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Symbiotic interactions between rhizobia and legumes result in the formation of root nodules, which fix nitrogen that can be used for plant growth. Rhizobia usually invade legume roots through a plant-made tunnel-like structure called an infection thread (IT). RPG (Rhizobium-directed polar growth) encodes a coiled-coil protein that has been identified in Medicago truncatula as required for root nodule infection, but the function of RPG remains poorly understood. In this study, we identified and characterized RPG in Lotus japonicus and determined that it is required for IT formation. RPG was induced by Mesorhizobium loti or purified Nodulation factor and displayed an infection-specific expression pattern. Nodule inception (NIN) bound to the RPG promoter and induced its expression. We showed that RPG displayed punctate subcellular localization in L. japonicus root protoplasts and in root hairs infected by M. loti. The N-terminal predicted C2 lipid-binding domain of RPG was not required for this subcellular localization or for function. CERBERUS, a U-box E3 ligase which is also required for rhizobial infection, was found to be localized similarly in puncta. RPG co-localized and directly interacted with CERBERUS in the early endosome (TGN/EE) compartment and near the nuclei in root hairs after rhizobial inoculation. Our study sheds light on an RPG-CERBERUS protein complex that is involved in an exocytotic pathway mediating IT elongation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In the last three years, several gene editing techniques have been developed for both model and crop legumes. CRISPR-Cas9-based tools, in particular, are outpacing other comparable gene editing technologies used in legume hosts and their microbial symbionts to understand the molecular basis of symbiotic nitrogen-fixation. Gene editing has helped identify new gene functions, validate genetic screens, resolve gene redundancy, examine the role of tandemly duplicated genes, and investigate symbiotic signaling networks in non-model plants. In this review, we discuss the advances made in understanding the legume-rhizobia symbiosis through the use of gene editing and highlight studies conducted under varying environmental conditions. We reason that future climate-hardy legumes must be able to better integrate environmental signals with nitrogen fixation by fine-tuning long distance signaling, continuing to select efficient rhizobial partners, and adjusting their molecular circuitry to function optimally under variable light and nutrient availability and rising atmospheric carbon dioxide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Symbiotic nitrogen fixation by Rhizobium bacteria in the cells of legume root nodules alleviates the need for nitrogen fertilizers. Nitrogen fixation requires the endosymbionts to differentiate into bacteroids which can be reversible or terminal. The latter is controlled by the plant, it is more beneficial and has evolved in multiple clades of the Leguminosae family. The plant effectors of terminal differentiation in inverted repeat-lacking clade legumes (IRLC) are nodule-specific cysteine-rich (NCR) peptides, which are absent in legumes such as soybean where there is no terminal differentiation of rhizobia. It was assumed that NCRs co-evolved with specific transcription factors, but our work demonstrates that expression of NCR genes does not require NCR-specific transcription factors. Introduction of the Medicago truncatula NCR169 gene under its own promoter into soybean roots resulted in its nodule-specific expression, leading to bacteroid changes associated with terminal differentiation. We identified two AT-Hook Motif Nuclear Localized (AHL) transcription factors from both M. truncatula and soybean nodules that bound to AT-rich sequences in the NCR169 promoter inducing its expression. Whereas mutation of NCR169 arrested bacteroid development at a late stage, the absence of MtAHL1 or MtAHL2 completely blocked bacteroid differentiation indicating that they also regulate other NCR genes required for the development of nitrogen-fixing nodules. Regulation of NCRs by orthologous transcription factors in non-IRLC legumes opens up the possibility of increasing the efficiency of nitrogen fixation in legumes lacking NCRs., (© 2023. The Author(s).)

CLE (CLAVATA3/ENDOSPERM SURROUNDING REGION-related) peptides are systemic regulators of legume-rhizobium symbiosis that negatively control the number of nitrogen-fixing nodules. CLE peptides are produced in the root in response to rhizobia inoculation and/or nitrate treatment and are transported to the shoot where they are recognized by the CLV1-like (CLAVATA1-like) receptor kinase. As a result, a shoot-derived signaling pathway is activated that inhibits subsequent nodule development in the root. In Medicago truncatula, MtCLE35 is activated in response to rhizobia and nitrate treatment and the overexpression of this gene systemically inhibits nodulation. The inhibitory effect of MtCLE35 overexpression is dependent on the CLV1-like receptor kinase MtSUNN (SUPER NUMERIC NODULES), suggesting that MtSUNN could be involved in the reception of the MtCLE35 peptide. Yet little is known about the downstream genes regulated by a MtCLE35-activated response in the root. In order to identify genes whose expression levels could be regulated by the MtCLE35-MtSUNN pathway, we performed a MACE-Seq (Massive Analysis of cDNA Ends) transcriptomic analysis of MtCLE35-overexpressing roots. Among upregulated genes, the gene MtSUNN that encodes a putative receptor of MtCLE35 was detected. Moreover, we found that MtSUNN, as well as several other differentially expressed genes, were upregulated locally in MtCLE35-overexpressing roots whereas the MtTML1 and MtTML2 genes were upregulated systemically. Our data suggest that MtCLE35 has both local and systemic effects on target genes in the root., Competing Interests: Declaration of competing interest The authors 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 © 2023 Elsevier GmbH. All rights reserved.)

Transport systems are crucial in many plant processes, including plant-microbe interactions. Nodule formation and function in legumes involve the expression and regulation of multiple transport proteins, and many are still uncharacterized, particularly for nitrogen transport. Amino acids originating from the nitrogen-fixing process are an essential form of nitrogen for legumes. This work evaluates the role of MtN21 (henceforth MtUMAMIT14), a putative transport system from the MtN21/EamA-like/UMAMIT family, in nodule formation and nitrogen fixation in Medicago truncatula. To dissect this transporter's role, we assessed the expression of MtUMAMIT14 using GUS staining, localized the corresponding protein in M. truncatula root and tobacco leaf cells, and investigated two independent MtUMAMIT14 mutant lines. Our results indicate that MtUMAMIT14 is localized in endosomal structures and is expressed in both the infection zone and interzone of nodules. Comparison of mutant and wild-type M. truncatula indicates MtUMAMIT14, the expression of which is dependent on the presence of NIN, DNF1, and DNF2, plays a role in nodule formation and nitrogen-fixation. While the function of the transporter is still unclear, our results connect root nodule nitrogen fixation in legumes with the UMAMIT family., (© 2023. The Author(s).)

