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Symbiotic nodules comprise two classes, indeterminate and determinate, defined by the presence/absence of apical meristem and developmental zonation. Why meristem and zonation are absent from determinate nodules remains unclear. Here, we define cell types in developing soybean nodules, highlighting the undifferentiated infection zones and differentiated nitrogen-fixation zones. Auxin governs infection zone maintenance. GRETCHEN HAGEN 3 (GH3) enzymes deactivate auxin by conjugation and promote cell differentiation. gh3 mutants increased undifferentiated cells and enlarged infection zones. The central symbiosis-transcription factor NIN2a activates GH3.1 to reduce auxin levels and facilitates cell differentiation. High auxin promotes NIN2a protein accumulation and enhances signaling, further deactivating auxin and depleting infection zones. Our findings shed light on the NIN2a-GH3-auxin module that drives soybean nodule cell differentiation. This study challenges our understanding of determinate nodule development and proposes that the regulation of nodule zonation offers valuable insights into broader mechanisms of cell differentiation across plant species., Competing Interests: Declaration of interests Patents entitled “Soybean auxin conjugating enzyme GRETCHEN HAGEN 3 (GH3) gene family and their applications (202410456123.X)” have been applied for in China. X.C., T.T., Z.G., L.H., and J.C. are listed as inventors., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The phosphoenolpyruvate carboxylase kinase of Medicago sativa L. (MsPPCK1) modulates the phosphorylation status and activity of the C4 pathway phosphoenolpyruvate carboxylase enzyme, which is pivotal for photosynthetic carbon assimilation in plants. This study investigated the role of MsPPCK1 in alfalfa by creating transgenic plants overexpressing MsPPCK1 under the control of the CaMV35S promoter. The enhanced alkali tolerance of transgenic plants indicated an important role of MsPPCK1 gene in regulating plant alkali tolerance. Transgenic plants exhibited heightened antioxidant activity (SOD, POD, and CAT), reduced MDA, H 2 O 2 , OFR and REC% content, increased activity of key photosynthetic enzymes (PEPC, PPDK, NADP-ME, and NADP-MDH), and enhanced photosynthetic parameters (Pn, E, Gs, and Ci). Moreover, MsPPCK1 overexpression increased the content of organic acids (oxaloacetic, malic, citric, and succinic acids) in the plants. The upregulation of MsPPCK1 under rhizobial inoculation showcased its other role in nodule development. In transgenic plants, MsDMI2, MsEnod12, and MsNODL4 expression increased, facilitating root nodule development and augmenting plant nodulation. Accelerated root nodule growth positively influences plant growth and yield and enhances alfalfa resistance to alkali stress. This study highlights the pivotal role of MsPPCK1 in fortifying plant alkali stress tolerance and improving yield, underscoring its potential as a key genetic target for developing alkali-tolerant and high-yielding alfalfa varieties., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hua Cai reports financial support was provided by National Natural Science Foundation of China with grant number (32071866). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Leguminous plants provide carbon to symbiotic rhizobia in root nodules to fuel the energy-consuming process of nitrogen fixation. The carbon investment pattern from the acquired sources is crucial for shaping the growth regime of the host plants. The autoregulation of nodulation (AON) signaling pathway tightly regulates the number of nodules that form. AON disruption leads to excessive nodule formation and stunted shoot growth. However, the physiological role of AON in adjusting the carbon investment pattern is unknown. Here, we show that AON plays an important role in sustaining shoot water availability, which is essential for promoting carbon investment in shoot growth in Lotus japonicus. We found that AON-defective mutants exhibit substantial accumulation of nonstructural carbohydrates, such as sucrose. Consistent with this metabolic signature, resilience against water-deficit stress was enhanced in the shoots of the AON-defective mutants. Furthermore, the water uptake ability was attenuated in the AON-defective mutants, likely due to the increased ratio of nodulation zone, which is covered with hydrophobic surfaces, on the roots. These results increase our physiological understanding of legume-rhizobia symbiosis by revealing a trade-off between root nodule formation and shoot water availability., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., (© 2024. The Author(s).)

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.

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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (© 2024 The Authors New Phytologist © 2024 New Phytologist Foundation.)

Legumes establish symbiosis with rhizobia, forming nitrogen-fixing nodules. The central role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in nodule biology has been clearly established. Recently, hydrogen sulfide (H2S) and other reactive sulfur species (RSS) have emerged as novel signaling molecules in animals and plants. A major mechanism by which ROS, RNS, and RSS fulfil their signaling role is the post-translational modification of proteins. To identify possible functions of H2S in nodule development and senescence, we used the tag-switch method to quantify changes in the persulfidation profile of common bean (Phaseolus vulgaris) nodules at different developmental stages. Proteomic analyses indicate that persulfidation plays a regulatory role in plant and bacteroid metabolism and senescence. The effect of a H2S donor on nodule functioning and on several proteins involved in ROS and RNS homeostasis was also investigated. Our results using recombinant proteins and nodulated plants support a crosstalk among H2S, ROS, and RNS, a protective function of persulfidation on redox-sensitive enzymes, and a beneficial effect of H2S on symbiotic nitrogen fixation. We conclude that the general decrease of persulfidation levels observed in plant proteins of aging nodules is one of the mechanisms that disrupt redox homeostasis leading to senescence., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The conservation of GOLVEN (GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide encoding genes across plant genomes capable of forming roots or root-like structures underscores their potential significance in the terrestrial adaptation of plants. This study investigates the function and role of GOLVEN peptide-coding genes in Medicago truncatula. Five out of fifteen GLV/RGF genes were notably upregulated during nodule organogenesis and were differentially responsive to nitrogen deficiency and auxin treatment. Specifically, the expression of MtGLV9 and MtGLV10 at nodule initiation sites was contingent upon the NODULE INCEPTION transcription factor. Overexpression of these five nodule-induced GLV genes in hairy roots of M. truncatula and application of their synthetic peptide analogues led to a decrease in nodule count by 25-50%. Uniquely, the GOLVEN10 peptide altered the positioning of the first formed lateral root and nodule on the primary root axis, an observation we term 'noduletaxis'; this decreased the length of the lateral organ formation zone on roots. Histological section of roots treated with synthetic GOLVEN10 peptide revealed an increased cell number within the root cortical cell layers without a corresponding increase in cell length, leading to an elongation of the root likely introducing a spatiotemporal delay in organ formation. At the transcription level, the GOLVEN10 peptide suppressed expression of microtubule-related genes and exerted its effects by changing expression of a large subset of Auxin responsive genes. These findings advance our understanding of the molecular mechanisms by which GOLVEN peptides modulate root morphology, nodule ontogeny, and interactions with key transcriptional pathways., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Legumes attract symbiotic bacteria and create de novo root organs called nodules. Nodule development consists of bacterial infection of root epidermis and subsequent primordium formation in root cortex, steps that need to be spatiotemporally coordinated. The Lotus japonicus mutant “ daphne ” has uncoupled symbiotic events in epidermis and cortex, in that it promotes excessive bacterial infection in epidermis but does not produce nodule primordia in cortex. Therefore, daphne should be useful for exploring unknown signals that coordinate these events across tissues. Here, we conducted time-course RNA sequencing using daphne after rhizobial infection. We noticed that IAA carboxyl methyltransferase 1 (IAMT1) , which encodes the enzyme that converts auxin (IAA) into its methyl ester (MeIAA), is transiently induced in wild-type roots at early stages of infection but shows different expression dynamics in daphne . IAMT1 serves an important function in shoot development of Arabidopsis , a nonsymbiotic plant, but the function of IAMT1 in roots has not been reported. Phylogenetic tree analysis suggests a gene duplication of IAMT1 in the legume lineage, and we found that one of the two IAMT1s (named IAMT1a ) was induced in roots by epidermal infection. IAMT1a knockdown inhibited nodule development in cortex; however, it had no effect on epidermal infection. The amount of root MeIAA increased with rhizobial infection. Application of MeIAA, but not IAA , significantly induced expression of the symbiotic gene NIN in the absence of rhizobial infection. Our results provide evidence for the role of auxin methylation in an early stage of root nodule development.

