorylation at Tyr-109 in orchestrating symbiotic processes. Conversely, expression of the phospho-mimic NIRE1Y109E results in the formation of spontaneous nodules in L. japonicus, further emphasizing the critical role of the phosphorylation-dependent functional switch in NIRE1. In summary, these findings uncover a fine-tuned symbiotic mechanism that a single E3 ligase could undergo a phosphorylation-dependent functional switch to dynamically and precisely regulate NF receptor protein levels.

Combined dynamic transcriptome and flavonoid metabolome reveal the role of Mo nanoparticles in the nodulation process in soybean.

Symbiotic nitrogen fixation can reduce the impact of agriculture on the environment by reducing fertilizer input. The rapid development of nanomaterials in agriculture provides a new prospect for us to improve the biological nitrogen fixation ability of leguminous crops. Molybdenum is an important component of nitrogenase, and the potential application of MoO3NPs in agriculture is largely unexplored. In this study, on the basis of verifying that MoO3NPs can improve the nitrogen fixation ability of soybean, the effects of MoO3NPs on the symbiotic nitrogen fixation process of soybean were investigated by using dynamic transcriptome and targeted metabolome techniques. Here we showed that compared with conventional molybdenum fertilizer, minute concentrations of MoO3NPs (0.01-0.1 mg kg-1) could promote soybean growth and nitrogen fixation efficiency. The nodules number, fresh nodule weight and nitrogenase activity of 0.1 mg kg-1 were increased by 17 %, 14 % and 27 %, and plant nitrogen accumulation increased by 17 %. Compared with conventional molybdenum fertilizer, MoO3NPs had a greater effect on apigenin, kaempferol and other flavonoid, and the expression of nodulation related genes such as ENOD93, F3'H. Based on WGCNA analysis, we identified a core gene GmCHS9 that was positively responsive to molybdenum and was highly expressed during MoO3NPs induced nodulation. MoO3NPs could improve the nitrogen fixation ability of soybean by promoting the secretion of flavonoids and the expression of key genes. This study provided a new perspective for the nano-strengthening strategy of nodules development and flavonoid biosynthesis by molybdenum.

The role of GmHSP23.9 in regulating soybean nodulation under elevated CO2 condition.

Legume-rhizobia symbiosis offers a unique approach to increase leguminous crop yields. Previous studies have indicated that the number of soybean nodules are increased under elevated CO2 concentration. However, the underlying mechanism behind this phenomenon remains elusive. In this study, transcriptome analysis was applied to identify candidate genes involved in regulating soybean nodulation mediated by elevated CO2 concentration. Among the different expression genes (DEGs), we identified a gene encoding small heat shock protein (sHSP) called GmHSP23.9, which mainly expressed in soybean roots and nodules, and its expression was significantly induced by rhizobium USDA110 infection at 14 days after inoculation (DAI) under elevated CO2 conditions. We further investigated the role of GmHSP23.9 by generating transgenic composite plants carrying GmHSP23.9 overexpression (GmHSP23.9-OE), RNA interference (GmHSP23.9-RNAi), and CRISPR-Cas9 (GmHSP23.9-KO), and these modifications resulted in notable changes in nodule number and the root hairs deformation and suggesting that GmHSP23.9 function as an important positive regulator in soybean. Moreover, we found that altering the expression of GmHSP23.9 influenced the expression of genes involved in the Nod factor signaling pathway and AON signaling pathway to modulate soybean nodulation. Interestingly, we found that knocking down of GmHSP23.9 prevented the increase in the nodule number of soybean in response to elevated CO2 concentration. This research has successfully identified a crucial regulator that influences soybean nodulation under elevated CO2 level and shedding new light on the role of sHSPs in legume nodulation.

Regulatory roles of the second messenger c-di-GMP in beneficial plant-bacteria interactions.

The rhizosphere system of plants hosts a diverse consortium of bacteria that confer beneficial effects on plant, such as plant growth-promoting rhizobacteria (PGPR), biocontrol agents with disease-suppression activities, and symbiotic nitrogen fixing bacteria with the formation of root nodule. Efficient colonization in planta is of fundamental importance for promoting of these beneficial activities. However, the process of root colonization is complex, consisting of multiple stages, including chemotaxis, adhesion, aggregation, and biofilm formation. The secondary messenger, c-di-GMP (cyclic bis-(3'-5') dimeric guanosine monophosphate), plays a key regulatory role in a variety of physiological processes. This paper reviews recent progress on the actions of c-di-GMP in plant beneficial bacteria, with a specific focus on its role in chemotaxis, biofilm formation, and nodulation.

Chickpea (Cicer arietinum) PHO1 family members function redundantly in Pi transport and root nodulation.