Iron (Fe) is essential for DNA synthesis, photosynthesis and respiration of plants. The demand for Fe substantially increases during legumes-rhizobia symbiotic nitrogen fixation because of the synthesis of leghemoglobin in the host and Fe-containing proteins in bacteroids. However, the mechanism by which plant controls iron transport to nodules remains largely unknown. Here we demonstrate that GmYSL7 serves as a key regulator controlling Fe uptake from root to nodule and distribution in soybean nodules. GmYSL7 is Fe responsive and GmYSL7 transports iron across the membrane and into the infected cells of nodules. Alterations of GmYSL7 substantially affect iron distribution between root and nodule, resulting in defective growth of nodules and reduced nitrogenase activity. GmYSL7 knockout increases the expression of GmbHLH300, a transcription factor required for Fe response of nodules. Overexpression of GmbHLH300 decreases nodule number, nitrogenase activity and Fe content in nodules. Remarkably, GmbHLH300 directly binds to the promoters of ENOD93 and GmLbs, which regulate nodule number and nitrogenase activity, and represses their transcription. Our data reveal a new role of GmYSL7 in controlling Fe transport from host root to nodule and Fe distribution in nodule cells, and uncover a molecular mechanism by which Fe affects nodule number and nitrogenase activity., (© 2022 Institute of Botany, Chinese Academy of Sciences.)

Key Message: Compatible interaction between rhizobial Nod factors and host receptors enables initial recognition and signaling events during legume-rhizobia symbiosis. Molecular communication is a new paradigm of information relay, which uses chemical signals or molecules as dialogues for communication and has been witnessed in prokaryotes, plants as well as in animal kingdom. Understanding this fascinating relay of signals between plants and rhizobia during the establishment of a synergistic relationship for biological nitrogen fixation represents one of the hotspots in plant biology research. Predominantly, their interaction is initiated by flavonoids exuding from plant roots, which provokes changes in the expression profile of rhizobial genes. Compatible interactions promote the secretion of Nod factors (NFs) from rhizobia, which are recognised by cognate host receptors. Perception of NFs by host receptors initiates the symbiosis and ultimately leads to the accommodation of rhizobia within root nodules via a series of mutual exchange of signals. This review elucidates the bacterial and plant perspectives during the early stages of symbiosis, explicitly emphasizing the significance of NFs and their cognate NF receptors., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The effect of phosphorus deficiency on plant growth, nodulation, and symbiotic nitrogen fixation as well as, the nodulated-roots oxygen consumption, nodule permeability and conductance to the oxygen diffusion of Medicago truncatula-Sinorhizobium meliloti symbiosis were studied. Three lines, namely TN6.18, originated from local populations, F83005.5 originated from Var (France) and Jemalong 6, a reference cultivar from Australia, were hydroponically grown in nutrient solution supplied with 5 µmol (P deficient) and 15 µmol (P sufficient: Control), under semi-controlled conditions in a glasshouse. A genotypic variation in tolerance to P deficiency was found: TN6.18 was the most tolerant line whereas F83005.5 was the most sensitive. The relative tolerance of TN6.18 was concomitant with the greater P requirement, the higher N2 fixation, the stimulation of nodule respiration and the less increases of conductance to the oxygen diffusion in nodules tissues. The higher P use efficiency for nodule growth and for symbiotic nitrogen fixation was detected in the tolerant line. Results suggest that the tolerance to P deficiency seems to depend on thehost plant ability to reallocate P from both leaves and roots to their nodules. P is needed in high energy demand conditions to maintain adequate nodule activity and prevent negative effects of the O2 excess on the nitrogenase.

Legumes have adaptive mechanisms that regulate nodulation in response to the amount of nitrogen in the soil. In Lotus japonicus, two NODULE INCEPTION (NIN)-LIKE PROTEIN (NLP) transcription factors, LjNLP4 and LjNLP1, play pivotal roles in the negative regulation of nodulation by controlling the expression of symbiotic genes in high nitrate conditions. Despite an improved understanding of the molecular basis for regulating nodulation, how nitrate plays a role in the signaling pathway to negatively regulate this process is largely unknown. Here, we show that nitrate transport via NITRATE TRANSPORTER 2.1 (LjNRT2.1) is a key step in the NLP signaling pathway to control nodulation. A mutation in the LjNRT2.1 gene attenuates the nitrate-induced control of nodulation. LjNLP1 is necessary and sufficient to induce LjNRT2.1 expression, thereby regulating nitrate uptake/transport. Our data suggest that LjNRT2.1-mediated nitrate uptake/transport is required for LjNLP4 nuclear localization and induction/repression of symbiotic genes. We further show that LjNIN, a positive regulator of nodulation, counteracts the LjNLP1-dependent induction of LjNRT2.1 expression, which is linked to a reduction in nitrate uptake. These findings suggest a plant strategy in which nitrogen acquisition switches from obtaining nitrogen from the soil to symbiotic nitrogen fixation., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

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.)

