Nitrogen-fixing Root Nodules Research Articles

Peptide Signals Regulate Nitrogen Deficiency Adaptation of Dicotyledonous Model Plants.

Nitrogen is a crucial macroelement essential for plant growth and development. In Arabidopsis Thaliana, classical phytohormones such as auxin and cytokinin orchestrate local and systemic signalling networks coordinate plant growth and development in response to nitrogen deficiency. Nowadays, emerging signalling pathways involving small peptides like CLAVATA3/EMBRYO SURROUNDINGR REGION (CLE) and C-TERMINALLY ENCODED PEPTIDE (CEP) and their corresponding kinase receptors, also regulate Arabidopsis' adaptation to nitrogen scarcity. Unlike Arabidopsis, which adapts to nitrogen deficiency by changing root development, legumes have the unique ability to form nitrogen-fixing root nodules through symbiotic interactions with soil rhizobia. During the symbiotic nodulation in Medicago, CLE and CEP peptides and their receptors consist of an autoregulatory network governing the number of nodules in accordance with the soil nitrogen level. Additionally, other plant peptides, such as phytosulfokine (PSK) and root meristem growth factors (RGF), have been identified as new regulators of leguminous root nodule development under nitrogen-limited condition. However, the precise mechanism by which these peptides coordinate nitrogen deficiency response and the development of nitrogen-fixing organs remains to be fully elucidated. This review summarises the adaptive strategies of dicotyledons to nitrogen deficiency, with a particular focus on the regulation of Medicago nitrogen-fixing nodule development by the peptides.

The CEP peptide-CRA2 receptor module promotes arbuscular mycorrhizal symbiosis.

C-terminally encoded peptides (CEPs) are small secreted signaling peptides that promote nitrogen-fixing root nodulation symbiosis in legumes, depending on soil mineral nitrogen availability.1 In Medicago truncatula, their action is mediated by the leucine-rich repeat receptor-like protein kinase COMPACT ROOT ARCHITECTURE 2 (CRA2).2,3,4 Like most land plants, under inorganic phosphate limitation, M.truncatula establishes another root endosymbiotic interaction with arbuscular fungi, the arbuscular mycorrhizal symbiosis (AMS). Because this interaction is beneficial for the plant but has a high energetic cost, it is tightly controlled by host plants to limit fungal infections mainly depending on phosphate availability.5 We show in this study that the expression of a subset of CEP-encoding genes is enhanced in the low-phosphate conditions and that overexpression of the low-phosphate-induced MtCEP1 gene, previously shown to promote the nitrogen-fixing root nodulation symbiosis, enhances AMS from the initial entry point of the fungi. Conversely, a loss-of-function mutation of the CRA2 receptor required for mediating CEP peptide action2 decreases the endomycorrhizal interaction from the same initial fungal entry stage. Transcriptomic analyses revealed that the cra2 mutant is negatively affected in the regulation of key phosphate transport and response genes as well as in the biosynthesis of strigolactone hormones that are required for establishing AMS. Accordingly, strigolactone contents were drastically decreased in cra2 mutant roots. Overall, we showed that the CEP/CRA2 pathway promotes both root nodulation and AMS in legume plants, depending on soil mineral nutrient availability.

Hot Spots of Site-Specific Integration into the Sinorhizobium meliloti Chromosome.

The diversity of phage-related sequences (PRSs) and their site-specific integration into the genomes of nonpathogenic, agriculturally valuable, nitrogen-fixing root nodule bacteria, such as Sinorhizobium meliloti, were evaluated in this study. A total of 314 PRSs, ranging in size from 3.24 kb to 88.98 kb, were identified in the genomes of 27 S. meliloti strains. The amount of genetic information foreign to S. meliloti accumulated in all identified PRSs was 6.30 Mb. However, more than 53% of this information was contained in prophages (Phs) and genomic islands (GIs) integrated into genes encoding tRNAs (tRNA genes) located on the chromosomes of the rhizobial strains studied. It was found that phiLM21-like Phs were predominantly abundant in the genomes of S. meliloti strains of distant geographical origin, whereas RR1-A- and 16-3-like Phs were much less common. In addition, GIs predominantly contained fragments of phages infecting bacteria of distant taxa, while rhizobiophage-like sequences were unique. A site-specific integration analysis revealed that not all tRNA genes in S. meliloti are integration sites, but among those in which integration occurred, there were "hot spots" of integration into which either Phs or GIs were predominantly inserted. For the first time, it is shown that at these integration "hot spots", not only is the homology of attP and attB strictly preserved, but integrases in PRSs similar to those of phages infecting the Proteobacteria genera Azospirillum or Pseudomonas are also present. The data presented greatly expand the understanding of the fate of phage-related sequences in host bacterial genomes and also raise new questions about the role of phages in bacterial-phage coevolution.

