Nodulation Genes Research Articles

Genome wide identification and characterization of nodulation related genes in Arachis hypogaea.

Nitrogen is an important plant nutrient that has a significant role in crop yield. Hence, to fulfill the needs of sustainable agriculture, it is necessary to improve biological nitrogen fixation in leguminous crops. Nod inducing gene families plays a crucial role in the interaction between rhizobia and legumes, leading to biological nitrogen fixation. However, nod inducing genes identification and characterization has not yet been performed in Arachis hypogaea. In this study, identification and genome-wide analysis of nod inducing genes are performed so that to explore their potential functions in the Arachis hypogaea for the first time. Nod genes were comprehensively analyzed by phylogenetic clustering analysis, gene structure determination, detection of conserved motifs, subcellular localization, conserved motifs, cis-acting elements and promoter region analysis. This study identified 42 Nod inducing genes in Arachis hypogaea, their sequences were submitted to NCBI and accession numbers were obtained. Potential involvement of these genes in biological nitrogen fixation has been unraveled, such as, phylogenetic analysis revealed that nod inducing genes evolved independently in Arachis hypogaea, the amino acid structures exhibited 20 highly conserved motifs, the proteins are present at different locations in cells and the gene structures revealed that all the genes are full-length genes with upstream intronic regions. Further, the promoter analysis determined a large number of cis-regulatory elements involved in nodulation. Moreover, this study not only provides identification and characterization of genes underlying developmental and functional stages of nodulation and biological nitrogen fixation but also lays the foundation for further revelation of nod inducing gene family. Besides, identification and structural analysis of these genes in Arachis hypogaea may provide a theoretical basis for the study of evolutionary relationships in future analysis.

Whole genome sequencing of mesorhizobia isolated from northern Canada.

Rhizobia are soil-dwelling bacteria that can form N2-fixing symbioses with legume plant species (Fabaceae). These bacteria are globally distributed; however, few studies have examined the genomics of rhizobia that live in cold environments. Here, we isolated and characterized three rhizobial strains from legume nodules collected at a pair of distant low Arctic tundra and boreal forest sites in northern Canada. Phylogenetic and average nucleotide identity measurements suggested that the three strains are members of the genus Mesorhizobium, and that each strain represents a novel genospecies. Intriguingly, whereas most mesorhizobia contain the classical determinants of nodulation and nitrogen fixation on their chromosome, whole genome sequencing revealed that all three strains carry these genes on large symbiotic megaplasmids of ∼750 to ∼1000kb. Phylogenetic and sequence analyses of the common nodulation genes revealed highly conserved alleles amongst these northern mesorhizobia, leading us to propose that they belong to a novel symbiovar that we termed symbiovar oxytropis. Interestingly, these nod gene alleles are uncommon in mesorhizobia isolated from similar plant hosts in other climatic regions, suggesting potential functional adaptive differences.

The Rhizobium tropici CIAT 899 NodD2 protein promotes symbiosis and extends rhizobial nodulation range by constitutive nodulation factor synthesis.

In the symbiotic associations between rhizobia and legumes, the NodD regulators orchestrate the transcription of the specific nodulation genes. This set of genes is involved in the synthesis of nodulation factors, which are responsible for initiating the nodulation process. Rhizobium tropici CIAT 899 is the most successful symbiont of Phaseolus vulgaris and can nodulate a variety of legumes. Among the five NodD regulators present in this rhizobium, only NodD1 and NodD2 seem to have a role in the symbiotic process. However, the individual role of each NodD in the absence of the other proteins has remained elusive. In this work, we show that the CIAT 899 NodD2 does not require activation by inducers to promote the synthesis of nodulation factors. A CIAT 899 strain overexpressing nodD2, but lacking all additional nodD genes, can nodulate three different legumes as efficiently as the wild type. Interestingly, CIAT 899 NodD2-mediated gain of nodulation can be extended to another rhizobial species, since its overproduction in Sinorhizobium fredii HH103 not only increases the number of nitrogen-fixing nodules in two host legumes but also results in nodule development in incompatible legumes. These findings potentially open exciting opportunities to develop rhizobial inoculants and increase legume crop production.

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.

