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

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

Occurrence and diversity of stem nodulation in Aeschynomene and Sesbania legumes from wetlands of Madagascar.

Legumes have the ability to establish a nitrogen-fixing symbiosis with soil rhizobia that they house in specific organs, the nodules. In most rhizobium-legume interactions, nodulation occurs on the root. However, certain tropical legumes growing in wetlands possess a unique trait: the capacity to form rhizobia-harbouring nodules on the stem. Despite the originality of the stem nodulation process, its occurrence and diversity in waterlogging-tolerant legumes remains underexplored, impeding a comprehensive analysis of its genetics and biology. Here, we aimed at filling this gap by surveying stem nodulation in legume species-rich wetlands of Madagascar. Stem nodulation was readily observed in eight hydrophytic species of the legume genera, Aeschynomene and Sesbania, for which significant variations in stem nodule density and morphology was documented. Among these species, A. evenia, which is used as genetic model to study the rhizobial symbiosis, was found to be frequently stem-nodulated. Two other Aeschynomene species, A. cristata and A. uniflora, were evidenced to display a profuse stem-nodulation as occurs in S. rostrata. These findings extend our knowledge on legumes species that are endowed with stem nodulation and further indicate that A. evenia, A. cristata, A. uniflora and S. rostrata are of special interest for the study of stem nodulation. As such, these legume species represent opportunities to investigate different modalities of the nitrogen-fixing symbiosis and this knowledge could provide cues for the engineering of nitrogen-fixation in non-legume crops.

OROSOMUCOID PROTEIN 1 regulation of sphingolipid synthesis is required for nodulation in Aeschynomene evenia.

Legumes establish symbiotic interactions with nitrogen-fixing rhizobia that are accommodated in root-derived organs known as nodules. Rhizobial recognition triggers a plant symbiotic signaling pathway that activates 2 coordinated processes: infection and nodule organogenesis. How these processes are orchestrated in legume species utilizing intercellular infection and lateral root base nodulation remains elusive. Here, we show that Aeschynomene evenia OROSOMUCOID PROTEIN 1 (AeORM1), a key regulator of sphingolipid biosynthesis, is required for nodule formation. Using A. evenia orm1 mutants, we demonstrate that alterations in AeORM1 function trigger numerous early aborted nodules, defense-like reactions, and shorter lateral roots. Accordingly, AeORM1 is expressed during lateral root initiation and elongation, including at lateral root bases where nodule primordium form in the presence of symbiotic bradyrhizobia. Sphingolipidomics revealed that mutations in AeORM1 lead to sphingolipid overaccumulation in roots relative to the wild type, particularly for very long-chain fatty acid-containing ceramides. Taken together, our findings reveal that AeORM1-regulated sphingolipid homeostasis is essential for rhizobial infection and nodule organogenesis, as well as for lateral root development in A. evenia.

Exploring the cellular surface polysaccharide and root nodule symbiosis characteristics of the rpoN mutants of Bradyrhizobium sp. DOA9 using synchrotron-based Fourier transform infrared microspectroscopy in conjunction with X-ray absorption spectroscopy.