Nodule Inception (NIN) is one of the most important root nodule symbiotic genes as it is required for both infection and nodule organogenesis in legumes. Unlike most legumes with a sole NIN gene, there are four putative orthologous NIN genes in soybean (Glycine max). Whether and how these NIN genes contribute to soybean-rhizobia symbiotic interaction remain unknown. In this study, we found that all four GmNIN genes are induced by rhizobia and that conserved CE and CYC binding motifs in their promoter regions are required for their expression in the nodule formation process. By generation of multiplex Gmnin mutants, we found that the Gmnin1a nin2a nin2b triple mutant and Gmnin1a nin1b nin2a nin2b quadruple mutant displayed similar defects in rhizobia infection and root nodule formation, Gmnin2a nin2b produced fewer nodules but displayed a hyper infection phenotype compared to wild type (WT), while the Gmnin1a nin1b double mutant nodulated similar to WT. Overexpression of GmNIN1a, GmNIN1b, GmNIN2a, and GmNIN2b reduced nodule numbers after rhizobia inoculation, with GmNIN1b overexpression having the weakest effect. In addition, overexpression of GmNIN1a, GmNIN2a, or GmNIN2b, but not GmNIN1b, produced malformed pseudo-nodule-like structures without rhizobia inoculation. In conclusion, GmNIN1a, GmNIN2a, and GmNIN2b play functionally redundant yet complicated roles in soybean nodulation. GmNIN1b, although expressed at a comparable level with the other homologs, plays a minor role in root nodule symbiosis. Our work provides insight into the understanding of the asymmetrically redundant function of GmNIN genes in soybean., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Nodulation and symbiotic nitrogen fixation are important factors that determine legume growth. A pot experiment was carried out to determine the effects of Zn-Pb contamination on nodule apoplast (cell walls and intercellular spaces) of bird's foot trefoil (Lotus corniculatus L.) that spontaneously colonized old calamine wastes. The plants were grown in pots filled with sterile calamine substrate (M, metal treated) or expanded clay (NM, untreated) and inoculated with calamine-derived Lotus-nodulating Bradyrhizobium liaoningense. Apoplast reorganization in the nodules was examined using specific dyes for cellulose, pectin and lignin detection, and immuno-histochemical techniques based on monoclonal antibodies against xyloglucan (Lm25), pectins (Jim5 and Jim7), and structural proteins (arabinogalactan protein - Lm14 and extensin - Jim12). Microscopic analysis of metal-treated nodules revealed changes in the apoplast structure and composition of nodule cortex tissues and infected cells. Wall thickening was accompanied by intensified deposition of cellulose, xyloglucan, esterified pectin, arabinogalactan protein and extensin. The metal presence redirected also lignin and suberin deposition in the walls of the nodule cortex tissues. Our results showed reorganization of the apoplast of cortex tissues and infected cells of Lotus nodules under Zn-Pb presence. These changes in the apoplast structure and composition may have created actual barriers for the toxic ions. For this reason, they can be regarded as an element of legume defense strategy against metal stress that enables effective functioning of L. corniculatus-rhizobia symbiosis on Zn-Pb polluted calamine tailings., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Legumes (soybeans, peas, lentils, etc.) play important roles in agriculture on Earth because of their food value and their ability to form a mutualistic beneficial association with rhizobia bacteria. In this association, the host plant benefits from atmospheric nitrogen fixation by rhizobia. The presence of nitrogen in the Mars atmosphere offers the possibility to take advantage of this important plant-microbe association. While some studies have shown that Mars soil simulants can support plant growth, none have investigated if these soils can support the legume-rhizobia symbiosis. In this study, we investigated the establishment of the legume-rhizobia symbiosis on different Mars soil simulants (different grades of the Mojave Mars Simulant (MMS)-1: Coarse, Fine, Unsorted, Superfine, and the MMS-2 simulant). We used the model legume, Medicago truncatula, and its symbiotic partners, Sinorhizobium meliloti and Sinorhizobium medicae, in these experiments. Our results show that root nodules could develop on M. truncatula roots when grown on these Mars soil simulants and were comparable to those formed on plants that were grown on sand. We also detected nifH (a reporter gene for nitrogen fixation) expression inside these nodules. Our results indicate that the different Mars soil simulants used in this study can support legume-rhizobia symbiosis. While the average number of lateral roots and nodule numbers were comparable on plants grown on the different soil simulants, total plant mass was higher in plants grown on MMS-2 soil than on MMS-1 soil and its variants. Our results imply that the chemical composition of the simulants is more critical than their grain size for plant mass. Based on these results, we recommend that the MMS-2 Superfine soil simulant is a better fit than the MMS-1 soil and it's variants for future studies. Our findings can serve as an excellent resource for future studies investigating beneficial plant-microbe associations for sustainable agriculture on Mars., Competing Interests: The authors have declared that no competing interests exist.