Genomic Diversity of Bradyrhizobium from the Tree Legumes Inga and Lysiloma (Caesalpinioideae-Mimosoid Clade)

We identified diverse bradyrhizobia having distinct ERIC-PCR genomic fingerprints from native American trees Inga vera and Lysiloma spp. In addition, two nodule isolates recovered from Lysiloma divaricatum seedlings inoculated with soil from the tropical house of a UK botanical garden were also identified as Bradyrhizobium. Genomes were obtained (with sizes around 9 Mb each) from nine Inga and Lysiloma bradyrhizobial isolates; plasmids were detected in two of the Inga strains analyzed. Average Nucleotide Identity of whole genomes revealed five novel Bradyrhizobium genomospecies from Mexican trees, while the UK isolates were identified as Bradyrhizobium cajani and Bradyrhizobium brasilense. Inga vera and Lysiloma isolates, despite their genetic distances and different hosts, shared a common set of nod genes that suggested that I. vera and Lysiloma bradyrhizobia produce fucosylated, methylated and carbamylated lipochitooligosaccharides. Uptake hydrogenase hup, hyp and secretion system genes were found in some of the isolates. Lysiloma strains were found to be ineffective on I. vera. Some of the isolates may be used as plant inoculants.

A newly evolved chimeric lysin motif receptor-like kinase in Medicago truncatula spp. tricycla R108 extends its Rhizobia symbiotic partnership.

Rhizobial lipochitooligosaccharidic Nod factors (NFs), specified by nod genes, are the primary determinants of host specificity in the legume-Rhizobia symbiosis. We examined the nodulation ability of Medicago truncatula cv Jemalong A17 and M. truncatula ssp. tricycla R108 with the Sinorhizobium meliloti nodF/nodL mutant, which produces modified NFs. We then applied genetic and functional approaches to study the genetic basis and mechanism of nodulation of R108 by this mutant. We show that the nodF/nodL mutant can nodulate R108 but not A17. Using genomics and reverse genetics, we identified a newly evolved, chimeric LysM receptor-like kinase gene in R108, LYK2bis, which is responsible for the phenotype and can allow A17 to gain nodulation with the nodF/nodL mutant. We found that LYK2bis is involved in nodulation by mutants producing nonO-acetylated NFs and interacts with the key receptor protein NFP. Many, but not all, natural S. meliloti and S. medicae strains tested require LYK2bis for efficient nodulation of R108. Our findings reveal that a newly evolved gene in R108, LYK2bis, extends nodulation specificity to mutants producing nonO-acetylated NFs and is important for nodulation by many natural Sinorhizobia. Evolution of this gene may present an adaptive advantage to allow nodulation by a greater variety of strains.

Mesorhizobium ventifaucium sp. nov. and Mesorhizobium escarrei sp. nov., two novel root-nodulating species isolated from Anthyllis vulneraria

Ten mesorhizobial strains isolated from root-nodules of Anthyllis vulneraria by trapping using soils from southern France were studied to resolve their taxonomy. Their 16S rDNA sequences were identical and indicated that they are affiliated to the genus Mesorhizobium within the group M. prunaredense/M. delmotii/M. temperatum/M. mediterraneum/M. wenxiniae and M. robiniae as the closest defined species. Their evolutionary relationships with validated species were further characterized by multilocus sequence analysis (MLSA) using 4 protein-coding housekeeping genes (recA, atpD, glnII and dnaK), that divides the strains in two groups, and suggest that they belong to two distinct species. These results were well-supported by MALDI-TOF mass spectrometry analyses, wet-lab DNA-DNA hybridization (≤58%), and genome-based species delineation methods (ANI < 96%, in silico DDH < 70%), confirming their affiliation to two novel species. Based on these differences, Mesorhizobium ventifaucium (STM4922T = LMG 29643T = CFBP 8438T) and Mesorhizobium escarrei (type strain STM5069T = LMG 29642T = CFBP 8439T) are proposed as names for these two novel species. The phylogeny of nodulation genes nodC and nodA allocated the type strains into symbiovar anthyllidis as well as those of M. metallidurans STM2683T, M. delmotii STM4623T and M. prunaredense STM4891T, all recovered from the same legume species.

