Nodulation Genes Research Articles

Rhizobium wuzhouense sp. nov., isolated from roots of Oryza officinalis.

Three bacterial isolates, designated W44T, W15 and W11, were isolated from the root of Oryza officinalis grown in Wuzhou, China. These isolates were Gram-negative, aerobic, motile and rod-shaped; demonstrated cellulase and urea activities; and formed cream-coloured colonies. The 16S rRNA gene sequence analysis indicated that the similarities between strain W44T and strains W15 and W11 were 100 %; all of them belonged to the genus Rhizobium and had the highest sequence similarity to Rhizobium rosettiformans W3T (98.7 %), followed by Rhizobium ipomoeae shin9-1T (98.2 %). Sequencing of housekeeping genes (recA, atpD, rpoB and glnA) of the novel isolates revealed similarities to members of established Rhizobium species to be less than 94.3 %. The values of DNA-DNA hybridization between strain W44T and the reference strains (R. rosettiformans W3T and R. ipomoeae shin9-1T) were 41.3 and 29.2 %, respectively. The major cellular fatty acid of strain W44T was summed feature 8 (C18 : 1ω9t and/or C18 : 1ω9c and/or C18 : 1ω7c). The polar lipid profile of strain W44T consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, two unidentified lipids and two unidentified aminophospholipids. The G+C content of strain W44T was 62.4 mol%. In nodulation tests, none of the three strains could induce nodule formation in Glycine max, Phaseolus vulgaris or Medicago sativa. The nodulation gene (nodA), nitrogenase reductase gene (nifH) and virulence gene (virC) were not detected by PCR in these strains. Based on the above results and phenotypic features, a novel species, Rhizobium wuzhouense sp. nov., is proposed, with strain W44T (=CCTCC AB 2017179T=GDMCC 1.1257T=KCTC 62194T) as the type strain.

In silico structural homology modeling of nif A protein of rhizobial strains in selective legume plants.

Symbiosis is a complex genetic regulatory biological evolution which is highly specific pertaining to plant species and microbial strains. Biological nitrogen fixation in legumes is a functional combination of nodulation by nod genes and regulation by nif, fix genes. Three rhizobial strains (Rhizobium leguminosarum, Bradyrhizobium japonicum, and Mesorhizobium ciceri) that we considered for in silico analysis of nif A are proved to be the best isolates with respect to N2 fixing for ground nut, chick pea and soya bean (in vitro) out of 47 forest soil samples. An attempt has been made to understand the structural characteristics and variations of nif genes that may reveal the factors influencing the nitrogen fixation. The primary, secondary and tertiary structure of nif A protein was analyzed by using multiple bioinformatics tools such as chou-Fasman, GOR, ExPasy ProtParam tools, Prosa -web. Literature shows that the homology modeling of nif A protein have not been explored yet which insisted the immediate development for better understanding of nif A structure and its influence on biological nitrogen fixation. In the present predicted 3D structure, the nif A protein was analyzed by three different software tools (Phyre2, Swiss model, Modeller) and validated accordingly which can be considered as an acceptable model. However further in silico studies are suggested to determine the specific factors responsible for nitrogen fixing in the present three rhizobial strains.

Comparative symbiotic plasmid analysis indicates that symbiosis gene ancestor type affects plasmid genetic evolution.

This study compared 24 rhizobial symbiotic plasmids that included four genera and 11 species, illuminating the functional gene composition and symbiosis gene ancestor types of symbiotic plasmids from higher taxonomy. It provides valuable insights into mosaic structures and the evolution of symbiotic plasmids.

Phylogenetic analyses of Bradyrhizobium symbionts associated with invasive Crotalaria zanzibarica and its coexisting legumes in Taiwan