To overcome nitrogen deficiency, legume roots establish symbiotic interactions with nitrogen-fixing rhizobia that are fostered in specialized organs (nodules). Similar to other organs, nodule formation is determined by a local maximum of the phytohormone auxin at the primordium site. However, how auxin regulates nodule development remains poorly understood. Here, we found that in soybean, (Glycine max), dynamic auxin transport driven by PIN-FORMED (PIN) transporter GmPIN1 is involved in nodule primordium formation. GmPIN1 was specifically expressed in nodule primordium cells and GmPIN1 was polarly localized in these cells. Two nodulation regulators, (iso)flavonoids trigger expanded distribution of GmPIN1b to root cortical cells, and cytokinin rearranges GmPIN1b polarity. Gmpin1abc triple mutants generated with CRISPR-Cas9 showed the impaired establishment of auxin maxima in nodule meristems and aberrant divisions in the nodule primordium cells. Moreover, overexpression of GmPIN1 suppressed nodule primordium initiation. GmPIN9d, an ortholog of Arabidopsis thaliana PIN2, acts together with GmPIN1 later in nodule development to acropetally transport auxin in vascular bundles, fine-tuning the auxin supply for nodule enlargement. Our findings reveal how PIN-dependent auxin transport modulates different aspects of soybean nodule development and suggest that the establishment of auxin gradient is a prerequisite for the proper interaction between legumes and rhizobia., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Auxin is essential for root development, and its regulatory action is exerted at different steps from perception of the hormone up to transcriptional regulation of target genes. In legume plants there is an overlap between the developmental programs governing lateral root and N 2 -fixing nodule organogenesis, the latter induced as the result of the symbiotic interaction with rhizobia. Here we report the characterization of a member of the L. japonicus TIR1/AFB auxin receptor family, LjAFB6 . A preferential expression of the LjAFB6 gene in the aerial portion of L. japonicus plants was observed. Significant regulation of the expression was not observed during the symbiotic interaction with Mesorhizobium loti and the nodule organogenesis process. In roots, the LjAFB6 expression was induced in response to nitrate supply and was mainly localized in the meristematic regions of both primary and lateral roots. The phenotypic analyses conducted on two independent null mutants indicated a specialized role in the control of primary and lateral root elongation processes in response to auxin, whereas no involvement in the nodulation process was found. We also report the involvement of LjAFB6 in the hypocotyl elongation process and in the control of the expression profile of an auxin-responsive gene.

Carbonic anhydrase (CA) plays a vital role in photosynthetic tissues of higher plants, whereas its non-photosynthetic role in the symbiotic root nodule was rarely characterized. In this study, 13 CA genes were identified in the model legume Lotus japonicus by comparison with Arabidopsis CA genes. Using qPCR and promoter-reporter fusion methods, three previously identified nodule-enhanced CA genes ( Lj αCA2 , Lj αCA6 , and Lj βCA1 ) have been further characterized, which exhibit different spatiotemporal expression patterns during nodule development. Lj αCA2 was expressed in the central infection zone of the mature nodule, including both infected and uninfected cells. Lj αCA6 was restricted to the vascular bundle of the root and nodule. As for Lj βCA1 , it was expressed in most cell types of nodule primordia but only in peripheral cortical cells and uninfected cells of the mature nodule. Using CRISPR/Cas9 technology, the knockout of Lj βCA1 or both LjαCA2 and its homolog, LjαCA1 , did not result in abnormal symbiotic phenotype compared with the wild-type plants, suggesting that LjβCA1 or LjαCA1/2 are not essential for the nitrogen fixation under normal symbiotic conditions. Nevertheless, the nodule-enhanced expression patterns and the diverse distributions in different types of cells imply their potential functions during root nodule symbiosis, such as CO 2 fixation, N assimilation, and pH regulation, which await further investigations.

Legume and rhizobium species can establish a nitrogen-fixing nodule symbiosis. Previous studies have shown that several transcription factors that play a role in (lateral) root development are also involved in nodule development. Chromatin remodeling factors, like transcription factors, are key players in regulating gene expression. However, studies have not investigated whether chromatin remodeling genes that are essential for root development are also involved in nodule development. Here, we studied the role of Medicago (Medicago truncatula) histone deacetylases (MtHDTs) in nodule development. Arabidopsis (Arabidopsis thaliana) orthologs of HDTs have been shown to play a role in root development. MtHDT expression is induced in nodule primordia and is maintained in the nodule meristem and infection zone. Conditional, nodule-specific knockdown of MtHDT expression by RNAi blocks nodule primordium development. A few nodules may still form, but their nodule meristems are smaller, and rhizobial colonization of the cells derived from the meristem is markedly reduced. Although the HDTs are expressed during nodule and root development, transcriptome analyses indicate that HDTs control the development of each organ in a different manner. During nodule development, the MtHDTs positively regulate 3-hydroxy-3-methylglutaryl coenzyme a reductase 1 (MtHMGR1). Decreased expression of MtHMGR1 is sufficient to explain the inhibition of primordium formation., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Nitrogen fixation by rhizobia is a highly energy-demanding process. Therefore, nodule initiation in legumes is tightly regulated. Environmental nitrate is a potent inhibitor of nodulation. However, the precise mechanism by which this agent (co)regulates the inhibition of nodulation is not fully understood. Here, we demonstrate that in Medicago truncatula the lipo-chitooligosaccharide-induced accumulation of cytokinins is reduced in response to the application of exogenous nitrate. Under permissive nitrate conditions, perception of rhizobia-secreted signalling molecules leads to an increase in the level of four cytokinins (i.e., iP, iPR, tZ, and tZR). However, under high-nitrate conditions, this increase in cytokinins is reduced. The ethylene-insensitive mutant Mtein2 / sickle , as well as wild-type plants grown in the presence of the ethylene biosynthesis inhibitor 2-aminoethoxyvinyl glycine (AVG), is resistant to the inhibition of nodulation by nitrate. This demonstrates that ethylene biosynthesis and perception are required to inhibit nodule organogenesis under high-nitrate conditions.

Legumes control their nodule numbers through the autoregulation of nodulation (AON). Rhizobia infection stimulates the production of root-derived CLE peptide hormones that are translocated to the shoot where they regulate a new signal. We used soybean to demonstrate that this shoot-derived signal is miR2111, which is transported via phloem to the root where it targets transcripts of Too Much Love (TML), a negative regulator of nodulation. Shoot perception of rhizobia-induced CLE peptides suppresses miR2111 expression, resulting in TML accumulation in roots and subsequent inhibition of nodule organogenesis. Feeding synthetic mature miR2111 via the petiole increased nodule numbers per plant. Likewise, elevating miR2111 availability by over-expression promoted nodulation, while target mimicry of TML induced the opposite effect on nodule development in wild-type plants and alleviated the supernodulating and stunted root growth phenotypes of AON-defective mutants. Additionally, in non-nodulating wild-type plants, ectopic expression of miR2111 significantly enhanced lateral root emergence with a decrease in lateral root length and average root diameter. In contrast, hairy roots constitutively expressing the target mimic construct exhibited reduced lateral root density. Overall, these findings demonstrate that miR2111 is both the critical shoot-to-root factor that positively regulates root nodule development and also acts to shape root system architecture., (© 2021 John Wiley & Sons Ltd.)