The functional significance of rpoN genes that encode two sigma factors in the Bradyrhizobium sp. strain DOA9 has been reported to affect colony formation, root nodulation characteristics, and symbiotic interactions with Aeschynomene americana. rpoN mutant strains are defective in cellular surface polysaccharide (CSP) production compared with the wild-type (WT) strain, and they accordingly exhibit smaller colonies and diminished symbiotic effectiveness. To gain deeper insights into the changes in CSP composition and the nodules of rpoN mutants, we employed synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy and X-ray absorption spectroscopy. FTIR analysis of the CSP revealed the absence of specific components in the rpoN mutants, including lipids, carboxylic groups, polysaccharide-pyranose rings, and ß-galactopyranosyl residues. Nodules formed by DOA9WT exhibited a uniform distribution of lipids, proteins, and carbohydrates; mutant strains, particularly DOA9∆rpoNp:ΩrpoNc, exhibited decreased distribution uniformity and a lower concentration of C=O groups. Furthermore, Fe K-edge X-ray absorption near-edge structure and extended X-ray absorption fine structure analyses revealed deficiencies in the nitrogenase enzyme in the nodules of DOA9∆rpoNc and DOA9∆rpoNp:ΩrpoNc mutants; nodules from DOA9WT and DOA9∆rpoNp exhibited both leghemoglobin and the nitrogenase enzyme. IMPORTANCE This work provides valuable insights into how two rpoN genes affect the composition of cellular surface polysaccharides (CSPs) in Bradyrhizobium sp., which subsequently dictates root nodule chemical characteristics and nitrogenase production. We used advanced synchrotron methods, including synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy and X-ray absorption spectroscopy (XAS), for the first time in this field to analyze CSP components and reveal the biochemical changes occurring within nodules. These cutting-edge techniques confer significant advantages by providing detailed molecular information, enabling the identification of specific functional groups, chemical bonds, and biomolecule changes. This research not only contributes to our understanding of plant-microbe interactions but also establishes a foundation for future investigations and potential applications in this field. The combined use of the synchrotron-based FTIR and XAS techniques represents a significant advancement in facilitating a comprehensive exploration of bacterial CSPs and their implications in plant-microbe interactions.

Widespread Bradyrhizobium distribution of diverse Type III effectors that trigger legume nodulation in the absence of Nod factor.

The establishment of the rhizobium-legume symbiosis is generally based on plant perception of Nod factors (NFs) synthesized by the bacteria. However, some Bradyrhizobium strains can nodulate certain legume species, such as Aeschynomene spp. or Glycine max, independently of NFs, and via two different processes that are distinguished by the necessity or not of a type III secretion system (T3SS). ErnA is the first known type III effector (T3E) triggering nodulation in Aeschynomene indica. In this study, a collection of 196 sequenced Bradyrhizobium strains was tested on A. indica. Only strains belonging to the photosynthetic supergroup can develop a NF-T3SS-independent symbiosis, while the ability to use a T3SS-dependent process is found in multiple supergroups. Of these, 14 strains lacking ernA were tested by mutagenesis to identify new T3Es triggering nodulation. We discovered a novel T3E, Sup3, a putative SUMO-protease without similarity to ErnA. Its mutation in Bradyrhizobium strains NAS96.2 and WSM1744 abolishes nodulation and its introduction in an ernA mutant of strain ORS3257 restores nodulation. Moreover, ectopic expression of sup3 in A. indica roots led to the formation of spontaneous nodules. We also report three other new T3Es, Ubi1, Ubi2 and Ubi3, which each contribute to the nodulation capacity of strain LMTR13. These T3Es have no homology to known proteins but share with ErnA three motifs necessary for ErnA activity. Together, our results highlight an unsuspected distribution and diversity of T3Es within the Bradyrhizobium genus that may contribute to their symbiotic efficiency by participating in triggering legume nodulation.

Role of two RpoN in Bradyrhizobium sp. strain DOA9 in symbiosis and free-living growth.

RpoN is an alternative sigma factor (sigma 54) that recruits the core RNA polymerase to promoters of genes. In bacteria, RpoN has diverse physiological functions. In rhizobia, RpoN plays a key role in the transcription of nitrogen fixation (nif) genes. The Bradyrhizobium sp. DOA9 strain contains a chromosomal (c) and plasmid (p) encoded RpoN protein. We used single and double rpoN mutants and reporter strains to investigate the role of the two RpoN proteins under free-living and symbiotic conditions. We observed that the inactivation of rpoNc or rpoNp severely impacts the physiology of the bacteria under free-living conditions, such as the bacterial motility, carbon and nitrogen utilization profiles, exopolysaccharide (EPS) production, and biofilm formation. However, free-living nitrogen fixation appears to be under the primary control of RpoNc. Interestingly, drastic effects of rpoNc and rpoNp mutations were also observed during symbiosis with Aeschynomene americana. Indeed, inoculation with rpoNp, rpoNc, and double rpoN mutant strains resulted in decreases of 39, 64, and 82% in the number of nodules, respectively, as well as a reduction in nitrogen fixation efficiency and a loss of the bacterium's ability to survive intracellularly. Taken together, the results show that the chromosomal and plasmid encoded RpoN proteins in the DOA9 strain both play a pleiotropic role during free-living and symbiotic states.