Legume-rhizobia symbiosis enables biological nitrogen fixation to improve crop production for sustainable agriculture. Small heat shock proteins (sHSPs) are involved in multiple environmental stresses and plant development processes. However, the role of sHSPs in nodule development in soybean remains largely unknown. In the present study, we identified a nodule-localized sHSP, called GmHSP17.9, in soybean, which was markedly up-regulated during nodule development. GmHSP17.9 was specifically expressed in the infected regions of the nodules. GmHSP17.9 overexpression and RNAi in transgenic composite plants and loss of function in CRISPR-Cas9 gene-editing mutant plants in soybean resulted in remarkable alterations in nodule number, nodule fresh weight, nitrogenase activity, contents of poly β-hydroxybutyrate bodies (PHBs), ureide and total nitrogen content, which caused significant changes in plant growth and seed yield. GmHSP17.9 was also found to act as a chaperone for its interacting partner, GmNOD100, a sucrose synthase in soybean nodules which was also preferentially expressed in the infected zone of nodules, similar to GmHSP17.9. Functional analysis of GmNOD100 in composite transgenic plants revealed that GmNOD100 played an essential role in soybean nodulation. The hsp17.9 lines showed markedly more reduced sucrose synthase activity, lower contents of UDP-glucose and acetyl coenzyme A (acetyl-CoA), and decreased activity of succinic dehydrogenase (SDH) in the tricarboxylic acid (TCA) cycle in nodules due to the missing interaction with GmNOD100. Our findings reveal an important role and an unprecedented molecular mechanism of sHSPs in nodule development and nitrogen fixation in soybean., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

In legumes interacting with rhizobia, the formation of symbiotic organs involved in the acquisition of atmospheric nitrogen gas (N2) is dependent on the plant nitrogen (N) demand. We used Medicago truncatula plants cultivated in split-root systems to discriminate between responses to local and systemic N signaling. We evidenced a strong control of nodule formation by systemic N signaling but obtained no clear evidence of a local control by mineral nitrogen. Systemic signaling of the plant N demand controls numerous transcripts involved in root transcriptome reprogramming associated with early rhizobia interaction and nodule formation. SUPER NUMERIC NODULES (SUNN) has an important role in this control, but we found that major systemic N signaling responses remained active in the sunn mutant. Genes involved in the activation of nitrogen fixation are regulated by systemic N signaling in the mutant, explaining why its hypernodulation phenotype is not associated with higher nitrogen fixation of the whole plant. We show that the control of transcriptome reprogramming of nodule formation by systemic N signaling requires other pathway(s) that parallel the SUNN/CLE (CLAVATA3/EMBRYO SURROUNDING REGION-LIKE PEPTIDES) pathway., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The critical role of cytokinin in early nodulation in legumes is well known. In our study, exogenous cytokinin application to roots of the important crop legume, chickpea (Cicer arietinum L.), led to the formation of pseudo-nodules even in the absence of rhizobia. Hence, a genome-wide analysis of the cytokinin signalling, two-component system (TCS) genes, was conducted in chickpea, Medicago and Cajanus cajan. The integrated phylogenetic, evolutionary and expression analysis of the TCS genes was carried out, which revealed that histidine kinases (HKs) were highly conserved, whereas there was diversification leading to neofunctionalization at the level of response regulators (RRs) especially the TypeB RRs. Further, the functional role of the CaHKs in nodulation was established by complementation of the sln1Δ mutant of yeast and cre1 mutants of (Medicago) which led to restoration of the nodule-deficient phenotype. Additionally, the highest expressing TypeB RR of chickpea, CaRR13, was functionally characterized. Its localization in the nucleus and its Y1H assay-based interaction with the promoter of the early nodulation gene CaNSP2 indicated its role as a transcription factor regulating early nodulation. Overexpression, RNAi lines and complementation of cre1 mutants with CaRR13 revealed its critical involvement as an important signalling molecule regulating early events of nodule organogenesis in chickpea., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Autoregulation of nodulation (AON) plays a central role in nodulation by inhibiting the formation of excess number of legume root nodules. In this study, the effect of hydroxymethylglutaryl-coenzyme A reductase 1 (GmHMGR1) gene expression on nodulation and the AON system in Glycine max (L.) Merr was investigated. Wild-type soybean (cultivar Bragg) and its near-isogenic supernodulating mutant (nitrate tolerant symbiotic) nts1007 were selected to identify the expression pattern of this gene in rootlets after inoculation by its microsymbiont Bradyrhizobium. For further analysis, the full length of GmHMGR1 and its promoter were cloned after amplification by inverse-PCR and BAC library screening. Also, we constructed an intron hairpin RNA interference (ihpRNAi) and a GmHMGR1 promoter: β-glucuronidase fusion constructs, consequently for suppression of GmHMGR1 and histochemical analysis in transgenic soybean hairy roots induced by Agrobacterium rhizogenes strain K599. The GmHMGR1 gene was functional during the early stages of nodulation with the AON system having a negative effect on GmHMGR1 expression and nodule formation in wild-type rootlets. GmHMGR1 was particularly expressed in the developing phloem within the root, nodules and nodule lenticels. Expression of GmHMGR1 in transgenic hairy roots was suppressed by RNAi silencing approximately 85% as compared to empty vector controls. This suggests that the GmHMGR1 gene has an important role in triggering nodule formation as its suppression caused a reduction of nodule formation in nts mutant lines with a deficient AON system., (Crown Copyright © 2021. Published by Elsevier GmbH. All rights reserved.)