When engaging in symbiosis with legume hosts, rhizobia are confronted with environmental changes, including nutrient availability and stress exposure. Genetic circuits allow responding to these environmental stimuli to optimize physiological adaptations during the switch from the free-living to the symbiotic life style. A pivotal regulatory system of the nitrogen-fixing soybean endosymbiont Bradyrhizobium diazoefficiens for efficient symbiosis is the general stress response (GSR), which relies on the alternative sigma factor σ EcfG However, the GSR-controlled process required for symbiosis has not been identified. Here, we demonstrate that biosynthesis of trehalose is under GSR control, and mutants lacking the respective biosynthetic genes otsA and/or otsB phenocopy GSR-deficient mutants under symbiotic and selected free-living stress conditions. The role of trehalose as a cytoplasmic chemical chaperone and stress protectant can be functionally replaced in an otsA or otsB mutant by introducing heterologous genetic pathways for biosynthesis of the chemically unrelated compatible solutes glycine betaine and (hydroxy)ectoine. Alternatively, uptake of exogenously provided trehalose also restores efficient symbiosis and tolerance to hyperosmotic and hyperionic stress of otsA mutants. Hence, elevated cytoplasmic trehalose levels resulting from GSR-controlled biosynthesis are crucial for B. diazoefficiens cells to overcome adverse conditions during early stages of host infection and ensure synchronization with root nodule development. IMPORTANCE The Bradyrhizobium -soybean symbiosis is of great agricultural significance and serves as a model system for fundamental research in bacterium-plant interactions. While detailed molecular insight is available about mutual recognition and early nodule organogenesis, our understanding of the host-imposed conditions and the physiology of infecting rhizobia during the transition from a free-living state in the rhizosphere to endosymbiotic bacteroids is currently limited. In this study, we show that the requirement of the rhizobial general stress response (GSR) during host infection is attributable to GSR-controlled biosynthesis of trehalose. Specifically, trehalose is crucial for an efficient symbiosis by acting as a chemical chaperone to protect rhizobia from osmostress during host infection., (Copyright © 2021 Ledermann et al.)

Legume plants form a root-nodule symbiosis with rhizobia. This symbiosis establishment generally relies on rhizobium-produced Nod factors (NFs) and their perception by leguminous receptors (NFRs) that trigger nodulation. However, certain rhizobia hijack leguminous nodulation signalling via their type III secretion system, which functions in pathogenic bacteria to deliver effector proteins into host cells. Here, we report that rhizobia use pathogenic-like effectors to hijack legume nodulation signalling. The rhizobial effector Bel2-5 resembles the XopD effector of the plant pathogen Xanthomonas campestris and could induce nitrogen-fixing nodules on soybean nfr mutant. The soybean root transcriptome revealed that Bel2-5 induces expression of cytokinin-related genes, which are important for nodule organogenesis and represses ethylene- and defense-related genes that are deleterious to nodulation. Remarkably, Bel2-5 introduction into a strain unable to nodulate soybean mutant affected in NF perception conferred nodulation ability. Our findings show that rhizobia employ and have customized pathogenic effectors to promote leguminous nodulation signalling.

Legumes utilize a shoot-mediated signaling system to maintain a mutualistic relationship with nitrogen-fixing bacteria in root nodules. In Lotus japonicus, shoot-to-root transfer of microRNA miR2111 that targets TOO MUCH LOVE, a nodulation suppressor in roots, has been proposed to explain the mechanism underlying nodulation control from shoots. However, the role of shoot-accumulating miR2111s for the systemic regulation of nodulation was not clearly shown. Here, we find L. japonicus has seven miR2111 loci, including those mapped through RNA-seq. MIR2111-5 expression in leaves is the highest among miR2111 loci and repressed after rhizobial infection depending on a shoot-acting HYPERNODULATION ABERRANT ROOT FORMATION1 (HAR1) receptor. MIR2111-5 knockout mutants show significantly decreased nodule numbers and miR2111 levels. Furthermore, grafting experiments using transformants demonstrate scions with altered miR2111 levels influence nodule numbers in rootstocks in a dose-dependent manner. Therefore, miR2111 accumulation in leaves through MIR2111-5 expression is required for HAR1-dependent systemic optimization of nodule number.

Gibberellins (GAs), a class of phytohormones, act as an essential natural regulator of plant growth and development. Many studies have shown that GA is related to rhizobial infection and nodule organogenesis in legume species. However, thus far, GA metabolism and signaling components are largely unknown in the model legume Medicago truncatula . In this study, a genome-wide analysis of GA metabolism and signaling genes was carried out. In total 29 components, including 8 MtGA20ox genes, 2 MtGA3ox genes, 13 MtGA2ox genes, 3 MtGID1 genes, and 3 MtDELLA genes were identified in M. truncatula genome. Expression profiles revealed that most members of MtGAox , MtGID1 , and MtDELLA showed tissue-specific expression patterns. In addition, the GA biosynthesis and deactivation genes displayed a feedback regulation on GA treatment, respectively. Yeast two-hybrid assays showed that all the three MtGID1s interacted with MtDELLA1 and MtDELLA2, suggesting that the MtGID1s are functional GA receptors. More importantly, M. truncatula exhibited increased plant height and biomass by ectopic expression of the MtGA20ox1 , suggesting that enhanced GA response has the potential for forage improvement.

The mitogen-activated protein kinase (MAPK) LjMPK6 is a phosphorylation target of SIP2, a MAPK kinase that interacts with SymRK (symbiosis receptor-like kinase) for regulation of legume-rhizobia symbiosis. Both LjMPK6 and SIP2 are required for nodulation in Lotus japonicus . However, the dephosphorylation of LjMPK6 and its regulatory components in nodule development remains unexplored. By yeast two-hybrid screening, we identified a type 2C protein phosphatase, LjPP2C, that specifically interacts with and dephosphorylates LjMPK6 in vitro. Physiological and biochemical assays further suggested that LjPP2C phosphatase is required for dephosphorylation of LjMPK6 in vivo and for fine-tuning nodule development after rhizobial inoculation. A non-phosphorylatable mutant variant LjMPK6 (T224A Y226F) could mimic LjPP2C functioning in MAPK dephosphorylation required for nodule development in hairy root transformed plants. Collectively, our study demonstrates that interaction with LjPP2C phosphatase is required for dephosphorylation of LjMPK6 to fine tune nodule development in L. japonicus .

Soybean nodulation is a highly controlled process that involves complex gene regulation at both transcriptional and post-transcriptional levels. In the present study, we profiled gene expression changes, alternative splicing events, and DNA methylation patterns during nodule formation, development, and senescence. The transcriptome data uncovered key transcription patterns of nodule development that included 9669 core genes and 7302 stage-specific genes. Alternative splicing analysis uncovered a total of 2323 genes that undergo alternative splicing events in at least one nodule developmental stage, with activation of exon skipping and repression of intron retention being the most common splicing events in nodules compared to roots. Approximately 40% of the differentially spliced genes were also differentially expressed at the same nodule developmental stage, implying a substantial association between gene expression and alternative splicing. Genome-wide-DNA methylation analysis revealed dynamic changes in nodule methylomes that were specific to each nodule stage, occurred in a sequence-specific manner, and impacted the expression of 1864 genes. An attractive hypothesis raised by our data is that increased DNA methylation may contribute to the efficiency of alternative splicing. Together, our results provide intriguing insights into the associations between gene expression, alternative splicing, and DNA methylation that may shape transcriptome complexity and proteome specificity in developing soybean nodules., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

The mutualistic association between leguminous plants and endosymbiotic rhizobial bacteria is a paradigmatic example of a symbiosis driven by metabolic exchanges. Here, we report the reconstruction and modelling of a genome-scale metabolic network of Medicago truncatula (plant) nodulated by Sinorhizobium meliloti (bacterium). The reconstructed nodule tissue contains five spatially distinct developmental zones and encompasses the metabolism of both the plant and the bacterium. Flux balance analysis (FBA) suggests that the metabolic costs associated with symbiotic nitrogen fixation are primarily related to supporting nitrogenase activity, and increasing N 2 -fixation efficiency is associated with diminishing returns in terms of plant growth. Our analyses support that differentiating bacteroids have access to sugars as major carbon sources, ammonium is the main nitrogen export product of N 2 -fixing bacteria, and N 2 fixation depends on proton transfer from the plant cytoplasm to the bacteria through acidification of the peribacteroid space. We expect that our model, called 'Virtual Nodule Environment' (ViNE), will contribute to a better understanding of the functioning of legume nodules, and may guide experimental studies and engineering of symbiotic nitrogen fixation.