Assessing the effects of nickel on, e.g., Medicago sativa L. nodules using multidisciplinary approach.

Industrial wastes and fertilizers can introduce excessive levels of nickel (Ni) into the environment, potentially causing threats to plants, animals, as well as human beings. However, the number of studies on the effects of Ni toxicity on nodules is fairly limited. To address this issue, the effects of increasing Ni concentration on alfalfa nodules were assessed at chemical, biochemical, and transcriptomic levels. For this purpose, plants were grown in soils supplied with Ni (control, 0 mg/kg; C1, 50 mg/kg; C2, 150 mg/kg; C3, 250 mg/kg; and C4, 500 mg/kg) for 90 days. Ni loads in leaves, roots, and nodules were monitored after the exposure period. A set of biochemical biomarkers of oxidative stress was determined in nodules including antioxidants and metal homeostasis as well as lipid peroxidation. Gene expression levels of the main targets involved in oxidative stress and metal homeostasis were assessed. Our data indicated a high concentration of Ni in leaves, roots, and nodules where values reached 25.64 ± 3.04 mg/kg, 83.23 ± 5.16 mg/kg, and 125.71 ± 4.53 mg/kg in dry weight, respectively. Moreover, a significant increase in nodule biomass was observed in plants exposed to C4 in comparison to control treatment and percentage increased by 63%. Then, lipid peroxidation increased with a rate of 95% in nodules exposed to C4. Enzymatic activities were enhanced remarkably, suggesting the occurrence of oxidative stress, with increased superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX). Our results showed also a significant upregulation of SOD, GR and APX genes in nodules. Nodule homoglutathione (HGSH) levels increased with the different Ni concentrations, with a remarkable decrease of glutathione S-transferase (GST) activity and glutathione (GSH) content for the highest Ni concentration with 43% and 52% reduction, respectively. The phytochelatin (PC) and metallothionein (MT) concentrations increased in nodules, which implied the triggering of a cellular protection mechanism for coping with Ni toxicity. The results suggested that Ni promotes a drastic oxidative stress in alfalfa nodules, yet the expression of MT and PC to reduce Ni toxicity could be used as Ni stress bioindicators. Our findings provide new insights into the central role of alfalfa nodules in limiting the harmful effects of soil pollution. Therefore, nodules co-expressing antioxidant enzymes may have high phytoremediation potential.

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.

MtEFD and MtEFD2: Two transcription factors with distinct neofunctionalization in symbiotic nodule development.

Rhizobium-legume nitrogen-fixing symbiosis involves the formation of a specific organ, the root nodule, which provides bacteria with the proper cellular environment for atmospheric nitrogen fixation. Coordinated differentiation of plant and bacterial cells is an essential step of nodule development, for which few transcriptional regulators have been characterized. Medicago truncatula ETHYLENE RESPONSE FACTOR REQUIRED FOR NODULE DIFFERENTIATION (MtEFD) encodes an APETALA2/ETHYLENE RESPONSIVE FACTOR (ERF) transcription factor, the mutation of which leads to both hypernodulation and severe defects in nodule development. MtEFD positively controls a negative regulator of cytokinin signaling, the RESPONSE REGULATOR 4 (MtRR4) gene. Here we showed that that the Mtefd-1 mutation affects both plant and bacterial endoreduplication in nodules, as well as the expression of hundreds of genes in young and mature nodules, upstream of known regulators of symbiotic differentiation. MtRR4 expressed with the MtEFD promoter complemented Mtefd-1 hypernodulation but not the nodule differentiation phenotype. Unexpectedly, a nonlegume homolog of MtEFD, AtERF003 in Arabidopsis (Arabidopsis thaliana), could efficiently complement both phenotypes of Mtefd-1, in contrast to the MtEFD paralog MtEFD2 expressed in the root and nodule meristematic zone. A domain swap experiment showed that MtEFD2 differs from MtEFD by its C-terminal fraction outside the DNA binding domain. Furthermore, clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9)mutagenesis of MtEFD2 led to a reduction in the number of nodules formed in Mtefd-1, with downregulation of a set of genes, including notably NUCLEAR FACTOR-YA1 (MtNF-YA1) and MtNF-YB16, which are essential for nodule meristem establishment. We, therefore, conclude that nitrogen-fixing symbiosis recruited two proteins originally expressed in roots, MtEFD and MtEFD2, with distinct functions and neofunctionalization processes for each of them.