In this study, the genetic diversity and identification of Bradyrhizobium symbionts of Crotalaria zanzibarica, the most widely-distributed invasive legume in Taiwan, and other sympatric legume species growing along riverbanks of Taiwan were evaluated for the first time. In total, 59 and 54 Bradyrhizobium isolates were obtained from C. zanzibarica and its coexisting legume species, respectively. Based on the multilocus sequence analysis (MLSA) of concatenated four housekeeping genes (dnaK-glnII-recA-rpoB gene sequences, 1901bp), the 113 isolates displayed 53 unique haplotypes and grouped into 21 clades. Of these clades, 11 were found to be congruent to already defined Bradyrhizobium species, while the other 10 clades were found to not be congruent to any defined species. In particular, the C. zanzibarica isolates belong to 14 MLSA clades, six of which overlapped with the isolates of coexisting legumes. According to the nodA gene sequences (555bp) obtained from the 105 isolates, these isolates were classified into three known nodA clades, III.2, III.3 and VII and were further clustered into 10 groups. Furthermore, the C. zanzibarica isolates were clustered into 8 nodA groups, five of which overlapped with the isolates from coexisting legumes. Additionally, the nodA genes of the isolates from native species were dominated by Asian origin, while those from C. zanzibarica were dominated by American origin. In conclusion, C. zanzibarica is a promiscuous host capable of recruiting diverse Bradyrhizobium symbionts, some of which are phylogenetically similar to the symbionts of coexisting legumes in Taiwan.

Structural Polymorphism of Sinorhizobium meliloti Genes Related to Virulence and Salt Tolerance

Analysis of the structural polymorphism of eight genes in Sinorhizobium meliloti (nodA, nodB, nodC, and nodH, as well as betA, betB, betC, and betB2) involved in virulence control and salt tolerance, respectively, was carried out in native populations from two geographically distant areas of alfalfa diversity. These areas are located in the North Caucasian gene center of cultivated plants (NCG) and in the modern center of introgressive hybridization of alfalfa located next to the Aral Sea area (PAG) subjected to salinization. RFLP types (alleles) of the nod and bet genes, similar to those in the reference strain Rm1021 (A-type) and different from them (divergent, or D-type alleles) were revealed. The combinations for A- and D-type alleles of the aforementioned genes (analysis of the linkage disequilibrium, LD) were studied in both populations. It was shown that D-type alleles of the nod genes were two times more frequent in the NCG population, while D-type alleles of the bet genes were predominantly identified in the PAG population. At the same time, different combinations of D-type alleles of both the nod and bet genes prevailed in populations. For instance, in the case of the glycine betaine metabolism pathway, these were the betC and betB2 genes in NCG population and betB and betA genes in PAG population. The state of linkage disequilibrium was shown for 60.7% of combinations of alleles of the nod and bet genes in the S. meliloti strains from NCG and more than twice less in strains from the PAG population. It was concluded that clonal lines prevailed in NCG, while the PAG population of S. meliloti had a panmictic structure with revealed single clonal lines.

Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses

Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete Frankia as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using Parasponia species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between Parasponia andersonii and its nonnodulating relative Trema tomentosa demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.

QTLs underlying the genetic interrelationship between efficient compatibility of Bradyrhizobium strains with soybean and genistein secretion by soybean roots.

Soybean plants establish symbiotic relationships with soil rhizobia which form nodules on the plant roots. Nodule formation starts when the plant roots exudate isoflavonoids that induce nod gene expression of a specific Bradyrhizobium. We examined the specific indigenous rhizobia that form nodules with the soybean cultivars Peking and Tamahomare in different soils. PCR-RFLP analysis targeted to the 16S-23S rRNA gene internal transcribed spacer (ITS) region of the bacterial type of each root nodule showed that Bradyrhizobium japonicum (USDA110-type) and Bradyrhizobium elkanii (USDA94-type) had high compatibility with the Tamahomare and Peking cultivars, respectively. We grew 93 recombinant inbred lines (RIL) of soybean seeds derived from the cross between Peking and Tamahomare in three different field soils and identified the indigenous rhizobia nodulating each line using the same PCR-RFLP analysis. QTL analysis identified one QTL region in chromosome-18 with a highly significant additive effect that controls compatibility with both B. japonicum USDA110 and B. elkanii USDA94. We also measured the amount of daidzein and genistein secretion from roots of the 93 RILs by HPLC analysis. QTL analysis showed one QTL region in chromosome-18 controlling genistein secretion from roots and coinciding with that regulating compatibility of specific indigenous rhizobia with soybean. The amount of genistein may be a major regulatory factor in soybean-rhizobium compatibility.

Metabolomics and Transcriptomics Identify Multiple Downstream Targets of Paraburkholderia phymatum σ54 During Symbiosis with Phaseolus vulgaris.