Lotus japonicus is an important model legume for studying symbiotic nitrogen fixation as well as plant development. A genomic sequence of L. japonicus (MG20) has been available for more than ten years. However, the low quality of the genome limits its application in functional genomic studies. Therefore, it is necessary to assemble high-quality chromosome sequences of L. japonicus using new sequencing technology to facilitate the study of functional genomics. In this report, we used the third-generation sequencing combined with the Illumina HiSeq platform to sequence the genome of L. japonicus (MG20). We obtained 544 Mb of genomic sequence using third-generation assembly. Based on sequence analysis, 357 Mb of repeats, 28,251 genes, 626 tRNAs, 1409 rRNAs, and 1233 pseudogenes were predicted in the genome. A total of 27,991 genes were annotated into databases. Compared to the previously published data, the new genome database contains complete L. japonicus sequences in the proper order and orientation with a contig N50 2.81Mb and an excellent genome coverage, which provides more accurate genome information and more precise assembly for functional genomic study.

Leguminous plants establish endosymbiotic associations with rhizobia and form root nodules in which the rhizobia fix atmospheric nitrogen. The host plant and intracellular rhizobia strictly control this symbiotic nitrogen fixation. We recently reported a Lotus japonicus Fix - mutant, apn1 ( aspartic peptidase nodule-induced 1 ), that impairs symbiotic nitrogen fixation. APN1 encodes a nodule-specific aspartic peptidase involved in the Fix - phenotype in a rhizobial strain-specific manner. This host-strain specificity implies that some molecular interactions between host plant APN1 and rhizobial factors are required, although the biological function of APN1 in nodules and the mechanisms governing the interactions are unknown. To clarify how rhizobial factors are involved in strain-specific nitrogen fixation, we explored transposon mutants of Mesorhizobium loti strain TONO, which normally form Fix - nodules on apn1 roots, and identified TONO mutants that formed Fix + nodules on apn1 The identified causal gene encodes an autotransporter, part of a protein secretion system of Gram-negative bacteria. Expression of the autotransporter gene in M. loti strain MAFF3030399, which normally forms Fix + nodules on apn1 roots, resulted in Fix - nodules. The autotransporter of TONO functions to secrete a part of its own protein (a passenger domain) into extracellular spaces, and the recombinant APN1 protein cleaved the passenger protein in vitro. The M. loti autotransporter showed the activity to induce the genes involved in nodule senescence in a dose-dependent manner. Therefore, we conclude that the nodule-specific aspartic peptidase, APN1, suppresses negative effects of the rhizobial autotransporter in order to maintain effective symbiotic nitrogen fixation in root nodules., Competing Interests: The authors declare no competing interest.

The rhizobial type III secretion system secretes effector proteins into host plant cells, which may either promote or inhibit symbiosis with legumes. We herein demonstrated that the type III secretion system of Bradyrhizobium sp. SUTN9-2 obstructed symbiosis with Lotus japonicus Miyakojima, L. japonicus Gifu, and Lotus burttii. A mutant of SUTN9-2 that is unable to secrete effector proteins showed better nodulation and plant growth promotion than wild-type SUTN9-2 when paired with these Lotus spp. We propose that SUTN9-2 is a useful strain for understanding the mechanisms by which effector proteins obstruct symbiosis between Bradyrhizobium and Lotus spp.

The nifV gene encodes homocitrate synthase, the enzyme that catalyzes the formation of homocitrate, which is essential for arranging the FeMo-cofactor in the catalytic center of nitrogenase. Some host plants, such as Lotus japonicus, supply homocitrate to their symbionts, in this case, Mesorhizobium loti, which lacks nifV. In contrast, Bradyrhizobium ORS285, a symbiont of Aeschynomene cross-inoculation (CI) groups 2 and 3, requires nifV for symbiosis with Aeschynomene species that belong to CI group 3, and some species belonging to CI group 2. However, it currently remains unclear whether rhizobial nifV is required for symbiosis with Aeschynomene species belonging to CI group 1 or with other legumes. We generated nifV-disruption (ΔnifV) mutants of two wide-host-range rhizobia, Bradyrhizobium SUTN9-2 and DOA9, to investigate whether they require nifV for symbiosis. Both ΔnifV mutant strains showed significantly less nitrogenase activity in a free-living state than the respective wild-type strains. The symbiotic phenotypes of SUTN9-2, DOA9, and their ΔnifV mutants were examined with four legumes, Aeschynomene americana, Stylosanthes hamata, Indigofera tinctoria, and Desmodium tortuosum. nifV was required for the efficient symbiosis of SUTN9-2 with A. americana (CI group 1), but not for that of DOA9. SUTN9-2 established symbiosis with all three other legumes; nifV was required for symbiosis with I. tinctoria and D. tortuosum. These results suggest that, in addition to Aeschynomene CI groups 2 and 3, CI group 1 and several other legumes require the rhizobial nifV for symbiosis.

Background: Legumes can utilize atmospheric nitrogen by hosting nitrogen-fixing bacteria in special lateral root organs, called nodules. Legume nodules have a unique ontology, despite similarities in the gene networks controlling nodule and lateral root development. It has been shown that Medicago truncatula NODULE ROOT1 (MtNOOT1) is required for the maintenance of nodule identity, preventing the conversion to lateral root development. MtNOOT1 and its orthologs in other plant species -collectively called the NOOT-BOP-COCH-LIKE (NBCL) family- specify boundary formation in various aerial organs. However, MtNOOT1 is not only expressed in nodules and aerial organs, but also in developing roots, where its function remains elusive., Results: We show that Mtnoot1 mutant seedlings display accelerated root elongation due to an enlarged root apical meristem. Also, Mtnoot1 mutant roots are thinner than wild-type and are delayed in xylem cell differentiation. We provide molecular evidence that the affected spatial development of Mtnoot1 mutant roots correlates with delayed induction of genes involved in xylem cell differentiation. This coincides with a basipetal shift of the root zone that is susceptible to rhizobium-secreted symbiotic signal molecules., Conclusions: Our data show that MtNOOT1 regulates the size of the root apical meristem and vascular differentiation. Our data demonstrate that MtNOOT1 not only functions as a homeotic gene in nodule development but also coordinates the spatial development of the root.