Three separate pathways in Rhizobium leguminosarum maintain phosphatidylcholine biosynthesis, which is required for symbiotic nitrogen fixation with clover.

Understanding the molecular mechanisms of symbiotic nitrogen fixation has important implications for sustainable agriculture. The presence of the phospholipid phosphatidylcholine (PC) in the membrane of rhizobia is critical for the establishment of productive nitrogen-fixing root nodules on legume plants. The reasons for the PC requirement are unknown. Here, we employed Rhizobium leguminosarum and clover as model system for a beneficial plant-microbe interaction. We found that R. leguminosarum produces PC by three distinct pathways. The relative contribution of these pathways to PC formation was determined in an array of single, double, and triple mutant strains. Several of the PC biosynthesis enzymes were purified and biochemically characterized. Most importantly, we demonstrated the essential role of PC formation by R. leguminosarum in nitrogen fixation and pinpointed a specific enzyme indispensable for plant-microbe interaction. Our study offers profound insights into bacterial PC biosynthesis and its pivotal role in biological nitrogen fixation.

Pea leaf weevil, Sitona lineatus (L.) (Coleoptera: Curculionidae), adult feeding preference and larval development on pulse crop varieties in Montana.

Montana accounts for approximately 45% of US dry pea production and the pea leaf weevil (PLW; Sitona lineatus (L.)) is the most common insect pest in this region. After crop emergence adult PLW feed on the foliage to mature and subsequently mate, and the soil-dwelling larvae feed and develop on the nitrogen-fixing root nodules. Producers commonly apply prophylactic insecticide treatments to the seed at planting as well as one or two post-emergent insecticide sprays to control PLW damage. To develop alternative management strategies based on integrated pest management (IPM), this field study evaluated pulse crops grown in Montana for adult feeding preference and larval development. Ten different field pea varieties, along with two faba bean, lentil and chickpea varieties, were evaluated during the 2020 and 2021 field seasons at the Montana State University Arthur H. Post Agronomy Farm. Significant PLW pest pressure was observed within the research plots during both experimental years. Field pea and faba bean were preferred by the foliage feeding adult stage, with all but one variety averaging 39.2 to 86.3 average notches per plant. The pea variety Lifter was significantly preferred over all other comparisons, averaging 142.4 and 95.0 notches per plant in 2020 and 2021, respectively. Adult PLW feeding on lentil and chickpea was minimal, averaging 3.3 to 8.2 and 0.5 to 1.6 notches per plant, respectively. Numbers of larvae were highest on the roots of pea varieties, a known reproductive host, and almost nil on lentil and chickpea roots. Faba bean is also known as reproductive host, but, unexpectedly, larval populations were also low on the two faba bean varieties. The results from this study provide some limited evidence for alternative IPM strategies for field peas based on host plant tolerance or resistance within the range of varieties tested. Adult preference and larval development of PLW varied between the different pulse crops with field peas and faba beans being the most susceptible and lentils and chickpeas being the least susceptible. Host plant resistance against PLW could provide more sustainable IPM approaches in the future. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genetically Modified Legume Plants as a Basis for Studying the Signal Regulation of Symbiosis with Nodule Bacteria

The development of legume–rhizobial symbiosis results in the formation of nitrogen-fixing root nodules. In response to rhizobial molecules, Nod factors, signal transduction is mediated by the interaction of activated receptors with downstream signaling proteins. Previously, some new regulators of the signal pathway, such as phospholipases D, which regulate the level of phosphatidic acid (PA), as well as mitogen-activated protein kinases (MAPKs), have been identified in legumes. Since PA is an important signal messenger, we tested the hypothesis that increasing the level of proteins involved in the reversible binding of PA in plant tissues may have a positive effect on symbiosis. Our findings showed that overexpression of MtSPHK1-PA, encoding the PA-binding domain of sphingosine kinase 1 (SPHK1), stimulated plant growth and nodule development in legume plants. Furthermore, the influence of MAPK6 on the development of symbiosis was studied. Using genetic engineering methods, we increased MAPK6 transcriptional activity in transgenic roots, leading to an increase in the number of nodules and the biomass of pea plants. Therefore, new approaches to obtain plants with an increased efficiency of symbiosis were tested. We report here that both genes that encode signaling proteins may be used as potential targets for future modification using biotechnological approaches.