RpoN (or σ54) is the key sigma factor for the regulation of transcription of nitrogen fixation genes in diazotrophic bacteria, which include α- and β-rhizobia. Our previous studies showed that an rpoN mutant of the β-rhizobial strain Paraburkholderia phymatum STM815T formed root nodules on Phaseolus vulgaris cv. Negro jamapa, which were unable to reduce atmospheric nitrogen into ammonia. In an effort to further characterize the RpoN regulon of P. phymatum, transcriptomics was combined with a powerful metabolomics approach. The metabolome of P. vulgaris root nodules infected by a P. phymatum rpoN Fix− mutant revealed statistically significant metabolic changes compared to wild-type Fix+ nodules, including reduced amounts of chorismate and elevated levels of flavonoids. A transcriptome analysis on Fix− and Fix+ nodules—combined with a search for RpoN binding sequences in promoter regions of regulated genes—confirmed the expected control of σ54 on nitrogen fixation genes in nodules. The transcriptomic data also allowed us to identify additional target genes, whose differential expression was able to explain the observed metabolite changes in numerous cases. Moreover, the genes encoding the two-component regulatory system NtrBC were downregulated in root nodules induced by the rpoN mutant, and contained a putative RpoN binding motif in their promoter region, suggesting direct regulation. The construction and characterization of an ntrB mutant strain revealed impaired nitrogen assimilation in free-living conditions, as well as a noticeable symbiotic phenotype, as fewer but heavier nodules were formed on P. vulgaris roots.

Horizontal gene transfer among rhizobia of the Core Cape Subregion of southern Africa

This study determined the extent of horizontal gene transfer among rhizobial genera in the Core Cape Subregion (CCR) of South Africa and reconstructed the ancestral symbionts of the legumes. Phylogenies of two chromosomal genes (16S rRNA and recA) and one nodulation gene (nodA) of rhizobia, isolated from diverse legumes in the CCR, were reconstructed using Bayesian and Maximum Likelihood techniques. A cophylogenetic analysis was used to test for congruence between the chromosomal and the nodA phylogenies. Five genera of rhizobia (Bradyrhizobium, Burkholderia, Mesorhizobium, Rhizobium and Sinorhizobium) were studied. A phylogeny of the legumes was reconstructed from matK and rbcL DNA sequences and it was used to reconstruct their ancestral rhizobia, using Bayesian methods. The chromosomal phylogeny of the rhizobia was mostly incongruent with that of nodA, indicating potential horizontal inheritance of the latter. The nodA genes of Burkholderia, Mesorhizobium and Rhizobium had different evolutionary histories from their counterparts in other parts of the globe. Burkholderia was reconstructed as the ancestral symbionts of the CCR legumes. Evidence of co-diversification between the legumes and their symbionts was observed and this highlights a potential role of the legume-rhizobia interaction to the high diversity of legumes in the CCR. Finally, the availability of compatible rhizobia and their competitive ability are discussed as possible drivers for the lack of shared legumes between the Fynbos biome and the Kwongan of Australia.

Phosphate Limitation Alters Medicago–Sinorhizobium Signaling: Flavonoid Synthesis and AHL Production

The legume plant Medicago truncatula Gaertn. can establish a symbiotic interaction with Sinorhizobium meliloti. One of the most limiting factors for symbiosis is phosphate (P) deficiency. Therefore, legumes and their symbiotic partners, rhizobia, have developed mechanisms to adapt to P restriction. In the non-symbiotic state, plants would up-regulate flavonoid biosynthesis via increasing the expression of chalcone synthase (chs), catalyzing the first step of flavonoid synthesis. Simultaneously, bacterial quorum sensing (QS) pathway can regulate the expression of certain genes involved in symbiotic functions of bacteria in response to P availability as well as bacterial population. Since both flavonoids and QS signaling molecules (N-acyl homoserine lactones, AHL) play important roles in the rhizobia-legume symbiosis, we evaluated these processes in the symbiotic state under different P concentrations and bacterial populations. In this study, by using real-time PCR and HPLC, we showed the expression of pt1 (phosphate transporter 1) and chs as well as luteolin production increased, in a time dependent manner, in plants following P limitation. Nod gene inducing flavonoids can up-regulate the bacterial QS pathway which results in an increase in AHL production, possibly to enhance symbiotic behaviors of rhizobia. It has been estimated that there is a feedback loop from bacterial AHL to flavonoid production pathway in legume plants.