Phytohormones regulate the mutualistic symbiotic interaction between legumes and rhizobia, nitrogen-fixing soil bacteria, notably by controlling the formation of the infection thread in the root hair (RH). At the cellular level, the formation of the infection thread is promoted by the translocation of plasma membrane microdomains at the tip of the RH. We hypothesize that phytohormones regulate the translocation of plasma membrane microdomains to regulate infection thread formation. Accordingly, we treated with hormone and hormone inhibitors transgenic soybean roots expressing fusions between the Green Fluorescent Protein (GFP) and Gm FWL1 or Gm FLOT2/4, two microdomain-associated proteins translocated at the tip of the soybean RH in response to rhizobia. Auxin and cytokinin treatments are sufficient to trigger or inhibit the translocation of Gm FWL1 and Gm FLOT2/4 to the RH tip independently of the presence of rhizobia, respectively. Unexpectedly, the application of salicylic acid, a phytohormone regulating the plant defense system, also promotes the translocation of Gm FWL1 and Gm FLOT2/4 to the RH tip regardless of the presence of rhizobia. These results suggest that phytohormones are playing a central role in controlling the early stages of rhizobia infection by regulating the translocation of plasma membrane microdomains. They also support the concept of crosstalk of phytohormones to control nodulation.

Main Conclusion: A homologue of the ribosomal protein L22e, Rpf84, regulates root nodule symbiosis by mediating the infection process of rhizobia and preventing bacteroids from degradation in Robinia pseudoacacia. Ribosomal proteins (RPs) are known to have extraribosomal functions, including developmental regulation and stress responses; however, the effects of RPs on symbiotic nodulation of legumes are still unclear. Ribosomal protein 22 of the large 60S subunit (RPL22), a non-typical RP that is only found in eukaryotes, has been shown to function as a tumour suppressor in animals. Here, a homologue of RPL22, Rpf84, was identified from the leguminous tree R. pseudoacacia. Subcellular localization assays showed that Rpf84 was expressed in the cytoplasm and nucleus. Knockdown of Rpf84 by RNA interference (RNAi) technology impaired the infection process and nodule development. Compared with the control, root and stem length, dry weight and nodule number per plant were drastically decreased in Rpf84-RNAi plants. The numbers of root hair curlings, infection threads and nodule primordia were also significantly reduced. Ultrastructure analyses showed that Rpf84-RNAi nodules contained fewer infected cells with fewer bacteria. In particular, remarkable deformation of bacteroids and fusion of multiple symbiosomes occurred in infected cells. By contrast, overexpression of Rpf84 promoted nodulation, and the overexpression nodules maintained a larger infection/differentiation region and had more infected cells filled with bacteroids than the control at 45 days post inoculation, suggesting a retarded ageing process in nodules. These results indicate for the first time that RP regulates the symbiotic nodulation of legumes and that RPL22 may function in initiating the invasion of rhizobia and preventing bacteroids from degradation in R. pseudoacacia.

Boron (B) deficiency affects the development of Pisum sativum nodules and Arabidopsis thaliana root meristems. Both organs show an alteration of cell differentiation that result in the development of tumor-like structures. The fact that B in plants is not only able to interact with components of the cell wall but also with membrane-associated glycoconjugates, led us to analyze changes in high mannose type N-glycans (HMNG). The affinoblots with concanavalin A revealed alterations in the N-glycosylation pattern during early development of nodules and roots under B deprivation. Besides, there is increasing evidence of a B role in animal physiology that brought us to investigate the impact of B deficiency on Danio rerio (zebrafish) development. When B deficiency was induced prior to early cleavage stages, embryos developed as an abnormal undifferentiated mass of cells. Additionally, when B was removed at post-hatching, larvae undergo aberrant organogenesis. Resembling the phenomenon described in plants, alteration of the N-glycosylation pattern occurred in B-deficient zebrafish larvae prior to organogenesis. Overall, these results support a common function of B in plants and animals associated with glycosylation that might be important for cell signaling and cell fate determination during development., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Legume-rhizobia symbiosis is a time-limited process due to the onset of senescence, which results in the degradation of host plant cells and symbiosomes. A number of transcription factors, proteases, and functional genes have been associated with nodule senescence; however, whether other proteases or transcription factors are involved in nodule senescence remains poorly understood. In this study, we identified an early nodule senescence mutant in Medicago truncatula , denoted basic helix-loop-helix transcription factor2 ( bhlh2 ), that exhibits decreased nitrogenase activity, acceleration of plant programmed cell death (PCD), and accumulation of reactive oxygen species (ROS). The results suggest that MtbHLH2 plays a negative role in nodule senescence. Nodules of wild-type and bhlh2 -TALEN mutant plants at 28 d postinoculation were used for transcriptome sequencing. The transcriptome data analysis identified a papain-like Cys protease gene, denoted MtCP77 , that could serve as a potential target of MtbHLH2. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis demonstrated that MtbHLH2 directly binds to the promoter of MtCP77 to inhibit its expression. MtCP77 positively regulates nodule senescence by accelerating plant PCD and ROS accumulation. In addition, the expression of MtbHLH2 in the nodules gradually decreased from the meristematic zone to the nitrogen fixation zone, whereas the expression of MtCP77 showed enhancement. These results indicate that MtbHLH2 and MtCP77 have opposite functions in the regulation of nodule senescence. These results reveal significant roles for MtbHLH2 and MtCP77 in plant PCD, ROS accumulation, and nodule senescence, and improve our understanding of the regulation of the nodule senescence process., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Hopanoids are steroid-like bacterial lipids that enhance membrane rigidity and promote bacterial growth under diverse stresses. Hopanoid biosynthesis genes are conserved in nitrogen-fixing plant symbionts, and we previously found that the extended (C 35 ) class of hopanoids in Bradyrhizobium diazoefficiens are required for efficient symbiotic nitrogen fixation in the tropical legume host Aeschynomene afraspera . Here, we demonstrate that the nitrogen-fixation defect conferred by extended hopanoid loss can be fully explained by a reduction in root nodule sizes rather than per-bacteroid nitrogen-fixation levels. Using a single-nodule tracking approach to quantify A. afraspera nodule development, we provide a quantitative model of root nodule development in this host, uncovering both the baseline growth parameters for wild-type nodules and a surprising heterogeneity of extended hopanoid mutant developmental phenotypes. These phenotypes include a delay in root nodule initiation and the presence of a subpopulation of nodules with slow growth rates and low final volumes, which are correlated with reduced motility and surface attachment in vitro and lower bacteroid densities in planta, respectively. This work provides a quantitative reference point for understanding the phenotypic diversity of ineffective symbionts in A. afraspera and identifies specific developmental stages affected by extended hopanoid loss for future mechanistic work.