Genetically modified legume plants as a basis for studying the signal regulation of symbiosis with nodule bacteria

The development of legume-rhizobium symbiosis is based on signal exchange between partners, which leads to the formation of nitrogen-fixing root nodules. Under the influence of rhizobial signal molecules, the Nod factors, the signal transduction cascade is activated, where the LysM-type receptor kinases and a complex of intracellular regulators, a significant part of which are still unknown, can play an important role.
 Using transcriptomic and proteomic analysis, we searched for new regulators of the signal pathway in pea Pisum sativum L., which are activated under the influence of Nod factors. Phospholipases C and D, GTPases, calcium-dependent protein kinases, and mitogen-activated protein kinases (MAPKs) have been identified among such novel regulators. The influence of one of such regulators, the MAPK6, on the development of symbiosis was studied in more detail. Using genetic engineering approaches, we increased the transcriptional activity of MAPK6 in transgenic roots, which led to an increase in the number of nodules and the biomass of pea plants. A similar effect was also found for the homologous MAPK6 gene in another legume Medicago truncatula, which has a type of nodulation similar to that one in pea. New approaches have been developed to obtain stable pea transformants with enhanced level of MAPK6 transcription using the constitutive p35S promoter. A comprehensive study of such plants inoculated with rhizobia was carried out. In addition, the approaches for genome editing of pea plants have been designed using the CRISPR/Cas system, when the MAPK6 gene was used as a target. Thus, the possibility of using genetic engineering methods to obtain plants with increased symbiosis efficiency was investigated.

Conserved structured domains in plant non-coding RNA enod40, their evolution and recruitment of sequences from transposable elements.

Plant long noncoding RNA enod40 is involved in the regulation of symbiotic associations with bacteria, in particular, in nitrogen-fixing root nodules of legumes, and with fungi in phosphate-acquiring arbuscular mycorrhizae formed by various plants. The presence of enod40 genes in plants that do not form such symbioses indicates its other roles in cell physiology. The molecular mechanisms of enod40 RNA function are poorly understood. Enod40 RNAs form several structured domains, conserved to different extents. Due to relatively low sequence similarity, identification of enod40 sequences in plant genomes is not straightforward, and many enod40 genes remain unannotated even in complete genomes. Here, we used comparative structure analysis and sequence similarity searches in order to locate enod40 genes and determine enod40 RNA structures in nitrogen-fixing clade plants and in grasses. The structures combine conserved features with considerable diversity of structural elements, including insertions of structured domain modules originating from transposable elements. Remarkably, these insertions contain sequences similar to tandem repeats and several stem-loops are homologous to microRNA precursors.

Identification and evolution of nsLTPs in the root nodule nitrogen fixation clade and molecular response of Frankia to AgLTP24

Non-specific lipid transfer proteins (nsLTPs) are antimicrobial peptides, involved in several plant biological processes including root nodule nitrogen fixation (RNF). Nodulating plants belonging to the RNF clade establish symbiosis with the nitrogen-fixing bacteria rhizobia (legumes symbiosis model) and Frankia (actinorhizal symbiosis model) leading to root nodule formation. nsLTPs are involved in processes active in early step of symbiosis and functional nodule in both models. In legumes, nsLTPs have been shown to regulate symbiont entry, promote root cortex infection, membrane biosynthesis, and improve symbiosis efficiency. More recently, a nsLTP, AgLTP24 has been described in the context of actinorhizal symbiosis between Alnus glutinosa and Frankia alni ACN14a. AgLTP24 is secreted at an early step of symbiosis on the deformed root hairs and targets the symbiont in the nitrogen-fixing vesicles in functional nodules. nsLTPs are involved in RNF, but their functions and evolutionary history are still largely unknown. Numerous putative nsLTPs were found up-regulated in functional nodules compared to non-infected roots in different lineages within the RNF clade. Here, results highlight that nodulating plants that are co-evolving with their nitrogen-fixing symbionts appear to have independently specialized nsLTPs for this interaction, suggesting a possible convergence of function, which opens perspectives to investigate nsLTPs functions in RNF.