Symbiotic nitrogen fixation is an important part of the nitrogen biogeochemical cycles and the main nitrogen source of the biosphere. As a classical model system for symbiotic nitrogen fixation, rhizobium-legume systems have been studied elaborately for decades. Details about the molecular mechanisms of the communication and coordination between rhizobia and host plants is becoming clearer. For more systematic insights, there is an increasing demand for new studies integrating multiomics information. Here, we present a comprehensive computational framework integrating the reconstructed protein interactome of B. diazoefficiens USDA110 with its transcriptome and proteome data to study the complex protein-protein interaction (PPI) network involved in the symbiosis system. We reconstructed the interactome of B. diazoefficiens USDA110 by computational approaches. Based on the comparison of interactomes between B. diazoefficiens USDA110 and other rhizobia, we inferred that the slow growth of B. diazoefficiens USDA110 may be due to the requirement of more protein modifications, and we further identified 36 conserved functional PPI modules. Integrated with transcriptome and proteome data, interactomes representing free-living cell and symbiotic nitrogen-fixing (SNF) bacteroid were obtained. Based on the SNF interactome, a core-sub-PPI-network for symbiotic nitrogen fixation was determined and nine novel functional modules and eleven key protein hubs playing key roles in symbiosis were identified. The reconstructed interactome of B. diazoefficiens USDA110 may serve as a valuable reference for studying the mechanism underlying the SNF system of rhizobia and legumes.

Chickpea shoot exogenously treated with cytokinin showed stunted phenotype of root, shoot and significantly reduced nodule numbers. Genome-wide identification of LRR-RLKs in chickpea and Medicago resulted in 200 and 371 genes respectively. Gene duplication analysis revealed that LRR-RLKs family expanded through segmental duplications in chickpea and tandem duplications in Medicago. Expression profiling of LRR-RLKs revealed their involvement in cytokinin signaling and plant organ development. Overexpression of KLAVIER ortholog of chickpea, Ca_LRR-RLK147, in roots revealed its localization in the membrane but showed no effect on root nodulation despite increased cle peptide levels. Two findings (i) drastic effect on nodule number by exogenous cytokinin treatment to only shoot and restoration to normal nodulation by treatment to both root and shoot tissue and (ii) no effect on nodule number by overexpression of Ca_LRR-RLK147 establishes the fact that despite presence of cle peptides in root, the function of Ca_LRR-RLK147 was shoot mediated during AON., (Copyright © 2021. Published by Elsevier Inc.)

Salt stress reduces N 2 fixation by causing a reduction in nodule number, nodule weight, and nitrogenase activity in legumes. Emerging evidence from He et al. now suggests that glycogen synthase kinase 3 (GSK3) phosphorylates nodulation signaling pathway 1 (NSP1) in response to salt stress, reducing its DNA-binding activity, and thereby causing a reduction in nodulation., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background and Aims: Nitrogen fixation in legumes requires tight control of carbon and nitrogen balance. Thus, legumes control nodule numbers via an autoregulation mechanism. 'Autoregulation of nodulation' mutants super-nodulate are thought to be carbon-limited due to the high carbon-sink strength of excessive nodules. This study aimed to examine the effect of increasing carbon supply on the performance of super-nodulation mutants., Methods: We compared the responses of Medicago truncatula super-nodulation mutants (sunn-4 and rdn1-1) and wild type to five CO2 levels (300-850 μmol mol-1). Nodule formation and nitrogen fixation were assessed in soil-grown plants at 18 and 42 d after sowing., Key Results: Shoot and root biomass, nodule number and biomass, nitrogenase activity and fixed nitrogen per plant of all genotypes increased with increasing CO2 concentration and reached a maximum at 700 μmol mol-1. While the sunn-4 mutant showed strong growth retardation compared with wild-type plants, elevated CO2 increased shoot biomass and total nitrogen content of the rdn1-1 mutant up to 2-fold. This was accompanied by a 4-fold increase in nitrogen fixation capacity in the rdn1-1 mutant., Conclusions: These results suggest that the super-nodulation phenotype per se did not limit growth. The additional nitrogen fixation capacity of the rdn1-1 mutant may enhance the benefit of elevated CO2 for plant growth and N2 fixation., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Leguminous plants produce nodules for nitrogen fixation; however, nodule production incurs an energy cost. Therefore, as an adaptive strategy, leguminous plants halt root nodule development when sufficient amounts of nitrogen nutrients, such as nitrate, are present in the environment. Although legume NODULE INCEPTION (NIN)-LIKE PROTEIN (NLP) transcription factors have recently been identified, understanding how nodulation is controlled by nitrate, a fundamental question for nitrate-mediated transcriptional regulation of symbiotic genes, remains elusive. Here, we show that two Lotus japonicus NLPs, NITRATE UNRESPONSIVE SYMBIOSIS 1 (NRSYM1)/LjNLP4 and NRSYM2/LjNLP1, have overlapping functions in the nitrate-induced control of nodulation and act as master regulators for nitrate-dependent gene expression. We further identify candidate target genes of LjNLP4 by combining transcriptome analysis with a DNA affinity purification-seq approach. We then demonstrate that LjNLP4 and LjNIN, a key nodulation-specific regulator and paralog of LjNLP4, have different DNA-binding specificities. Moreover, LjNLP4-LjNIN dimerization underlies LjNLP4-mediated bifunctional transcriptional regulation. These data provide a basic principle for how nitrate controls nodulation through positive and negative regulation of symbiotic genes., (© 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.

Legume biological nitrogen fixation (BNF) is the most important N source in agricultural ecosystems. Nodule organogenesis from the primordia to the development of mature nodules with the ability to fix N 2 largely determines BNF capacity. However, nodule growth is often limited by low phosphorus (P) availability, while the mechanisms underlying nodule development responses to P deficiency remain largely unknown. In this study, we found that nodule enlargement is severely inhibited by P deficiency, as reflected by the smaller individual nodule size from a soybean core collection in the field. Wide-ranging natural diversity in nodule size was further identified in soybeans reared in low P soils, with the FC-1 genotype outperforming FC-2 in assessments of nodulation under low P conditions. Among β-expansin members, GmINS1 expression is most abundantly enhanced by P deficiency in FC-1 nodules, and its transcript level is further displayed to be tightly associated with nodule enlargement. Four single nucleotide polymorphisms discovered in the GmINS1 promoter distinguished the FC-1 and FC-2 genotypes and accounted for the differential expression levels of GmINS1 responses to P deficiency. GmINS1 overexpression led to increases in nodule size, infected cell abundance, and N 2 fixation capacity and subsequently promoted increases in N and P content, soybean biomass, and yield. Our findings provide a candidate gene for optimizing BNF capacity responses to low P stress in soybean molecular breeding programs., (© 2021 Scandinavian Plant Physiology Society.)