During the legume-rhizobium symbiotic interaction, rhizobial invasion of legumes is primarily mediated by a plant-made tubular invagination called an infection thread (IT). Here, we identify a gene in Lotus japonicus encoding a Leu-rich repeat receptor-like kinase (LRR-RLK), RINRK1 ( Rhizobial Infection Receptor-like Kinase1 ), that is induced by Nod factors (NFs) and is involved in IT formation but not nodule organogenesis. A paralog, RINRK2, plays a relatively minor role in infection. RINRK1 is required for full induction of early infection genes, including Nodule Inception ( NIN ), encoding an essential nodulation transcription factor. RINRK1 displayed an infection-specific expression pattern, and NIN bound to the RINRK1 promoter, inducing its expression. RINRK1 was found to be an atypical kinase localized to the plasma membrane and did not require kinase activity for rhizobial infection. We propose RINRK1 is an infection-specific RLK, which may specifically coordinate output from NF signaling or perceive an unknown signal required for rhizobial infection., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Little is known about the genes participating in digalactosyldiacylglycerol (DGDG) synthesis during nodule symbiosis. Here, we identified full-length MtDGD1 , a synthase of DGDG, and characterized its effect on symbiotic nitrogen fixation in Medicago truncatula . Immunofluorescence and immunoelectron microscopy showed that MtDGD1 was located on the symbiosome membranes in the infected cells. β-Glucuronidase histochemical staining revealed that MtDGD1 was highly expressed in the infection zone of young nodules as well as in the whole mature nodules. Compared with the control, MtDGD1 -RNA interference transgenic plants exhibited significant decreases in nodule number, symbiotic nitrogen fixation activity, and DGDG abundance in the nodules, as well as abnormal nodule and symbiosome development. Overexpression of MtDGD1 resulted in enhancement of nodule number and nitrogen fixation activity. In response to phosphorus starvation, the MtDGD1 expression level was substantially upregulated and the abundance of nonphospholipid DGDG was significantly increased in the roots and nodules, accompanied by corresponding decreases in the abundance of phospholipids such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Overall, our results indicate that DGD1 contributes to effective nodule organogenesis and nitrogen fixation by affecting the synthesis and content of DGDG during symbiosis.

The development and functioning of the nitrogen fixing symbiosis between legume plants and soil bacteria collectively called rhizobia requires continuous chemical dialogue between the partners using different molecules such as flavonoids, lipo-chitooligosaccharides, polysaccharides and peptides. Agrobacterium rhizogenes mediated hairy root transformation of legumes is widely used to study the function of plant genes involved in the process. The identification of transgenic plant tissues is based on antibiotics/herbicide selection and/or the detection of different reporter genes that usually require special equipment such as fluorescent microscopes or destructive techniques and chemicals to visualize enzymatic activity. Here, we developed and efficiently used in hairy root experiments binary vectors containing the MtLAP1 gene driven by constitutive and tissue-specific promoters that facilitate the production of purple colored anthocyanins in transgenic tissues and thus allowing the identification of transformed roots by naked eye. Anthocyanin producing roots were able to establish effective symbiosis with rhizobia. Moreover, it was shown that species-specific allelic variations and a mutation preventing posttranslational acetyl modification of an essential nodule-specific cysteine-rich peptide, NCR169, do not affect the symbiotic interaction of Medicago truncatula cv. Jemalong with Sinorhizobium medicae strain WSM419. Based on the experiments, it could be concluded that it is preferable to use the vectors with tissue-specific promoters that restrict anthocyanin production to the root vasculature for studying biotic interactions of the roots such as symbiotic nitrogen fixation or mycorrhizal symbiosis.

Symbiotic nitrogen fixation is the main source of nitrogen for soybean growth. Since the genotypes of rhizobia and soybean germplasms vary, the nitrogen-fixing ability of soybean after inoculation also varies. A few studies have reported that quantitative trait loci (QTLs) control biological nitrogen fixation traits, even soybean which is an important crop. The present study reported that the Sinorhizobium fredii HH103 gene rhcJ belongs to the tts (type III secretion) cluster and that the mutant HH103ΩrhcJ can clearly decrease the number of nodules in American soybeans. However, few QTLs of nodule traits have been identified. This study used a soybean (Glycine max (L.) Merr.) 'Charleston' × 'Dongnong 594' (C × D, n = 150) recombinant inbred line (RIL). Nodule traits were analysed in the RIL population after inoculation with S. fredii HH103 and the mutant HH103ΩrhcJ. Plants were grown in a greenhouse with a 16-h light cycle at 26 °C and an 8-h dark cycle at 18 °C. Then, 4 weeks after inoculation, plants were harvested for evaluation of nodule traits. Through QTL mapping, 16 QTLs were detected on 8 chromosomes. Quantitative PCR (qRT-PCR) and RNA-seq analysis determined that the genes Glyma.04g060600, Glyma.18g159800 and Glyma.13g252600 might interact with rhcJ.

The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana , and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

With a view to introducing white lupin (Lupinus albus L.) for cultivation in Tunisian calcareous soils, compatible indigenous rhizobia for nitrogen-fixing symbiosis were investigated and characterized. Two L. albus varieties, Mekna and Lumen, were used to trap rhizobia in soil samples collected from 56 sites with high active lime contents (0-49%). Nodulation occurred in only 15 soils. The local variety, Mekna, developed significantly more root nodules and had a trapping capacity in more soils than the imported variety Lumen. A phylogenetic analysis based on the partial 16S-23S ribosomal RNA internal transcribed spacer region (ITS) and multi-locus sequence analysis (MLSA) of three chromosomal housekeeping genes, recA, atpD and dnaK, showed that strains were affiliated to Agrobacterium, Rhizobium, and Neorhizobium, with large internal diversity, including separate lineages. Infectivity tests highlighted some nodulation specificity at the plant variety level, since the strains originating from Mekna could only nodulate this variety, while strains trapped in Lumen could nodulate both varieties. When inoculated, almost all strains resulted in a significant increase in plant shoot dry weight on L. albus. Although Agrobacterium sp. strains isolated from L. albus could nodulate and had a plant growth promoting effect, no nodA and nodC genes could be amplified. This is discussed together with the absence of bradyrhizobia and the general infrequency of L. albus-nodulating rhizobia in Tunisian soils. The adapted and efficient rhizobial strains reported here were promising candidates for inoculant development and represent a contribution towards successful cultivation of L. albus in Tunisia, especially the most promising Mekna variety., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Key Message: Symbiotic nitrogen fixation in root nodules of grain legumes is essential for high yielding. Protein phosphorylation/dephosphorylation plays important role in root nodule development. Differences in the phosphoproteomes may either be developmental specific and related to nitrogen fixation activity. An iTRAQ-based quantitative phosphoproteomic analyses during nodule development enables identification of specific phosphorylation signaling in the Lotus-rhizobia symbiosis. During evolution, legumes (Fabaceae) have evolved a symbiotic relationship with rhizobia, which fix atmospheric nitrogen and produce ammonia that host plants can then absorb. Root nodule development depends on the activation of protein phosphorylation-mediated signal transduction cascades. To investigate possible molecular mechanisms of protein modulation during nodule development, we used iTRAQ-based quantitative proteomic analyses to identify root phosphoproteins during rhizobial colonization and infection of Lotus japonicus. 1154 phosphoproteins with 2957 high-confidence phosphorylation sites were identified. Gene ontology enrichment analysis of functional groups of these genes revealed that the biological processes mediated by these proteins included cellular processes, signal transduction, and transporter activity. Quantitative data highlighted the dynamics of protein phosphorylation during nodule development and, based on regulatory trends, seven groups were identified. RNA splicing and brassinosteroid (BR) signaling pathways were extensively affected by phosphorylation, and most Ser/Arg-rich (SR) proteins were multiply phosphorylated. In addition, many proposed kinase-substrate pairs were predicted, and in these MAPK6 substrates were found to be highly enriched. This study offers insights into the regulatory processes underlying nodule development, provides an accessible resource cataloging the phosphorylation status of thousands of Lotus proteins during nodule development, and develops our understanding of post-translational regulatory mechanisms in the Lotus-rhizobia symbiosis.