Diverse Interactions: Root-Nodule Formation and Herb-Layer Composition in Black Locust (Robinia pseudoacacia) Stands.

The black locust (Robinia pseudoacacia L.) is the second-most abundant deciduous tree in forest plantations, and one of the most important invasive woody species worldwide. The species has a strong transformer capacity, especially expressed by its nitrogen enrichment effect caused by nitrogen-fixing bacteria living in its root-nodules. The aim of this study was to explore the mutually interacting factors of nitrogen-fixing root-nodules, site characteristics, and herb-layer composition of 28 North Hungarian black locust stands. In the herb-layers of the study sites, a total of 121 plant species were identified, representing a relatively low species richness. The studied black locust stands showed high variability both in their herb-layer compositions and root-nodule formation, but no clear relationship could be demonstrated between these characteristics. The PCA component with which the species richness and Shannon-Wiener diversity index were strongly correlated was negatively associated with all root-nodule parameters (number, surface area, and weight), supporting the biodiversity-reducing effect of black locust by its nitrogen-fixing bacteria. All of the root-nodule parameters were negatively correlated with the PCA factor predominantly determined by stand age, confirming that the root-nodule biomass decreases as time progresses.

Diversity, Genomics and Symbiotic Characteristics of Sinorhizobia That Nodulate Desmanthus spp. in Northwest Argentina.

Desmanthus spp. are legumes with the ability to associate with diverse α-proteobacteria-a microsymbiont-in order to establish nitrogen-fixing root nodules. A previous investigation from our laboratory revealed that the main bacteria associated with Desmanthus paspalaceus in symbiosis in central Argentina (Province of Santa Fe) were quite diverse and belonged to the genera Rhizobium and Mesorhizobium. To achieve a more extensive view of the local microsymbionts associated with Desmanthus spp., we sampled three different sites in Jujuy and Salta, in northwest Argentina. Matrix-assisted Laser-Desorption-Ionization Time-of-Flight mass spectrometry (MALDI-TOF) typing, 16S-rDNA analysis, and genome sequencing demonstrated that the dominant root-nodule microsymbionts belonged to the genus Sinorhizobium, with some sequenced genomes related to Sinorhizobium mexicanum, Sinorhizobium chiapanecum, and Sinorhizobium psoraleae. An analysis of nodA and nodC markers indicated that, in some of the isolates, horizontal gene transfer appeared to be responsible for the lack of congruence between the phylogenies of the chromosome and of the symbiotic region. These results revealed diverse evolutionary strategies for reaching the current Desmanthus-microsymbiont diversity. What is remarkable beside their observed genetic diversity is that the tolerance profiles of these isolates to abiotic stresses (temperature, salt concentration, pH) were quite coincident with the separation of the sinorhizobia according to place of origin, suggesting possible ecoedaphic adaptations. This observation, together with the higher aerial dry-weight matter that some isolates generated in Desmanthus virgatus cv. Marc when compared to the biomass generated by the commercial strain Sinorhizobium terangae CB3126, distinguish the collected sinorhizobia as constituting valuable germplasm for evaluation in local fields to select for more efficient symbiotic pairs.

Comparative phylotranscriptomics reveals ancestral and derived root nodule symbiosis programmes

Symbiotic interactions such as the nitrogen-fixing root nodule symbiosis (RNS) have structured ecosystems during the evolution of life. Here we aimed at reconstructing ancestral and intermediate steps that shaped RNS observed in extant flowering plants. We compared the symbiotic transcriptomic responses of nine host plants, including the mimosoid legume Mimosa pudica for which we assembled a chromosome-level genome. We reconstructed the ancestral RNS transcriptome composed of most known symbiotic genes together with hundreds of novel candidates. Cross-referencing with transcriptomic data in response to experimentally evolved bacterial strains with gradual symbiotic proficiencies, we found the response to bacterial signals, nodule infection, nodule organogenesis and nitrogen fixation to be ancestral. By contrast, the release of symbiosomes was associated with recently evolved genes encoding small proteins in each lineage. We demonstrate that the symbiotic response was mostly in place in the most recent common ancestor of the RNS-forming species more than 90 million years ago.