Phosphorus (P), a macronutrient, plays key roles in plant growth, development, and yield. Phosphate (Pi) transporters (PHTs) and PHOSPHATE1 (PHO1) are central to Pi acquisition and distribution. Potentially, PHO1 is also involved in signal transduction under low P. The current study was designed to identify and functionally characterize the PHO1 gene family in chickpea (CaPHO1s). Five CaPHO1 genes were identified through a comprehensive genome-wide search. Phylogenetically, CaPHO1s formed two clades, and protein sequence analyses confirmed the presence of conserved domains. CaPHO1s are expressed in different plant organs including root nodules and are induced by Pi-limiting conditions. Functional complementation of atpho1 mutant with three CaPHO1 members, CaPHO1, CaPHO1;like, and CaPHO1;H1, independently demonstrated their role in root to shoot Pi transport, and their redundant functions. To further validate this, we raised independent RNA-interference (RNAi) lines of CaPHO1, CaPHO1;like, and CaPHO1;H1 along with triple mutant line in chickpea. While single gene RNAi lines behaved just like WT, triple knock-down RNAi lines (capho1/like/h1) showed reduced shoot growth and shoot Pi content. Lastly, we showed that CaPHO1s are involved in root nodule development and Pi content. Our findings suggest that CaPHO1 members function redundantly in root to shoot Pi export and root nodule development in chickpea.

Rhizobial-induced phosphatase GmPP2C61A positively regulates soybean nodulation.

Symbiotic nitrogen fixation (SNF) is crucial for legumes, providing them with the nitrogen necessary for plant growth and development. Nodulation is the first step in the establishment of SNF. However, the determinant genes in soybean nodulation and the understanding of the underlying molecular mechanisms governing nodulation are still limited. Herein, we identified a phosphatase, GmPP2C61A, which was specifically induced by rhizobia inoculation. Using transgenic hairy roots harboring GmPP2C61A::GUS, we showed that GmPP2C61A was mainly induced in epidermal cells following rhizobia inoculation. Functional analysis revealed that knockdown or knock-out of GmPP2C61A significantly reduced the number of nodules, while overexpression of GmPP2C61A promoted nodule formation. Additionally, GmPP2C61A protein was mainly localized in the cytoplasm and exhibited conserved phosphatase activity in vitro. Our findings suggest that phosphatase GmPP2C61A serves as a critical regulator in soybean nodulation, highlighting its potential significance in enhancing symbiotic nitrogen fixation.

High freezing sensitivity of legumes relative to other herbaceous species in northern temperate plant communities.

BACKGROUND AND AIMS: Reduced snow cover and increased air temperature variability are predicted to expose overwintering herbaceous plants to more severe freezing in some northern temperate regions. Legumes are a key functional group that may exhibit lower freezing tolerance than other species in these regions, but this trend has been observed only for non-native legumes. Our aim was to confirm if this trend is restricted to non-native legumes or whether native legumes in these regions also exhibit low freezing tolerance. METHODS: First, we transplanted legumes (five non-native species and four native species) into either an old field (non-native) or a prairie (native) and used snow removal to expose the plots to increased soil freezing. Second, we grew plants in mesocosms (old field) and pots (prairie species) and exposed them in controlled environment chambers to a range of freezing treatments (control, 0, -5 or -10 °C) in winter or spring. We assessed freezing responses by comparing differences in biomass, cover and nodulation between freezing (or snow removal) treatments and controls. KEY RESULTS: Among legume species, lower freezing tolerance was positively correlated with a lower proportion of nodulated plants and active nodules, and under controlled conditions, freezing-induced reductions in above-ground biomass were lower on average in native legumes than in non-native legumes. Nevertheless, both non-native and native legumes (except Desmodium canadense) exhibited greater reductions in biomass in response to increased freezing than their non-leguminous neighbours, both in controlled environments and in the field. CONCLUSIONS: These results demonstrate that both native and non-native legumes exhibit low freezing tolerance relative to other herbaceous species in northern temperate plant communities. By reducing legume biomass and nodulation, increased soil freezing could reduce nitrogen inputs into these systems.

GmNLP1 and GmNLP4 activate nitrate-induced CLE peptides NIC1a/b to mediate nitrate-regulated root nodulation.