A mutant-based analysis of the establishment of Nod-independent symbiosis in the legume Aeschynomene evenia.

Intensive research on nitrogen-fixing symbiosis in two model legumes has uncovered the molecular mechanisms, whereby rhizobial Nod factors activate a plant symbiotic signaling pathway that controls infection and nodule organogenesis. In contrast, the so-called Nod-independent symbiosis found between Aeschynomene evenia and photosynthetic bradyrhizobia, which does not involve Nod factor recognition nor infection thread formation, is less well known. To gain knowledge on how Nod-independent symbiosis is established, we conducted a phenotypic and molecular characterization of A. evenia lines carrying mutations in different nodulation genes. Besides investigating the effect of the mutations on rhizobial symbiosis, we examined their consequences on mycorrhizal symbiosis and in nonsymbiotic conditions. Analyzing allelic mutant series for AePOLLUX, Ca2+/calmodulin dependent kinase, AeCYCLOPS, nodulation signaling pathway 2 (AeNSP2), and nodule inception demonstrated that these genes intervene at several stages of intercellular infection and during bacterial accommodation. We provide evidence that AeNSP2 has an additional nitrogen-dependent regulatory function in the formation of axillary root hairs at lateral root bases, which are rhizobia-colonized infection sites. Our investigation of the recently discovered symbiotic actor cysteine-rich receptor-like kinase specified that it is not involved in mycorrhization; however, it is essential for both symbiotic signaling and early infection during nodulation. These findings provide important insights on the modus operandi of Nod-independent symbiosis and contribute to the general understanding of how rhizobial-legume symbioses are established by complementing the information acquired in model legumes.

An integrated approach reveals how lipo-chitooligosaccharides interact with the lysin motif receptor-like kinase MtLYR3.

N-acetylglucosamine containing compounds acting as pathogenic or symbiotic signals are perceived by plant-specific Lysin Motif Receptor-Like Kinases (LysM-RLKs). The molecular mechanisms of this perception are not fully understood, notably those of lipo-chitooligosaccharides (LCOs) produced during root endosymbioses with nitrogen-fixing bacteria or arbuscular mycorrhizal fungi. In Medicago truncatula, we previously identified the LysM-RLK LYR3 (MtLYR3) as a specific LCO-binding protein. We also showed that the absence of LCO binding to LYR3 of the non-mycorrhizal Lupinus angustifolius, (LanLYR3), was related to LysM3, which differs from that of MtLYR3 by several amino acids and, particularly, by a critical tyrosine residue absent in LanLYR3. Here, we aimed to define the LCO binding site of MtLYR3 by using molecular modelling and simulation approaches, combined with site-directed mutagenesis and LCO binding experiments. 3D models of MtLYR3 and LanLYR3 ectodomains were built, and homology modelling and molecular dynamics (MD) simulations were performed. Molecular docking and MD simulation on the LysM3 identified potential key residues for LCO binding. We highlighted by steered MD simulations that in addition to the critical tyrosine, two other residues were important for LCO binding in MtLYR3. Substitution of these residues in LanLYR3-LysM3 by those of MtLYR3-LysM3 allowed the recovery of high-affinity LCO binding in experimental radioligand-binding assays. An analysis of selective constraints revealed that the critical tyrosine has experienced positive selection pressure and is absent in some LYR3 proteins. These findings now pave the way to uncover the functional significance of this specific evolutionary pattern.

A glutamate synthase mutant of Bradyrhizobium sp. strain ORS285 is unable to induce nodules on Nod factor-independent Aeschynomene species.