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.

Forward and reverse genetics using the model legumes Lotus japonicus and Medicago truncatula have been instrumental in identifying the essential genes governing legume-rhizobia symbiosis. However, little information is known about the effects of intraspecific variation on symbiotic signalling. Here, we use quantitative trait locus sequencing (QTL-seq) to investigate the genetic basis of the differentiated phenotypic responses shown by the Lotus accessions Gifu and MG20 to inoculation with the Mesorhizobium loti exoU mutant that produces truncated exopolysaccharides. We identified through genetic complementation the Pxy gene as a component of this differential exoU response. Lotus Pxy encodes a leucine-rich repeat receptor-like kinase similar to Arabidopsis thaliana PXY, which regulates stem vascular development. We show that Lotus pxy insertion mutants displayed defects in root and stem vascular organisation, as well as lateral root and nodule formation. Our work links Pxy to de novo organogenesis in the root, highlights the genetic overlap between regulation of lateral root and nodule formation, and demonstrates that natural variation in Pxy affects nodulation signalling., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)

Yellow Stripe-Like (YSL) proteins are a family of plant transporters that are typically involved in transition metal homeostasis. Three of the four YSL clades (I, II and IV) transport metals complexed with the non-proteinogenic amino acid nicotianamine or its derivatives. No such capability has been shown for any member of clade III, but the link between these YSLs and metal homeostasis could be masked by functional redundancy. We studied the role of the clade III YSL protein MtSYL7 in Medicago truncatula nodules. MtYSL7, which encodes a plasma membrane-bound protein, is mainly expressed in the pericycle and cortex cells of the root nodules. Yeast complementation assays revealed that MtSYL7 can transport short peptides. M. truncatula transposon insertion mutants with decreased expression of MtYSL7 had lower nitrogen fixation rates and showed reduced plant growth whether grown in symbiosis with rhizobia or not. YSL7 mutants accumulated more copper and iron in the nodules, which is likely to result from the increased expression of iron uptake and delivery genes in roots. Taken together, these data suggest that MtYSL7 plays an important role in the transition metal homeostasis of nodules and symbiotic nitrogen fixation., (© 2021 John Wiley & Sons Ltd.)

The multimember CEP (C-terminally Encoded Peptide) gene family is a complex group that is involved in various physiological activities in plants. Previous studies demonstrated that MtCEP1 and MtCEP7 control lateral root formation or nodulation, but these studies were based only on gain of function or artificial miRNA (amiRNA)/RNAi approaches, never knockout mutants. Moreover, an efficient multigene editing toolkit is not currently available for Medicago truncatula. Our quantitative reverse transcription-PCR data showed that MtCEP1, 2, 4, 5, 6, 7, 8, 9, 12, and 13 were up-regulated under nitrogen starvation conditions and that MtCEP1, 2, 7, 9, and 12 were induced by rhizobial inoculation. Treatment with synthetic MtCEP peptides of MtCEP1, 2, 4, 5, 6, 8, and 12 repressed lateral root emergence and promoted nodulation in the R108 wild type but not in the cra2 mutant. We optimized CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9] genome editing system for M. truncatula, and thus created single mutants of MtCEP1, 2, 4, 6, and 12 and the double mutants Mtcep1/2C and Mtcep5/8C; however, these mutants did not exhibit significant differences from R108. Furthermore, a triple mutant Mtcep1/2/12C and a quintuple mutant Mtcep1/2/5/8/12C were generated and exhibited more lateral roots and fewer nodules than R108. Overall, MtCEP1, 2, and 12 were confirmed to be redundantly important in the control of lateral root number and nodulation. Moreover, the CRISPR/Cas9-based multigene editing protocol provides an additional tool for research on the model legume M. truncatula, which is highly efficient at multigene mutant generation., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

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.)

Legume plants form nitrogen (N)-fixing symbiotic nodules when mineral N is limiting in soils. As N fixation is energetically costly compared to mineral N acquisition, these N sources, and in particular nitrate, inhibit nodule formation and N fixation. Here, in the model legume Medicago truncatula, we characterized a CLAVATA3-like (CLE) signaling peptide, MtCLE35, the expression of which is upregulated locally by high-N environments and relies on the Nodule Inception-Like Protein (NLP) MtNLP1. MtCLE35 inhibits nodule formation by affecting rhizobial infections, depending on the Super Numeric Nodules (MtSUNN) receptor. In addition, high N or the ectopic expression of MtCLE35 represses the expression and accumulation of the miR2111 shoot-to-root systemic effector, thus inhibiting its positive effect on nodulation. Conversely, ectopic expression of miR2111 or downregulation of MtCLE35 by RNA interference increased miR2111 accumulation independently of the N environment, and thus partially bypasses the nodulation inhibitory action of nitrate. Overall, these results demonstrate that the MtNLP1-dependent, N-induced MtCLE35 signaling peptide acts through the MtSUNN receptor and the miR2111 systemic effector to inhibit nodulation., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