The Medicago SymCEP7 hormone increases nodule number via shoots without compromising lateral root number.

Legumes acquire soil nutrients through nitrogen-fixing root nodules and lateral roots. To balance the costs and benefits of nodulation, legumes negatively control root nodule number by autoregulatory and hormonal pathways. How legumes simultaneously coordinate root nodule and lateral root development to procure nutrients remains poorly understood. In Medicago (Medicago truncatula), a subset of mature C-TERMINALLY ENCODED PEPTIDE (CEP) hormones can systemically promote nodule number, but all CEP hormones tested to date negatively regulate lateral root number. Here we showed that Medicago CEP7 produces a mature peptide, SymCEP7, that promotes nodulation from the shoot without compromising lateral root number. Rhizobial inoculation induced CEP7 in the susceptible root nodulation zone in a Nod factor-dependent manner, and, in contrast to other CEP genes, its transcription level was elevated in the ethylene signaling mutant sickle. Using mass spectrometry, fluorescence microscopy and expression analysis, we demonstrated that SymCEP7 activity requires the COMPACT ROOT ARCHITECTURE 2 receptor and activates the shoot-to-root systemic effector, miR2111. Shoot-applied SymCEP7 rapidly promoted nodule number in the pM to nM range at concentrations up to five orders of magnitude lower than effects mediated by root-applied SymCEP7. Shoot-applied SymCEP7 also promoted nodule number in White Clover (Trifolium repens) and Lotus (Lotus japonicus), which suggests that this biological function may be evolutionarily conserved. We propose that SymCEP7 acts in the Medicago shoot to counter balance the autoregulation pathways induced rapidly by rhizobia to enable nodulation without compromising lateral root growth, thus promoting the acquisition of nutrients other than nitrogen to support their growth.

3-Pentanol glycosides from root nodules of the actinorhizal plant Alnus cremastogyne

Alnus cremastogyne Burkill (Betulaceae), an actinorhizal plant, can enter a mutualistic symbiosis with Frankia species that leads to the formation of nitrogen fixing root nodules. Some primary metabolites (carbohydrates, dicarboxylic acids, amino acids, citrulline and amides) involved in carbon and nitrogen metabolism in actinorhizal nodules have been identified, while specialized metabolites in A. cremastogyne root nodules are yet to be characterized. In this study, we isolated and identified three undescribed 3-pentanol glycosides, i.e., 3-pentyl α-l-arabinofuranosyl-(1′′→6′)-β-d-glucopyranoside, 3-pentyl α-l-rhamnopyranosyl-(1′′→6′)-β-d-glucopyranoside, and 3-pentyl 6′-(3-hydroxy3-methylglutaryl)-β-d-glucopyranoside, as well as seventeen known compounds from A. cremastogyne root nodules. 3-Pentanol glycosides are abundantly distributed in root nodules, while they are distributed in stems, roots, leaves and fruits at low/zero levels. A. cremastogyne plants treated by root nodule suspension emit 3-pentanol. This study enriches the knowledge about specialized metabolites in the actinorhizal host, and provides preliminarily information on the signal exchange in the actinorhizal symbiosis between A. cremastogyne and Frankia.

Synergism of Bradyrhizobium and Azospirillum baldaniorum improves growth and symbiotic performance in lima bean under salinity by positive modulations in leaf nitrogen compounds