Genetic variation in symbiotic islands of natural variant strains of soybean Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens differing in competitiveness and in the efficiency of nitrogen fixation.

Soybean is the most important legume cropped worldwide and can highly benefit from the biological nitrogen fixation (BNF) process. Brazil is recognized for its leadership in the use of inoculants and two strains, Bradyrhizobium japonicum CPAC 15 (=SEMIA 5079) and Bradyrhizobium diazoefficiens CPAC 7 (=SEMIA 5080) compose the majority of the 70 million doses of soybean inoculants commercialized yearly in the country. We studied a collection of natural variants of these two strains, differing in properties of competitiveness and efficiency of BNF. We sequenced the genomes of the parental strain SEMIA 566 of B. japonicum , of three natural variants of this strain (S 204, S 340 and S 370), and compared with another variant of this group, strain CPAC 15. We also sequenced the genome of the parental strain SEMIA 586 of B. diazoefficiens , of three natural variants of this strain (CPAC 390, CPAC 392 and CPAC 394) and compared with the genome of another natural variant, strain CPAC 7. As the main genes responsible for nodulation (nod, noe, nol) and BNF (nif, fix) in soybean Bradyrhizobium are located in symbiotic islands, our objective was to identify genetic variations located in this region, including single nucleotide polymorphisms (SNPs) and insertions and deletions (indels), that could be potentially related to their different symbiotic phenotypes. We detected 44 genetic variations in the B. japonicum strains and three in B. diazoefficiens . As the B. japonicum strains have gone through a longer period of adaptation to the soil, the higher number of genetic variations could be explained by survival strategies under the harsh environmental conditions of the Brazilian Cerrado biome. Genetic variations were detected in genes enconding proteins such as a dephospho-CoA kinase, related to the CoA biosynthesis; a glucosamine-fructose-6-phosphate aminotransferase, key regulator of the hexosamine biosynthetic pathway; a LysR family transcriptional regulator related to nodulation genes; and NifE and NifS proteins, directly related to the BNF process. We suggest potential genetic variations related to differences in the symbiotic phenotypes.

High-Resolution Translatome Analysis Reveals Cortical Cell Programs During Early Soybean Nodulation.

Nodule organogenesis in legumes is regulated temporally and spatially through gene networks. Genome-wide transcriptome, proteomic, and metabolomic analyses have been used previously to define the functional role of various plant genes in the nodulation process. However, while significant progress has been made, most of these studies have suffered from tissue dilution since only a few cells/root regions respond to rhizobial infection, with much of the root non-responsive. To partially overcome this issue, we adopted translating ribosome affinity purification (TRAP) to specifically monitor the response of the root cortex to rhizobial inoculation using a cortex-specific promoter. While previous studies have largely focused on the plant response within the root epidermis (e.g., root hairs) or within developing nodules, much less is known about the early responses within the root cortex, such as in relation to the development of the nodule primordium or growth of the infection thread. We focused on identifying genes specifically regulated during early nodule organogenesis using roots inoculated with Bradyrhizobium japonicum. A number of novel nodulation gene candidates were discovered, as well as soybean orthologs of nodulation genes previously reported in other legumes. The differential cortex expression of several genes was confirmed using a promoter-GUS analysis, and RNAi was used to investigate gene function. Notably, a number of differentially regulated genes involved in phytohormone signaling, including auxin, cytokinin, and gibberellic acid (GA), were also discovered, providing deep insight into phytohormone signaling during early nodule development.