The Bradyrhizobium sp. strain ORS285 is able to establish a nitrogen-fixing symbiosis with both Nod factor (NF) dependent and NF-independent Aeschynomene species. Here, we have studied the growth characteristics and symbiotic interaction of a glutamate synthase (GOGAT; gltD::Tn5) mutant of Bradyrhizobium ORS285. We show that the ORS285 gltD::Tn5 mutant is unable to use ammonium, nitrate and many amino acids as nitrogen source for growth and is unable to fix nitrogen under free-living conditions. Moreover, on several nitrogen sources, the growth rate of the gltB::Tn5 mutant was faster and/or the production of the carotenoid spirilloxanthin was much higher as compared to the wild-type strain. The absence of GOGAT activity has a drastic impact on the symbiotic interaction with NF-independent Aeschynomene species. With these species, inoculation with the ORS285 gltD::Tn5 mutant does not result in the formation of nodules. In contrast, the ORS285 gltD::Tn5 mutant is capable to induce nodules on NF-dependent Aeschynomene species, but these nodules were ineffective for nitrogen fixation. Interestingly, in NF-dependent and NF-independent Aeschynomene species inoculation with the ORS285 gltD::Tn5 mutant results in browning of the plant tissue at the site of the infection suggesting that the mutant bacteria induce plant defence responses.

Genetics of nodulation in Aeschynomene evenia uncovers mechanisms of the rhizobium-legume symbiosis.

Among legumes (Fabaceae) capable of nitrogen-fixing nodulation, several Aeschynomene spp. use a unique symbiotic process that is independent of Nod factors and infection threads. They are also distinctive in developing root and stem nodules with photosynthetic bradyrhizobia. Despite the significance of these symbiotic features, their understanding remains limited. To overcome such limitations, we conduct genetic studies of nodulation in Aeschynomene evenia, supported by the development of a genome sequence for A. evenia and transcriptomic resources for 10 additional Aeschynomene spp. Comparative analysis of symbiotic genes substantiates singular mechanisms in the early and late nodulation steps. A forward genetic screen also shows that AeCRK, coding a receptor-like kinase, and the symbiotic signaling genes AePOLLUX, AeCCamK, AeCYCLOPS, AeNSP2, and AeNIN are required to trigger both root and stem nodulation. This work demonstrates the utility of the A. evenia model and provides a cornerstone to unravel mechanisms underlying the rhizobium-legume symbiosis.

RibBX of Bradyrhizobium ORS285 Plays an Important Role in Intracellular Persistence in Various Aeschynomene Host Plants.

Bradyrhizobium ORS285 forms a nitrogen-fixating symbiosis with both Nod factor (NF)-dependent and NF-independent Aeschynomene spp. The Bradyrhizobium ORS285 ribBA gene encodes for a putative bifunctional enzyme with 3,4-dihydroxybutanone phosphate (3,4-DHBP) synthase and guanosine triphosphate (GTP) cyclohydrolase II activities, catalyzing the initial steps in the riboflavin biosynthesis pathway. In this study, we show that inactivating the ribBA gene does not cause riboflavin auxotrophy under free-living conditions and that, as shown for RibBAs from other bacteria, the GTP cyclohydrolase II domain has no enzymatic activity. For this reason, we have renamed the annotated ribBA as ribBX. Because we were unable to identify other ribBA or ribA and ribB homologs in the genome of Bradyrhizobium ORS285, we hypothesize that the ORS285 strain can use unconventional enzymes or an alternative pathway for the initial steps of riboflavin biosynthesis. Inactivating ribBX has a drastic impact on the interaction of Bradyrhizobium ORS285 with many of the tested Aeschynomene spp. In these Aeschynomene spp., the ORS285 ribBX mutant is able to infect the plant host cells but the intracellular infection is not maintained and the nodules senesce early. This phenotype can be complemented by reintroduction of the 3,4-DHBP synthase domain alone. Our results indicate that, in Bradyrhizobium ORS285, the RibBX protein is not essential for riboflavin biosynthesis under free-living conditions and we hypothesize that its activity is needed to sustain riboflavin biosynthesis under certain symbiotic conditions.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The rhizobial type III effector ErnA confers the ability to form nodules in legumes.