NIN (NODULE INCEPTION) is a transcription factor that plays a key role during root nodule initiation. However, its role in later nodule developmental stages is unclear. Both NIN mRNA and protein accumulated at the highest level in the proximal part of the infection zone in Medicago truncatula nodules. Two nin weak allele mutants, nin-13/16, form a rather normal nodule infection zone, whereas a fixation zone is not formed. Instead, a zone with defence responses and premature senescence occurred and symbiosome development gets arrested. Mutations in nin-13/16 resulted in a truncated NIN lacking the conserved PB1 domain. However, this did not cause the nodule phenotype as nin mutants expressing NIN ΔPB1 formed wild-type-like nodule. The phenotype is likely to be caused by reduced NIN mRNA levels in the cytoplasm. Transcriptome analyses of nin-16 nodules showed that expression levels of defence/senescence-related genes are markedly increased, whereas the levels of defence suppressing genes are reduced. Although defence/senescence seems well suppressed in the infection zone, the transcriptome is already markedly changed in the proximal part of infection zone. In addition to its function in infection and nodule organogenesis, NIN also plays a major role at the transition from infection to fixation zone in establishing a functional symbiosis., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)

Legumes play an important role in the soil nitrogen availability via symbiotic nitrogen fixation (SNF). Phosphate (Pi) deficiency severely impacts SNF because of the high Pi requirement of symbiosis. Whereas PHT1 transporters are involved in Pi uptake into nodules, it is unknown how Pi is transferred from the plant infected cells to nitrogen-fixing bacteroids. We hypothesized that Medicago truncatula genes homologous to Arabidopsis PHO1, encoding a vascular apoplastic Pi exporter, are involved in Pi transfer to bacteroids. Among the seven MtPHO1 genes present in M. truncatula, we found that two genes, namely MtPHO1.1 and MtPHO1.2, were broadly expressed across the various nodule zones in addition to the root vascular system. Expressions of MtPHO1.1 and MtPHO1.2 in Nicotiana benthamiana mediated specific Pi export. Plants with nodule-specific downregulation of both MtPHO1.1 and MtPHO1.2 were generated by RNA interference (RNAi) to examine their roles in nodule Pi homeostasis. Nodules of RNAi plants had lower Pi content and a three-fold reduction in SNF, resulting in reduced shoot growth. Whereas the rate of 33Pi uptake into nodules of RNAi plants was similar to control, transfer of 33Pi from nodule cells into bacteroids was reduced and bacteroids activated their Pi-deficiency response. Our results implicate plant MtPHO1 genes in bacteroid Pi homeostasis and SNF via the transfer of Pi from nodule infected cells to bacteroids., (© The Author(s) 2020. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Microbial symbiosis in legumes is achieved through nitrogen-fixing root nodules, and these are important for sustainable agriculture. The molecular mechanisms underlying development of root nodules in polyploid legume crops are largely understudied. Through map-based cloning and QTL-seq approaches, we identified a pair of homoeologous GRAS transcription factor genes, Nodulation Signaling Pathway 2 (AhNSP2-B07 or Nb) and AhNSP2-A08 (Na), controlling nodulation in cultivated peanut (Arachis hypogaea L.), an allotetraploid legume crop, which exhibited non-Mendelian and Mendelian inheritance, respectively. The segregation of nodulation in the progeny of Nananbnb genotypes followed a 3:1 Mendelian ratio, in contrast to the 5:3~1:1 non-Mendelian ratio for nanaNbnb genotypes. Additionally, a much higher frequency of the nb allele (13%) than the na allele (4%) exists in the peanut germplasm collection, suggesting that Nb is less essential than Na in nodule organogenesis. Our findings reveal the genetic basis of naturally occurred non-nodulating peanut plants, which can be potentially used for nitrogen fixation improvement in peanut. Furthermore, the results have implications for and provide insights into the evolution of homoeologous genes in allopolyploid species., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Organogenesis of legume root nodules begins with the nodulation factor-dependent stimulation of compatible root cells to initiate divisions, signifying an early nodule primordium formation event. This is followed by cellular differentiation, including cell expansion and vascular bundle formation, and we previously showed that Lotus japonicus NF-YA1 is essential for this process, presumably by regulating three members of the SHORT INTERNODES/STYLISH (STY) transcription factor gene family. In this study, we used combined genetics, genomics and cell biology approaches to characterize the role of STY genes during root nodule formation and to test a hypothesis that they mediate nodule development by stimulating auxin signalling. We show here that L. japonicus STYs are required for nodule emergence. This is attributed to the NF-YA1-dependent regulatory cascade, comprising STY genes and their downstream targets, YUCCA1 and YUCCA11, involved in a local auxin biosynthesis at the post-initial cell division stage. An analogous NF-YA1/STY regulatory module seems to operate in Medicago truncatula in association with the indeterminate nodule patterning. Our data define L. japonicus and M. truncatula NF-YA1 genes as important nodule emergence stage-specific regulators of auxin signalling while indicating that the inductive stage and subsequent formation of early nodule primordia are mediated through an independent mechanism(s)., (© 2020 Her Majesty the Queen in Right of Canada. New Phytologist © 2020 New Phytologist Foundation.)

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.