Rhizobium tropici CIAT 899 is a facultative symbiotic diazotroph able to deal with stressful concentrations of metals. Nevertheless the molecular mechanisms involved in metal tolerance have not been elucidated. Copper (Cu 2+ ) is a metal component essential for the heme-copper respiratory oxidases and enzymes that catalyse redox reactions, however, it is highly toxic when intracellular trace concentrations are surpassed. In this study, we report that R. tropici CIAT 899 is more tolerant to Cu 2+ than other Rhizobium and Sinorhizobium species. Through Tn5 random mutagenesis we identify a R. tropici mutant strain with a severe reduction in Cu 2+ tolerance. The Tn5 insertion disrupted the gene RTCIAT899_CH17575, encoding a putative heavy metal efflux P1B-1-type ATPase designated as copA. Phaseolus vulgaris plants inoculated with the copA::Tn5 mutant in the presence of toxic Cu 2+ concentrations showed a drastic reduction in plant and nodule dry weight, as well as nitrogenase activity. Nodules induced by the copA::Tn5 mutant present an increase in H2O2 concentration, lipoperoxidation and accumulate 40-fold more Cu 2+ than nodules formed by the wild-type strain. The copA::Tn5 mutant complemented with the copA gene recovered the wild-type symbiotic phenotypes. Therefore, the copA gene is essential for R. tropici CIAT 899 to survive in copper-rich environments in both free life and symbiosis with P. vulgaris plants.

Gibberellin (GA) plays a controversial role in the legume-rhizobium symbiosis. Recent studies have shown that the GA level in legumes must be precisely controlled for successful rhizobial infection and nodule organogenesis. However, regulation of the GA level via catabolism in legume roots has not been reported to date. Here, we investigate a novel GA inactivating C20-GA2-oxidase gene MtGA2ox10 in Medicago truncatula. RNA sequencing analysis and quantitative polymerase chain reaction revealed that MtGA2ox10 was induced as early as 6 h post-inoculation (hpi) of rhizobia and reached peak transcript abundance at 12 hpi. Promoter::β-glucuronidase fusion showed that the promoter activity was localized in the root infection/differentiation zone during the early stage of rhizobial infection and in the vascular bundle of the mature nodule. The CRISPR/Cas9-mediated deletion mutation of MtGA2ox10 suppressed infection thread formation, which resulted in reduced development and retarded growth of nodules on the Agrobacterium rhizogenes-transformed roots. Over-expression of MtGA2ox10 in the stable transgenic plants caused dwarfism, which was rescued by GA 3 application, and increased infection thread formation but inhibition of nodule development. We conclude that MtGA2ox10 plays an important role in the rhizobial infection and the development of root nodules through fine catabolic tuning of GA in M. truncatula.

Fabaceans produce two major classes of symbiotic nodules: the indeterminate type characterized by a persistent meristem, and the determinate type that lacks a persistent meristem. The class III homeodomain leucine zipper (HD-ZIP III) transcription factor family influence development of multiple lateral organs and meristem maintenance, but their role in determinate nodule development is not known. HD-ZIP III protein activity is post-translationally regulated by members of the small leucine zipper protein (ZPR) family in arabidopsis. We characterized the ZPR gene family in soybean and evaluated their ability to interact with two key members of GmHD-ZIP III family through yeast two-hybrid assays. GmZPR3d displayed the strongest interaction with GmHD-ZIP III-2 among the different pairs evaluated. GmHD-ZIP III-1, -2, and GmZPR3d showed overlapping expression patterns in the root stele and in nodule parenchyma tissues. Over-expression of GmZPR3d resulted in ectopic root secondary xylem formation, and enhanced expression of vessel-specific master switch genes in soybean. The nodules in ZPR3d over-expressing roots were larger in size, had a relatively larger central zone and displayed increased nodule vascular branching. The results from this study point to a key role for GmZPR3d in soybean root and nodule development.

Evidences for an involvement of the bacterial type IV secretion system (T4SS) in the symbiotic relationship between rhizobia and legumes have been pointed out by several recent studies. However, information regarding this secretion system in Mesorhizobium is still very scarce. The aim of the present study was to investigate the phylogeny and expression of the traG gene, which encodes a substrate receptor of the T4SS. In addition, the occurrence and genomic context of this and other T4SS genes, namely, genes from tra/trb and virB/virD4 complexes, were also analyzed in order to unveil the structural and functional organization of T4SS in mesorhizobia. The location of the T4SS genes in the symbiotic region of the analyzed rhizobial genomes, along with the traG phylogeny, suggests that T4SS genes could be horizontally transferred together with the symbiosis genes. Regarding the T4SS structural organization in Mesorhizobium , the virB/virD4 genes were absent in all chickpea ( Cicer arietinum L.) microsymbionts and in the Lotus symbiont Mesorhizobium japonicum MAFF303099 T . Interestingly, the presence of genes belonging to another secretion system (T3SS) was restricted to these strains lacking the virB/virD4 genes. The traG gene expression was detected in M. mediterraneum Ca36 T and M. ciceri LMS-1 strains when exposed to chickpea root exudates and also in the early nodules formed by M. mediterraneum Ca36 T , but not in older nodules. This study contributes to a better understanding of the importance of T4SS in mutualistic symbiotic bacteria.

Nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi (AMF) form symbioses with plant roots and these are established by precise regulation of symbiont accommodation within host plant cells. In model legumes such as Lotus japonicus and Medicago truncatula, rhizobia enter into roots through an intracellular invasion system that depends on the formation of a root-hair infection thread (IT). While IT-mediated intracellular rhizobia invasion is thought to be the most evolutionarily derived invasion system, some studies have indicated that a basal intercellular invasion system can replace it when some nodulation-related factors are genetically modified. In addition, intracellular rhizobia accommodation is suggested to have a similar mechanism as AMF accommodation. Nevertheless, our understanding of the underlying genetic mechanisms is incomplete. Here we identify a L. japonicus nodulation-deficient mutant, with a mutation in the LACK OF SYMBIONT ACCOMMODATION (LAN) gene, in which root-hair IT formation is strongly reduced, but intercellular rhizobial invasion eventually results in functional nodule formation. LjLAN encodes a protein that is homologous to Arabidopsis MEDIATOR 2/29/32 possibly acting as a subunit of a Mediator complex, a multiprotein complex required for gene transcription. We also show that LjLAN acts in parallel with a signaling pathway including LjCYCLOPS. In addition, the lan mutation drastically reduces the colonization levels of AMF. Taken together, our data provide a new factor that has a common role in symbiont accommodation process during root nodule and AM symbiosis., Competing Interests: The authors have declared that no competing interests exist.