Salinity causes serious agricultural losses, and inoculation with plant growth-promoting bacteria (PGPB) represents an alternative to mitigate this and other abiotic stresses. This study aimed to investigate the effectiveness of five different rhizobia strains (B. elkanii BR 2003, B. pachyrhizi BR 3262, B. yuanmingense BR 3267, B. paxllaeri BR 10398, and B. icense BR 10399) and one Azospirillum baldaniorum strain (Sp245), alone or in combination, in inducing salt stress tolerance in lima bean. Plants were grown from seeds and inoculated with different rhizobia strains, A. baldaniorum, or both (coinoculation) and exposed to 0 (control) and 75 mM NaCl. Uninoculated plants were used as absolute control. Salinity negatively affects the absolute growth rate, root length, plant biomass (shoot, root, and nodules), number of nodules, and photosynthetic pigments. However, plants coinoculated with rhizobia and A. baldaniorum showed the highest value for these parameters. Salt stress provokes negative alterations in nitrogen compounds (nitrate, free ammonia, free amino acids, proline, and soluble protein). Nevertheless, these responses were minimized in plants coinoculated with rhizobia and A. baldaniorum — mainly BR 2003 and A. baldaniorum or BR 10399 and A. baldaniorum. The highest total N values were registered in plants coinoculated with BR 3267 and A. baldaniorum or BR 10399 and A. baldaniorum. Under salt stress, coinoculated plants show a minor increase in sodium and the highest potassium content values, nitrogen derived from the atmosphere, and nitrogen fixation efficiency than plants inoculated with rhizobia. Our findings indicate that lima bean coinoculation with rhizobia and A. baldaniorum was beneficial under salinity and improved salt tolerance. Synergistic interactions between rhizobia and A. baldaniorum, mainly among BR 2003 and A. baldaniorum and BR 10399 and A. baldaniorum, attenuated the deleterious effects of salt stress on lima bean. Furthermore, nitrogen fixation in root nodules was improved in coinoculated plants. Our results may be useful to develop adequate combinations of inoculants with rhizobia and A. baldaniorum for improving the growth and symbiotic performance of lima bean and others legumes under stressful conditions.

A CCaMK/Cyclops response element in the promoter of Lotus japonicus calcium-binding protein 1 (CBP1) mediates transcriptional activation in root symbioses.

Early gene expression in arbuscular mycorrhiza (AM) and the nitrogen-fixing root nodule symbiosis (RNS) is governed by a shared regulatory complex. Yet many symbiosis-induced genes are specifically activated in only one of the two symbioses. The Lotus japonicus T-DNA insertion line T90, carrying a promoterless uidA (GUS) gene in the promoter of Calcium Binding Protein 1 (CBP1) is exceptional as it exhibits GUS activity in both root endosymbioses. To identify the responsible cis- and trans-acting factors, we subjected deletion/modification series of CBP1 promoter : reporter fusions to transactivation and spatio-temporal expression analysis and screened ethyl methanesulphonate (EMS)-mutagenized T90 populations for aberrant GUS expression. We identified one cis-regulatory element required for GUS expression in the epidermis and a second element, necessary and sufficient for transactivation by the calcium and calmodulin-dependent protein kinase (CCaMK) in combination with the transcription factor Cyclops and conferring gene expression during both AM and RNS. Lack of GUS expression in T90 white mutants could be traced to DNA hypermethylation detected in and around this element. We concluded that the CCaMK/Cyclops complex can contribute to at least three distinct gene expression patterns on its direct target promoters NIN (RNS), RAM1 (AM), and CBP1 (AM and RNS), calling for yet-to-be identified specificity-conferring factors.

Rhizobial HmuSpSym as a heme-binding factor is required for optimal symbiosis between Mesorhizobium amorphae CCNWGS0123 and Robinia pseudoacacia.

Nitrogen-fixing root nodules are formed by symbiotic association of legume hosts with rhizobia in nitrogen-deprived soils. Successful symbiosis is regulated by signals from both legume hosts and their rhizobial partners. HmuS is a heme degrading factor widely distributed in bacteria, but little is known about the role of rhizobial hmuS in symbiosis with legumes. Here, we found that inactivation of hmuSpSym in the symbiotic plasmid of Mesorhizobium amorphae CCNWGS0123 disrupted rhizobial infection, primordium formation, and nitrogen fixation in symbiosis with Robinia pseudoacacia. Although there was no difference in bacteroids differentiation, infected plant cells were shrunken and bacteroids were disintegrated in nodules of plants infected by the ΔhmuSpSym mutant strain. The balance of defence reaction was also impaired in ΔhmuSpSym strain-infected root nodules. hmuSpSym was strongly expressed in the nitrogen-fixation zone of mature nodules. Furthermore, the HmuSpSym protein could bind to heme but not degrade it. Inactivation of hmuSpSym led to significantly decreased expression levels of oxygen-sensing related genes in nodules. In summary, hmuSpSym of M. amorphae CCNWGS0123 plays an essential role in nodule development and maintenance of bacteroid survival within R. pseudoacacia cells, possibly through heme-binding in symbiosis.

Rhizobial infection of 4C cells triggers their endoreduplication during symbiotic nodule development in soybean.