Several Bradyrhizobium species nodulate the leguminous plant Aeschynomene indica in a type III secretion system-dependent manner, independently of Nod factors. To date, the underlying molecular determinants involved in this symbiotic process remain unknown. To identify the rhizobial effectors involved in nodulation, we mutated 23 out of the 27 effector genes predicted in Bradyrhizobium strain ORS3257. The mutation of nopAO increased nodulation and nitrogenase activity, whereas mutation of 5 other effector genes led to various symbiotic defects. The nopM1 and nopP1 mutants induced a reduced number of nodules, some of which displayed large necrotic zones. The nopT and nopAB mutants induced uninfected nodules, and a mutant in a yet-undescribed effector gene lost the capacity for nodule formation. This effector gene, widely conserved among bradyrhizobia, was named ernA for "effector required for nodulation-A." Remarkably, expressing ernA in a strain unable to nodulate A. indica conferred nodulation ability. Upon its delivery by Pseudomonas fluorescens into plant cells, ErnA was specifically targeted to the nucleus, and a fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy approach supports the possibility that ErnA binds nucleic acids in the plant nuclei. Ectopic expression of ernA in A. indica roots activated organogenesis of root- and nodule-like structures. Collectively, this study unravels the symbiotic functions of rhizobial type III effectors playing distinct and complementary roles in suppression of host immune functions, infection, and nodule organogenesis, and suggests that ErnA triggers organ development in plants by a mechanism that remains to be elucidated.

The Modification of the Flavonoid Naringenin by Bradyrhizobium sp. Strain ORS285 Changes the nod Genes Inducer Function to a Growth Stimulator.

As inducers of nodulation (nod) genes, flavonoids play an important role in the symbiotic interaction between rhizobia and legumes. However, in addition to the control of expression of nod genes, many other effects of flavonoids on rhizobial cells have been described. Here, we show that the flavonoid naringenin stimulates the growth of the photosynthetic Bradyrhizobium sp. strain ORS285. This growth-stimulating effect was still observed for strain ORS285 with nodD1, nodD2, or the naringenin-degrading fde operon deleted. Phenotypic microarray analysis indicates that in cells grown in the presence of naringenin, the glycerol and fatty acid metabolism is activated. Moreover, electron microscopic and enzymatic analyses show that polyhydroxy alkanoate metabolism is altered in cells grown in the presence of naringenin. Although strain ORS285 was able to degrade naringenin, a fraction was converted into an intensely yellow-colored molecule with an m/z (+) of 363.0716. Further analysis indicates that this molecule is a hydroxylated and O-methylated form of naringenin. In contrast to naringenin, this derivative did not induce nod gene expression, but it did stimulate the growth of strain ORS285. We hypothesize that the growth stimulation and metabolic changes induced by naringenin are part of a mechanism to facilitate the colonization and infection of naringenin-exuding host plants.

Transcriptomic dissection of Bradyrhizobium sp. strain ORS285 in symbiosis with Aeschynomene spp. inducing different bacteroid morphotypes with contrasted symbiotic efficiency.

To circumvent the paucity of nitrogen sources in the soil legume plants establish a symbiotic interaction with nitrogen-fixing soil bacteria called rhizobia. During symbiosis, the plants form root organs called nodules, where bacteria are housed intracellularly and become active nitrogen fixers known as bacteroids. Depending on their host plant, bacteroids can adopt different morphotypes, being either unmodified (U), elongated (E) or spherical (S). E- and S-type bacteroids undergo a terminal differentiation leading to irreversible morphological changes and DNA endoreduplication. Previous studies suggest that differentiated bacteroids display an increased symbiotic efficiency (E > U and S > U). In this study, we used a combination of Aeschynomene species inducing E- or S-type bacteroids in symbiosis with Bradyrhizobium sp. ORS285 to show that S-type bacteroids present a better symbiotic efficiency than E-type bacteroids. We performed a transcriptomic analysis on E- and S-type bacteroids formed by Aeschynomene afraspera and Aeschynomene indica nodules and identified the bacterial functions activated in bacteroids and specific to each bacteroid type. Extending the expression analysis in E- and S-type bacteroids in other Aeschynomene species by qRT-PCR on selected genes from the transcriptome analysis narrowed down the set of bacteroid morphotype-specific genes. Functional analysis of a selected subset of 31 bacteroid-induced or morphotype-specific genes revealed no symbiotic phenotypes in the mutants. This highlights the robustness of the symbiotic program but could also indicate that the bacterial response to the plant environment is partially anticipatory or even maladaptive. Our analysis confirms the correlation between differentiation and efficiency of the bacteroids and provides a framework for the identification of bacterial functions that affect the efficiency of bacteroids.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Complete Genome Sequence of Bradyrhizobium sp. ORS285, a Photosynthetic Strain Able To Establish Nod Factor-Dependent or Nod Factor-Independent Symbiosis with Aeschynomene Legumes.