Symbiotic nitrogen fixation is an energy-intensive process, to maintain the balance between growth and nitrogen fixation, high concentrations of nitrate inhibit root nodulation. However, the precise mechanism underlying the nitrate inhibition of nodulation in soybean remains elusive. In this study, CRISPR-Cas9-mediated knockout of GmNLP1 and GmNLP4 unveiled a notable nitrate-tolerant nodulation phenotype. GmNLP1b and GmNLP4a play a significant role in the nitrate-triggered inhibition of nodulation, as the expression of nitrate-responsive genes was largely suppressed in Gmnlp1b and Gmnlp4a mutants. Furthermore, we demonstrated that GmNLP1b and GmNLP4a can bind to the promoters of GmNIC1a and GmNIC1b and activate their expression. Manipulations targeting GmNIC1a and GmNIC1b through knockdown or overexpression strategies resulted in either increased or decreased nodule number in response to nitrate. Additionally, transgenic roots that constitutively express GmNIC1a or GmNIC1b rely on both NARK and hydroxyproline O-arabinosyltransferase RDN1 to prevent the inhibitory effects imposed by nitrate on nodulation. In conclusion, this study highlights the crucial role of the GmNLP1/4-GmNIC1a/b module in mediating high nitrate-induced inhibition of nodulation.

Genetically optimizing soybean nodulation improves yield and protein content.

Symbiotic nitrogen fixation in legume nodules requires substantial energy investment from host plants, and soybean (Glycine max (L.) supernodulation mutants show stunting and yield penalties due to overconsumption of carbon sources. We obtained soybean mutants differing in their nodulation ability, among which rhizobially induced cle1a/2a (ric1a/2a) has a moderate increase in nodule number, balanced carbon allocation, and enhanced carbon and nitrogen acquisition. In multi-year and multi-site field trials in China, two ric1a/2a lines had improved grain yield, protein content and sustained oil content, demonstrating that gene editing towards optimal nodulation improves soybean yield and quality.

A lateral organ boundaries domain transcription factor acts downstream of the auxin response factor 2 to control nodulation and root architecture in Medicago truncatula.

Legume plants develop two types of root postembryonic organs, lateral roots and symbiotic nodules, using shared regulatory components. The module composed by the microRNA390, the Trans-Acting SIRNA3 (TAS3) RNA and the Auxin Response Factors (ARF)2, ARF3, and ARF4 (miR390/TAS3/ARFs) mediates the control of both lateral roots and symbiotic nodules in legumes. Here, a transcriptomic approach identified a member of the Lateral Organ Boundaries Domain (LBD) family of transcription factors in Medicago truncatula, designated MtLBD17/29a, which is regulated by the miR390/TAS3/ARFs module. ChIP-PCR experiments evidenced that MtARF2 binds to an Auxin Response Element present in the MtLBD17/29a promoter. MtLBD17/29a is expressed in root meristems, lateral root primordia, and noninfected cells of symbiotic nodules. Knockdown of MtLBD17/29a reduced the length of primary and lateral roots and enhanced lateral root formation, whereas overexpression of MtLBD17/29a produced the opposite phenotype. Interestingly, both knockdown and overexpression of MtLBD17/29a reduced nodule number and infection events and impaired the induction of the symbiotic genes Nodulation Signaling Pathway (NSP) 1 and 2. Our results demonstrate that MtLBD17/29a is regulated by the miR390/TAS3/ARFs module and a direct target of MtARF2, revealing a new lateral root regulatory hub recruited by legumes to act in the root nodule symbiotic program.

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.

Legume rhizodeposition promotes nitrogen fixation by soil microbiota under crop diversification.

Biological nitrogen fixation by free-living bacteria and rhizobial symbiosis with legumes plays a key role in sustainable crop production. Here, we study how different crop combinations influence the interaction between peanut plants and their rhizosphere microbiota via metabolite deposition and functional responses of free-living and symbiotic nitrogen-fixing bacteria. Based on a long-term (8 year) diversified cropping field experiment, we find that peanut co-cultured with maize and oilseed rape lead to specific changes in peanut rhizosphere metabolite profiles and bacterial functions and nodulation. Flavonoids and coumarins accumulate due to the activation of phenylpropanoid biosynthesis pathways in peanuts. These changes enhance the growth and nitrogen fixation activity of free-living bacterial isolates, and root nodulation by symbiotic Bradyrhizobium isolates. Peanut plant root metabolites interact with Bradyrhizobium isolates contributing to initiate nodulation. Our findings demonstrate that tailored intercropping could be used to improve soil nitrogen availability through changes in the rhizosphere microbiome and its functions.

Nodulating another way: what can we learn from lateral root base nodulation in legumes?

Certain legumes provide a special pathway for rhizobia to invade the root and develop nitrogen-fixing nodules, a process known as lateral root base (LRB) nodulation. This pathway involves intercellular infection at the junction of the lateral roots with the taproot, leading to nodule formation in the lateral root cortex. Remarkably, this LRB pathway serves as a backbone for various adaptative symbiotic processes. Here, we describe different aspects of LRB nodulation and highlight directions for future research to elucidate the mechanisms of this as yet little known but original pathway that will help in broadening our knowledge on the rhizobium-legume symbiosis.