Ensifer meliloti L6-AK89, an Effective Inoculant of Medicago lupulina Varieties: Phenotypic and Deep-Genome Screening

This paper presents a deep analysis of the accessory genome of an economically promising strain of Ensifer (Sinorhizobium) meliloti, L6-AK89, obtained as a result of next-generation high-throughput sequencing (MiSeq, MinIon). Strain L6-AK89 is a StrR mutant of the native strain CIAM1775, a symbiont of Medicago lupulina that adapted to a saline and arid habitat in NW Kazakhstan. CIAM1775 is an effective inoculant of M. lupulina cv. Mira (fodder type standard), cultivated on moderately acid soils in the NW agricultural region of Russia. Strain L6-AK89 makes it possible to obtain the expected high (&gt;150%) increases in dry mass of the same plant variety in plant tests. The L6-AK89 genome has an increased proportion of sequences related to the accessory elements relative to reference strain Rm1021, 7.4% versus 4.8%. A set of 53 nod/noe/nol/nif/fdx/fix genes and 32 genes involved in stress tolerance together with 16S rRNA and recA–atpD–glnII–gyrB–dnaJ were evaluated. The high symbiotic efficiency of L6-АК89 with hop clover is most likely due to unique features of its genome, in combination with structural differences in its nod and stress-related genes, as well as unique clusters of quorum-sensing genes and osmoprotector synthesis.

Nod-Factor structure and functional redundancy of nod genes contribute the broad host range Bradyrhizobium sp. DOA9

Bradyrhizobium sp. DOA9 has a broad host range and a highly divergent nodulation (nod) gene on the megaplasmid (pDOA9). Duplicated copies of the general regulator nodD (nodD1 and nodD2) and nodA (nodA1 and nodA2) genes were found on pDOA9. The nodA gene is encoding an acyltransferase that controls the transfer of an acyl chain to the chitooligosaccharides (COs) structure and contribute to the determination of host specify. In general, compatible recognition between transcriptional regulators NodD and plant flavonoid inducer able to activate the function of other nodulation (nod) genes which responsible to produce lipo-chitooligosaccharides (LCOs), calls Nod-Factor, a major determinants of host range, infection, and nodulation. In this study, the function of the duplicated nodD (nodD1 and nodD2) and nodA (nodA1 and nodA2) genes were disrupted to determine their role on the symbiotic nodulation. The results showed that the mutation of nodD1 or nodD2 did not affected to the symbiotic nodulation, indicating the functional redundancy of the regulatory nodD1 and nodD2 genes in strain DOA9. Whereas nodA2 and nodB genes plays as a central role in the Nod-Factor biosynthesis and the symbiotic nodulation in all plant tests. Moreover, Nod-Factor produced from DOA9 under genistein induction shared a high similarity with the major Nod-Factor of Rhizobium sp. NGR234. Interestingly, mutation of nodA1 (ΔnodA1) did not affect to the nodulation but lead to induce the production of Nod-Factor belonged to the wild type, with the absence of Nod-Factor bearing the C18:1 acyl group. All these results suggest that the host range of the strain DOA9 might acquire from the functional redundancy of regulatory nodD genes and the Nod-Factor structure.

Analysis of Outer Membrane Vesicles Indicates That Glycerophospholipid Metabolism Contributes to Early Symbiosis Between Sinorhizobium fredii HH103 and Soybean.

Gram-negative bacteria can produce outer membrane vesicles (OMVs), and most functional studies of OMVs have been focused on mammalian-bacterial interactions. However, research on the OMVs of rhizobia is still limited. In this work, we isolated and purified OMVs from Sinorhizobium fredii HH103 under free-living conditions that were set as control (C-OMVs) and symbiosis-mimicking conditions that were induced by genistein (G-OMVs). The soybean roots treated with G-OMVs displayed significant deformation of root hairs. G-OMVs significantly induced the expression of nodulation genes related to early symbiosis, while they inhibited that of the defense genes of soybean. Proteomics analysis identified a total of 93 differential proteins between C-OMVs and G-OMVs, which are mainly associated with ribosome synthesis, flagellar assembly, two-component system, ABC transporters, oxidative phosphorylation, nitrogen metabolism, quorum sensing, glycerophospholipid metabolism, and peptidoglycan biosynthesis. A total of 45 differential lipids were identified through lipidomics analysis. Correlation analysis of OMV proteome and lipidome data revealed that glycerophospholipid metabolism is the enriched Kyoto Encyclopedia of Genes and Genomes metabolic pathway, and the expression of phosphatidylserine decarboxylase was significantly up-regulated in G-OMVs. The changes in three lipids related to symbiosis in the glycerophospholipid metabolism pathway were verified by enzyme-linked immunosorbent assay. Our results indicate that glycerophospholipid metabolism contributes to rhizobia-soybean symbiosis via OMVs.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Revealing the underlying mechanisms mediated by endophytic actinobacteria to enhance the rhizobia - chickpea (Cicer arietinum L.) symbiosis