Here, we report the complete genome sequence of Bradyrhizobium sp. strain ORS285, which is able to nodulate Aeschynomene legumes using two distinct strategies that differ in the requirement of Nod factors. The genome sequence information of this strain will help understanding of the different mechanisms of interaction of rhizobia with legumes.

The role of rhizobial (NifV) and plant (FEN1) homocitrate synthases in Aeschynomene/photosynthetic Bradyrhizobium symbiosis.

In the most studied rhizobium-legume interactions, the host plant supplies the symbiont with homocitrate, an essential co-factor of the nitrogenase enzyme complex, via the expression of a nodule-specific homocitrate synthase FEN1. Photosynthetic bradyrhizobia interacting with Nod factor (NF) dependent and NF-independent Aeschynomene legumes are able to synthesize homocitrate themselves as they contain a nifV gene encoding a homocitrate synthase. Here, we show that in the model strain ORS285, nifV is required for free-living and symbiotic dinitrogen fixation with NF-independent Aeschynomene species. In contrast, in symbiosis with NF-dependent Aeschynomene species, the nifV requirement for efficient nitrogen fixation was found to be host plant dependent. Interestingly, orthologs of FEN1 were found in both NF-dependent and NF-independent Aeschynomene species. However, a high nodule specific induction of FEN1 expression was only observed in A. afraspera, a host plant in which nifV is not required for symbiotic dinitrogen fixation. These data indicate that efficient symbiotic nitrogen fixation in many of the tested Aeschynomene species requires rhizobial homocitrate synthesis. Considering that more than 10% of the fully sequenced rhizobium strains do contain a nifV gene, the Aeschynomene/photosynthetic Bradyrhizobium interaction is likely not the only rhizobium/legume symbiosis where rhizobial nifV expression is required.

nifDK Clusters Located on the Chromosome and Megaplasmid of Bradyrhizobium sp. Strain DOA9 Contribute Differently to Nitrogenase Activity During Symbiosis and Free-Living Growth.

Bradyrhizobium sp. strain DOA9 contains two copies of the nifDK genes, nifDKc, located on the chromosome, and nifDKp, located on a symbiotic megaplasmid. Unlike most rhizobia, this bacterium displays nitrogenase activity under both free-living and symbiotic conditions. Transcriptional analysis using gusA reporter strains showed that both nifDK operons were highly expressed under symbiosis, whereas nifDKc was the most abundantly expressed under free-living conditions. During free-living growth, the nifDKp mutation did not affect nitrogenase activity, whereas nitrogenase activity was drastically reduced with the nifDKc mutant. This led us to suppose that nifDKc is the main contributor of nitrogenase activity in the free-living state. In contrast, during symbiosis, no effect of the nifDKc mutation was observed and the nitrogen-fixation efficiency of plants inoculated with the nifDKp mutant was reduced. This suggests that nifDKp plays the main role in nitrogenase enzyme activity during symbiosis. Together, these data suggest that Bradyrhizobium sp. strain DOA9 contains two functional copies of nifDK genes that are regulated differently and that, depending on their lifestyle, contribute differently to nitrogenase activity.

NodD1 and NodD2 Are Not Required for the Symbiotic Interaction of Bradyrhizobium ORS285 with Nod-Factor-Independent Aeschynomene Legumes.