Symbiotic nitrogen fixation carried out in legume root nodules requires transition metals. These nutrients are delivered by the host plant to the endosymbiotic nitrogen-fixing bacteria living within the nodule cells, a process in which vascular transport is essential. As members of the Yellow Stripe-Like (YSL) family of metal transporters are involved in root to shoot transport, they should also be required for root to nodule metal delivery. The genome of the model legume Medicago truncatula encodes eight YSL proteins, four of them with a high degree of similarity to Arabidopsis thaliana YSLs involved in long-distance metal trafficking. Among them, MtYSL3 is a plasma membrane protein expressed by vascular cells in roots and nodules and by cortical nodule cells. Reducing the expression level of this gene had no major effect on plant physiology when assimilable nitrogen was provided in the nutrient solution. However, nodule functioning was severely impaired, with a significant reduction of nitrogen fixation capabilities. Further, iron and zinc accumulation and distribution changed. Iron was retained in the apical region of the nodule, while zinc became strongly accumulated in the nodule veins in the ysl3 mutant. These data suggest a role for MtYSL3 in vascular delivery of iron and zinc to symbiotic nitrogen fixation., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Cyclins, together with highly conserved cyclin-dependent kinases (CDKs), play an important role in the process of cell cycle in plants, but less is known about the functions of cyclins in legume plants, especially Medicago truncatula . Our genome-wide analysis identified 58, 103, and 51 cyclin members in the M. truncatula , Glycine max , and Phaseolus vulgaris genomes. Phylogenetic analysis suggested that these cyclins could be classified into 10 types, and the CycB-like types (CycBL1-BL8) were the specific subgroups in M. truncatula , which was one reason for the expansion of the B-type in M. truncatula . All putative cyclin genes were mapped onto their own chromosomes of each genome, and 9 segmental duplication gene pairs involving 20 genes were identified in M. truncatula cyclins. Determined by quantitative real-time PCR, the expression profiling suggested that 57 cyclins in M. truncatula were differentially expressed in 9 different tissues, while a few genes were expressed in some specific tissues. Using the publicly available RNAseq data, the expression of Mtcyclins in the wild-type strain A17 and three nodule mutants during rhizobial infection showed that 23 cyclins were highly upregulated in the nodulation (Nod) factor-hypersensitive mutant sickle ( skl ) mutant after 12 h of rhizobium inoculation. Among these cyclins, six cyclin genes were also specifically expressed in roots and nodules, which might play specific roles in the various phases of Nod factor-mediated cell cycle activation and nodule development. Our results provide information about the cyclin gene family in legume plants, serving as a guide for further functional research on plant cyclins.

Nitrogen fixation in soybean consumes a tremendous amount of energy, leading to substantial differences in energy metabolism and mitochondrial activities between nodules and uninoculated roots. While C-to-U RNA editing and intron splicing of mitochondrial transcripts are common in plant species, their roles in relation to nodule functions are still elusive. In this study, we performed RNA-seq to compare transcript profiles and RNA editing of mitochondrial genes in soybean nodules and roots. A total of 631 RNA editing sites were identified on mitochondrial transcripts, with 12% or 74 sites differentially edited among the transcripts isolated from nodules, stripped roots, and uninoculated roots. Eight out of these 74 differentially edited sites are located on the matR transcript, of which the degrees of RNA editing were the highest in the nodule sample. The degree of mitochondrial intron splicing was also examined. The splicing efficiencies of several introns in nodules and stripped roots were higher than in uninoculated roots. These include nad1 introns 2/3/4, nad4 intron 3, nad5 introns 2/3, cox2 intron 1, and ccmFc intron 1. A greater splicing efficiency of nad4 intron 1, a higher NAD4 protein abundance, and a reduction in supercomplex I + III 2 were also observed in nodules, although the causal relationship between these observations requires further investigation.

Rhizobium leguminosarum bv. trifolii produces exopolysaccharide (EPS) composed of glucose, glucuronic acid, and galactose residues at a molar ratio 5:2:1. A majority of genes involved in the synthesis, modification, and export of exopolysaccharide are located in the chromosomal Pss-I region. In the present study, a Δ pssJ deletion mutant was constructed and shown to produce EPS lacking terminal galactose in the side chain of the octasaccharide subunit. The lack of galactose did not block EPS subunit translocation and polymerization. The in trans delivery of the pssJ gene restored the production of galactose-containing exopolysaccharide. The mutant was compromised in several physiological traits, e.g., motility and biofilm production. An impact of the pssJ mutation and changed EPS structure on the symbiotic performance was observed as improper signaling at the stage of molecular recognition, leading to formation of a significant number of non-infected empty nodules. Terminal galactosyltransferase PssJ was shown to display a structure typical for the GT-A class of glycosyltransferases and interact with other GTs and Wzx/Wzy system proteins. The latter, together with PssJ presence in soluble and membrane protein fractions indicated that the protein plays its role at the inner membrane interface and as a component of a larger complex.

Iron is an essential cofactor for symbiotic nitrogen fixation, required by many of the enzymes involved, including signal transduction proteins, O 2 homeostasis systems, and nitrogenase itself. Consequently, host plants have developed a transport network to deliver essential iron to nitrogen-fixing nodule cells. Ferroportin family members in model legume Medicago truncatula were identified and their expression was determined. Yeast complementation assays, immunolocalization, characterization of a tnt1 insertional mutant line, and synchrotron-based X-ray fluorescence assays were carried out in the nodule-specific M. truncatula ferroportin Medicago truncatula nodule-specific gene Ferroportin2 (MtFPN2) is an iron-efflux protein. MtFPN2 is located in intracellular membranes in the nodule vasculature and in inner nodule tissues, as well as in the symbiosome membranes in the interzone and early-fixation zone of the nodules. Loss-of-function of MtFPN2 alters iron distribution and speciation in nodules, reducing nitrogenase activity and biomass production. Using promoters with different tissular activity to drive MtFPN2 expression in MtFPN2 mutants, we determined that expression in the inner nodule tissues is sufficient to restore the phenotype, while confining MtFPN2 expression to the vasculature did not improve the mutant phenotype. These data indicate that MtFPN2 plays a primary role in iron delivery to nitrogen-fixing bacteroids in M. truncatula nodules., (© 2020 The Authors New Phytologist © 2020 New Phytologist Trust.)

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.

By analyzing successive lifestyle stages of a model Rhizobium- legume symbiosis using mariner-based transposon insertion sequencing (INSeq), we have defined the genes required for rhizosphere growth, root colonization, bacterial infection, N 2 -fixing bacteroids, and release from legume (pea) nodules. While only 27 genes are annotated as nif and fix in Rhizobium leguminosarum , we show 603 genetic regions (593 genes, 5 transfer RNAs, and 5 RNA features) are required for the competitive ability to nodulate pea and fix N 2 Of these, 146 are common to rhizosphere growth through to bacteroids. This large number of genes, defined as rhizosphere-progressive, highlights how critical successful competition in the rhizosphere is to subsequent infection and nodulation. As expected, there is also a large group (211) specific for nodule bacteria and bacteroid function. Nodule infection and bacteroid formation require genes for motility, cell envelope restructuring, nodulation signaling, N 2 fixation, and metabolic adaptation. Metabolic adaptation includes urea, erythritol and aldehyde metabolism, glycogen synthesis, dicarboxylate metabolism, and glutamine synthesis (GlnII). There are 17 separate lifestyle adaptations specific to rhizosphere growth and 23 to root colonization, distinct from infection and nodule formation. These results dramatically highlight the importance of competition at multiple stages of a Rhizobium- legume symbiosis., Competing Interests: The authors declare no competing interest.