PurposeThe effects of endophytic actinobacterial strains, Microbispora sp. CP56, Actinomadura sp. CP84B, Streptomyces spp. CP200B and CP21A, on the chickpea-Mesorhizobium symbiosis, were investigated in planta, with the aim of revealing the underlying mechanisms of action.MethodsThe actinobacterial endophytes were co-inoculated with Mesorhizobium ciceri onto chickpea seedlings to study the effect on plant growth parameters, nodulation development and grain yield. The role of actinobacterial exudates on rhizobial growth was investigated, as was the role of root exudates of actinobacteria-colonized plants on the expression of rhizobial nod factors and biofilm formation. Changes in expression of plant flavonoids and bacterial N-fixation genes resulting from actinobacterial co-inoculation were assessed using qPCR.ResultsApplication of actinobacterial endophytes, together with M. ciceri, showed growth promotion of chickpea with an increase in root nodule number and weight. Enhanced nodulation was accompanied by increases in total plant nitrogen, larger total plant weight and a 2–3-fold increase in grain yield. Factors associated with this tripartite symbiosis are promotion of rhizobial growth, earlier nodule formation, increased secondary root formation, up-regulated expression of genes related to flavonoid synthesis and nif genes. In addition, exudates of chickpea roots colonised with actinobacteria increased nodulation-related biological processes - rhizobial chemotaxis, biofilm formation and nod gene expression.ConclusionThese endophytic actinobacteria positively affect many aspects of the chickpea-Mesorhizobium symbiosis resulting in increases in grain yield. Similar improvements recorded in chickpea growing in potted field soils, shows the potential to enhance chickpea production in the field.

Genome of Hippophae rhamnoides provides insights into a conserved molecular mechanism in actinorhizal and rhizobial symbioses.

Sea buckthorn (Hippophae rhamnoides), a horticulturally multipurpose species in the family Elaeagnaceae, can build associations with Frankia actinomycetes to enable symbiotic nitrogen-fixing. Currently, no high-quality reference genome is available for an actinorhizal plant, which greatly hinders the study of actinorhizal symbiotic nodulation. Here, by combining short-read, long-read and Hi-C sequencing technologies, we generated a chromosome-level reference genome of H. rhamnoides (scaffold N50: 65 Mb, and genome size: 730 Mb) and predicted 30 812 protein-coding genes mainly on 12 pseudochromosomes. Hippophae rhamnoides was found to share a high proportion of symbiotic nodulation genes with Medicago truncatula, implying a shared molecular mechanism between actinorhizal and rhizobial symbioses. Phylogenetic analysis clustered the three paralogous NODULE INCEPTION (NIN) genes of H. rhamnoides with those of other nodulating species, forming the NIN group that most likely evolved from the ancestral NLP group. The genome of H. rhamnoides will help us to decipher the underlying genetic programming of actinorhizal symbiosis, and our high-quality genome and transcriptomic resources will make H. rhamnoides a new excellent model plant for actinorhizal symbiosis research.