Photosynthetic Bradyrhizobium strain ORS285 forms nitrogen-fixing nodules on the roots and stems of tropical aquatic legumes of the Aeschynomene genus. Depending on the Aeschynomene species, this symbiotic interaction does or does not rely on the synthesis of Nod-factors (NFs). However, whether during the interaction of Bradyrhizobium ORS285 with NF-independent Aeschynomene species the nod genes are expressed and if the general regulator NodD plays a symbiotic role is unknown. Expression studies showed that in contrast to the interaction with the NF-dependent Aeschynomene species, A. afraspera, the Bradyrhizobium ORS285 nod genes are not induced upon contact with the NF-independent host plant A. indica. Mutational analysis of the two nodD genes present in ORS285, showed that deletion of nodD1 and nodD2 did not affect the symbiotic interaction between Bradyrhizobium ORS285 and A. indica whereas the deletions had an effect on the symbiotic interaction with A. afraspera plants. In addition, when the expression of nod genes was artificially induced by adding naringenin to the plant growth medium, the nodulation of A. indica by Bradyrhizobium ORS285 is delayed and resulted in lower nodule numbers.

A Peptidoglycan-Remodeling Enzyme Is Critical for Bacteroid Differentiation in Bradyrhizobium spp. During Legume Symbiosis.

In response to the presence of compatible rhizobium bacteria, legumes form symbiotic organs called nodules on their roots. These nodules house nitrogen-fixing bacteroids that are a differentiated form of the rhizobium bacteria. In some legumes, the bacteroid differentiation comprises a dramatic cell enlargement, polyploidization, and other morphological changes. Here, we demonstrate that a peptidoglycan-modifying enzyme in Bradyrhizobium strains, a DD-carboxypeptidase that contains a peptidoglycan-binding SPOR domain, is essential for normal bacteroid differentiation in Aeschynomene species. The corresponding mutants formed bacteroids that are malformed and hypertrophied. However, in soybean, a plant that does not induce morphological differentiation of its symbiont, the mutation does not affect the bacteroids. Remarkably, the mutation also leads to necrosis in a large fraction of the Aeschynomene nodules, indicating that a normally formed peptidoglycan layer is essential for avoiding the induction of plant immune responses by the invading bacteria. In addition to exopolysaccharides, capsular polysaccharides, and lipopolysaccharides, whose role during symbiosis is well defined, our work demonstrates an essential role in symbiosis for yet another rhizobial envelope component, the peptidoglycan layer.

Rhizobium-legume symbiosis in the absence of Nod factors: two possible scenarios with or without the T3SS.

The occurrence of alternative Nod factor (NF)-independent symbiosis between legumes and rhizobia was first demonstrated in some Aeschynomene species that are nodulated by photosynthetic bradyrhizobia lacking the canonical nodABC genes. In this study, we revealed that a large diversity of non-photosynthetic bradyrhizobia, including B. elkanii, was also able to induce nodules on the NF-independent Aeschynomene species, A. indica. Using cytological analysis of the nodules and the nitrogenase enzyme activity as markers, a gradient in the symbiotic interaction between bradyrhizobial strains and A. indica could be distinguished. This ranged from strains that induced nodules that were only infected intercellularly to rhizobial strains that formed nodules in which the host cells were invaded intracellularly and that displayed a weak nitrogenase activity. In all non-photosynthetic bradyrhizobia, the type III secretion system (T3SS) appears required to trigger nodule organogenesis. In contrast, genome sequence analysis revealed that apart from a few exceptions, like the Bradyrhizobium ORS285 strain, photosynthetic bradyrhizobia strains lack a T3SS. Furthermore, analysis of the symbiotic properties of an ORS285 T3SS mutant revealed that the T3SS could have a positive or negative role for the interaction with NF-dependent Aeschynomene species, but that it is dispensable for the interaction with all NF-independent Aeschynomene species tested. Taken together, these data indicate that two NF-independent symbiotic processes are possible between legumes and rhizobia: one dependent on a T3SS and one using a so far unknown mechanism.