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 .

Legumes have evolved a symbiotic relationship with rhizobial bacteria and their roots form unique nitrogen-fixing organs called nodules. Studies have shown that abiotic and biotic stresses alter the profile of gene expression and transcript mobility in plants. However, little is known about the systemic transport of RNA between roots and shoots in response to rhizobial infection on a genome-wide scale during the formation of legume-rhizobia symbiosis. In our study, we found that two soybean (Glycine max) cultivars, Peking and Williams, show a high frequency of single nucleotide polymorphisms; this allowed us to characterize the origin and mobility of transcripts in hetero-grafts of these two cultivars. We identified 4,552 genes that produce mobile RNAs in soybean, and found that rhizobial infection triggers mass transport of mRNAs between shoots and roots at the early stage of nodulation. The majority of these mRNAs are of relatively low abundance and their transport occurs in a selective manner in soybean plants. Notably, the mRNAs that moved from shoots to roots at the early stage of nodulation were enriched in many nodule-related responsive processes. Moreover, the transcripts of many known symbiosis-related genes that are induced by rhizobial infection can move between shoots and roots. Our findings provide a deeper understanding of endogenous RNA transport in legume-rhizobia symbiotic processes.

Post-transcriptional control of gene expression allows plants to rapidly adapt to changes in their environment. Under low nitrogen conditions, legume plants engage into a symbiosis with soil bacteria that results in the formation of root nodules, where bacteria are allocated and fix atmospheric nitrogen for the plant's benefit. Recent studies highlighted the importance of small RNA-mediated mechanisms in the control of bacterial infection, nodule organogenesis, and the long-distance signaling that balances plant growth and nodulation. Examples of such mechanisms are shoot-to-root mobile microRNAs and small RNA fragments derived from degradation of bacterial transfer RNAs that repress complementary mRNAs in the host plant. Mechanisms of selective mRNA translation also contribute to rapidly modulate the expression of nodulation genes in a cell-specific manner during symbiosis. Here, the most recent advances made on the regulation of mRNA stability and translatability, and the emerging roles of long non-coding RNAs in symbiosis are summarized., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The plant receptor-like-kinase subfamily CrRLK1L has been widely studied, and CrRLK1Ls have been described as crucial regulators in many processes in Arabidopsis thaliana (L.), Heynh. Little is known, however, about the functions of these proteins in other plant species, including potential roles in symbiotic nodulation. We performed a phylogenetic analysis of CrRLK1L subfamily receptors of 57 different plant species and identified 1050 CrRLK1L proteins, clustered into 11 clades. This analysis revealed that the CrRLK1L subfamily probably arose in plants during the transition from chlorophytes to embryophytes and has undergone several duplication events during its evolution. Among the CrRLK1Ls of legumes and A. thaliana , protein structure, gene structure, and expression patterns were highly conserved. Some legume CrRLK1L genes were active in nodules. A detailed analysis of eight nodule-expressed genes in Phaseolus vulgaris L. showed that these genes were differentially expressed in roots at different stages of the symbiotic process. These data suggest that CrRLK1Ls are both conserved and underwent diversification in a wide group of plants, and shed light on the roles of these genes in legume-rhizobia symbiosis.

Legumes and actinorhizal plants are capable of forming root nodules symbiosis with rhizobia and Frankia bacteria. All these nodulating species belong to the nitrogen fixation clade. Most likely, nodulation evolved once in the last common ancestor of this clade. NIN (NODULE INCEPTION) is a transcription factor that is essential for nodulation in all studied species. Therefore, it seems probable that it was recruited at the start when nodulation evolved. NIN is the founding member of the NIN-like protein (NLP) family. It arose by duplication, and this occurred before nodulation evolved. Therefore, several plant species outside the nitrogen fixation clade have NLP(s), which is orthologous to NIN. In this review, we discuss how NIN has diverged from the ancestral NLP, what minimal changes would have been essential for it to become a key transcription controlling nodulation, and which adaptations might have evolved later.

Expression of Nodule Inception (NIN) is essential for initiation of legume-rhizobial symbiosis. An existing model regarding the regulation of NIN expression involves two GRAS transcription factors - NSP1 (Nodulation Signaling Pathway 1) and NSP2. NSP2 forms a complex with NSP1 to directly bind to NIN promoter. However, rhizobial treatment-induced NIN expression could still be detected in the nsp1 mutant plants, suggesting that other proteins must be involved in the regulation of NIN expression. A combination of molecular, biochemical and genetic analyses was used to investigate the molecular basis of IPN2 in regulating root development and NIN expression in Lotus japonicus. In this study, we identified that IPN2 is a close homolog of Arabidopsis APL (ALTERED PHLOEM DEVELOPMENT) with essential function in root development. However, Lotus IPN2 has a different expression pattern compared with the Arabidopsis APL gene. IPN2 binds to the IPN2-responsive cis element (IPN2-RE) of NIN promoter and activates NIN expression. IPN2, NSP1 and NSP2 form a protein complex to directly target NIN promoter and activate NIN expression in the legume-rhizobial symbiosis. Our data refine the regulatory model of NIN expression that NSP2 works together with NSP1 and IPN2 to activate the NIN gene allowing nodulation in L. japonicus., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)