Symbiosis between legumes and rhizobia results in the formation of nitrogen-fixing root nodules. Endoreduplication is essential for nodule development and efficient nitrogen fixation; however, the cellular mechanism by which rhizobial infection causes endoreduplication in symbiotic nodules and the roles of the resulting polyploid cells in nitrogen fixation remain largely unknown. Here, we developed a series of different approaches to separate infected cells (ICs) and uninfected cells (UCs) and determined their ploidy levels in soybean (Glycine max) developing nodules. We demonstrated that 4C nuclei exist in both UCs and ICs of developing nodules and that these 4C cells are primarily invaded by rhizobia and subsequently undergo endoreduplication. Furthermore, RNA-sequencing analysis of nuclei with different ploidy levels from soybean nodules at 12 d post-infection (dpi) and 20 dpi showed that 4C cells are predominantly ICs in 12-dpi nodules but UCs in 20-dpi nodules. We conclude that the infection of 4C cells by rhizobia is critical for initiating endoreduplication. These findings provide significant insight into rhizobial infection, nodule endoreduplication and nitrogen fixation in symbiotic nodules.

From Roots to Leaves: The Capacity of Micromonospora to Colonize Different Legume Tissues

An important number of Micromonospora strains have been reported from nitrogen-fixing root nodules of legume and actinorhizal plants. However, the question of whether this bacterium can also be found in other parts of these plants remains unanswered. More than 150 strains were recovered from different Lupinus angustifolius and Pisum sativum tissues, including leaves, stems, roots, and nodules. In all, 97% of the isolates were identified by 16S ribosomal RNA gene sequence in the target genus and were associated with 27 different Micromonospora spp. Plant-polymer-degrading enzymes are suspected to play a role in the colonization of plants. To this end, bacterial enzymatic activity assays for amylases, cellulases, chitinases, pectinases, and xylanases were determined. All strains produced xylanases and pectinases, while 98.6, 98, and 94.6% of them produced amylases, cellulases, and chitinases, respectively. The most productive strains included seven isolates from P. sativum and one from L. angustifolius. Strain Micromonospora lupini ML01-gfp was used to determine its capacity to reach and colonize different plant organs using P. sativum as the plant model. Stem and leaf samples were monitored by optical and fluorescence microscopy to locate the tagged strain. These results strongly suggest that Micromonospora is able not only to infect nitrogen-fixing nodules but also to reach other parts of the host plant, especially the leaves.

Amino Acid Polymorphisms in the VHIID Conserved Motif of Nodulation Signaling Pathways 2 Distinctly Modulate Symbiotic Signaling and Nodule Morphogenesis in Medicago truncatula.

Legumes establish an endosymbiotic association with nitrogen-fixing soil bacteria. Following the mutual recognition of the symbiotic partner, the infection process is controlled by the induction of the signaling pathway and subsequent activation of symbiosis-related host genes. One of the protein complexes regulating nitrogen-fixing root nodule symbiosis is formed by GRAS domain regulatory proteins Nodulation Signaling Pathways 1 and 2 (NSP1 and NSP2) that control the expression of several early nodulation genes. Here, we report on a novel point mutant allele (nsp2-6) affecting the function of the NSP2 gene and compared the mutant with the formerly identified nsp2-3 mutant. Both mutants carry a single amino acid substitution in the VHIID motif of the NSP2 protein. We found that the two mutant alleles show dissimilar root hair response to bacterial infection. Although the nsp2-3 mutant developed aberrant infection threads, rhizobia were able to colonize nodule cells in this mutant. The encoded NSP2 proteins of the nsp2-3 and the novel nsp2 mutants interact with NSP1 diversely and, as a consequence, the activation of early nodulin genes and nodule organogenesis are arrested in the new nsp2 allele. The novel mutant with amino acid substitution D244H in NSP2 shows similar defects in symbiotic responses as a formerly identified nsp2-2 mutant carrying a deletion in the NSP2 gene. Additionally, we found that rhizobial strains induce delayed nodule formation on the roots of the ns2-3 weak allele. Our study highlights the importance of a conserved Asp residue in the VHIID motif of NSP2 that is required for the formation of a functional NSP1-NSP2 signaling module. Furthermore, our results imply the involvement of NSP2 during differentiation of symbiotic nodule cells.