Defining the mutation sites in chickpea nodulation mutants PM233 and PM405

BackgroundLike most legumes, chickpeas form specialized organs called root nodules. These nodules allow for a symbiotic relationship with rhizobium bacteria. The rhizobia provide fixed atmospheric nitrogen to the plant in a usable form. It is of both basic and practical interest to understand the host plant genetics of legume root nodulation. Chickpea lines PM233 and PM405, which harbor the mutationally identified nodulation genes rn1 and rn4, respectively, both display nodulation-deficient phenotypes. Previous investigators identified the rn1 mutation with the chickpea homolog of Medicago truncatula nodulation gene NSP2, but were unable to define the mutant rn1 allele. We used Illumina and Nanopore sequencing reads to attempt to identify and characterize candidate mutation sites responsible for the PM233 and PM405 phenotypes.ResultsWe aligned Illumina reads to the available desi chickpea reference genome, and did a de novo contig assembly of Nanopore reads. In mutant PM233, the Nanopore contigs allowed us to identify the breakpoints of a ~ 35 kb deleted region containing the CaNSP2 gene, the Medicago truncatula homolog of which is involved in nodulation. In mutant PM405, we performed variant calling in read alignments and identified 10 candidate mutations. Genotyping of a segregating progeny population narrowed that pool down to a single candidate gene which displayed homology to M. truncatula nodulation gene NIN.ConclusionsWe have characterized the nodulation mutation sites in chickpea mutants PM233 and PM405. In mutant PM233, the rn1 mutation was shown to be due to deletion of the entire CaNSP2 nodulation gene, while in mutant PM405 the rn4 mutation was due to a single base deletion resulting in a frameshift mutation between the predicted RWP-RK and PB1 domains of the NIN nodulation gene. Critical to characterization of the rn1 allele was the generation of Nanopore contigs for mutant PM233 and its wild type parent ICC 640, without which the deletional boundaries could not be defined. Our results suggest that efforts of prior investigators were hampered by genomic misassemblies in the CaNSP2 region of both the desi and kabuli reference genomes.

Effect of induction moment on Bradyrhizobium-soybean (Glycine max) interaction

Introduction. Legume nodulation is regulated by the exchange of molecular signals between the plant and rhizobia. The bacterial nodulation genes are activated by flavonoids present in root exudates. As a result of this activation, the Nod factors are synthesized, which in turn participate in the morphogenesis and function of the nodules. It is then possible to regulate the induction of Nod factors and other signals related to the nodulation process, during the inoculants production. Objective. To determine if the moment to induce the nodulation genes in Bradyrhizobium inoculants affects the synthesis of compounds related to the nodulation process and its interaction with soybean plants. Materials and methods. The research was developed in Rizobacter S.A., Argentina and Fyteko S.A. laboratories, Belgium, and in a plant growth chamber of the first one, during May-June of 2017. Two Bradyrhizobium strains were induced with daidzein in three moments. The signals produced by the strains and the biological effect in soybean plants were studied. Results. Differences were detected in the molecules produced by the bacteria, but it did not affect the growing and plants nodulation related to the different moments of induction. Conclusion. The metabolites excreted by Bradyrhizobium changed with strain and induction moment, although the latter did not affect nodulation or soybean plants growth.

Fields with no recent legume cultivation have sufficient nitrogen-fixing rhizobia for crops of faba bean (Vicia faba L.)

Purpose(1) To assess the biological N fixation (BNF) potential of varieties of faba bean (Vicia faba L.) cropped with or without compost in an experimental field-scale rotation with no recent history of legumes, (2) to enumerate soil populations of Rhizobium leguminosarum sv. viciae (Rlv), and to genetically characterize the nodulating Rlv strains, (3) compare BNF with other sites in Britain.MethodsBNF was evaluated from 2012 to 2015 using 15 N natural abundance. Treatments were either PK fertilizer or compost. Soil rhizobial populations were determined using qPCR, the symbiotic rhizobia genotyped (16 S rRNA, nodA and nodD genes), and their BNF capacity assessed ex situ. The reliance of legumes on BNF at other British sites was estimated in a single season, and their nodulating symbionts examined.ResultsFaba bean obtained most of its N through BNF (>80%) regardless of variety or year. N-accumulation by cvs Babylon and Boxer increased with compost treatment in 2014/2015. Rhizobial populations were c. 105-106Rlv cells g−1 soil regardless of field or treatment. 157 Rlv microsymbionts grouped into two large nodAD clades; one mainly from V. faba, and the other from various legumes. All isolates nodulated, and some performed better than commercial inoculant strains.ConclusionsFaba bean can provide most of its nitrogen through BNF and leave economically valuable residual N for subsequent crops. Recent legume cropping in northern Europe is not essential for effective nodulation: rhizobia may persist in a range of farmland locations. Nevertheless, there is the potential to apply elite rhizobial strains as inoculants in some soils.