Morphological and Genetic Characterization of Non‐Nodulating Peanut Recombinant Inbred Lines

Peanut (Arachis hypogaea L.) nodulation is relatively diverged from other legumes. Further understanding of nodulation mechanisms would facilitate improvement of biological N fixation and yield in peanut. The objective of this study was to characterize the non‐nodulating (Nod−) peanut recombinant inbred lines (RILs) in comparison to their nodulating (Nod+) sister RILs morphologically and genetically. Two pairs of Nod− and Nod+ RILs were inoculated with rhizobia for morphological observation. Nodules and root hairs were absent in Nod− lines and present in Nod+ lines. The Nod− lines had a much smaller canopy with yellow leaves and fewer pods than Nod+ lines. Simple‐sequence repeat (SSR) marker genotyping of the two pairs of RILs and their parental lines revealed several chromosome regions differing between Nod− and Nod+ sister lines, which potentially harbor the genes controlling nodulation. Two F2 populations segregating for nodulation were constructed by crossing the two Nod− lines with their Nod+ sister lines. The segregation ratios of Nod−/Nod+ of the two populations followed 7:9 and 7:57, respectively, indicating that multiple genes were involved in controlling peanut nodulation. The results from this study provided important morphological and genetic information related to peanut nodulation, which paved the road for further mapping and cloning of peanut nodulation genes and for eventually developing peanut cultivars with improved N fixation efficiency.

A nodule endophytic Bacillus megaterium strain isolated from Medicago polymorpha enhances growth, promotes nodulation by Ensifer medicae and alleviates salt stress in alfalfa plants

AbstractA Gram‐positive, fast‐growing, endophytic bacterium was isolated from root nodules of Medicago polymorpha and identified as Bacillus megaterium. The isolate, named NMp082, co‐inhabited nodules with the symbiotic rhizobium Ensifer medicae. B. megaterium NMp082 contained nifH and nodD genes that were 100% identical to those of Ensifer meliloti, an unusual event that suggested previous lateral gene transfer from a different rhizobial species. Despite the presence of nodulation and nitrogen fixation genes, the endophyte was not able to form effective nodules; however, it induced nodule‐like unorganised structures in alfalfa roots. Axenic inoculation promoted plant growth in M. polymorpha, Medicago lupulina, Medicago truncatula and Medicago sativa, and co‐inoculation with E. medicae enhanced growth and nodulation of Medicago spp. plants compared with inoculation with either bacterium alone. B. megaterium NMp082 also induced tolerance to salt stress in alfalfa and Arabidopsis plants. The ability to produce indole acetic acid (IAA) and the 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase activity displayed by the endophyte in vitro might explain the observed plant growth promotion and salt stress alleviation. The isolate was also highly tolerant to salt stress, water deficit and to the presence of different heavy metals. The newly characterised endophytic bacterium possessed specific characteristics that point at potential applications to sustain plant growth and nodulation under abiotic stress.

Transcriptomic profiling of Burkholderia phymatum STM815, Cupriavidus taiwanensis LMG19424 and Rhizobium mesoamericanum STM3625 in response to Mimosa pudica root exudates illuminates the molecular basis of their nodulation competitiveness and symbiotic evolutionary history

BackgroundRhizobial symbionts belong to the classes Alphaproteobacteria and Betaproteobacteria (called “alpha” and “beta”-rhizobia). Most knowledge on the genetic basis of symbiosis is based on model strains belonging to alpha-rhizobia. Mimosa pudica is a legume that offers an excellent opportunity to study the adaptation toward symbiotic nitrogen fixation in beta-rhizobia compared to alpha-rhizobia. In a previous study (Melkonian et al., Environ Microbiol 16:2099–111, 2014) we described the symbiotic competitiveness of M. pudica symbionts belonging to Burkholderia, Cupriavidus and Rhizobium species.ResultsIn this article we present a comparative analysis of the transcriptomes (by RNAseq) of B. phymatum STM815 (BP), C. taiwanensis LMG19424 (CT) and R. mesoamericanum STM3625 (RM) in conditions mimicking the early steps of symbiosis (i.e. perception of root exudates). BP exhibited the strongest transcriptome shift both quantitatively and qualitatively, which mirrors its high competitiveness in the early steps of symbiosis and its ancient evolutionary history as a symbiont, while CT had a minimal response which correlates with its status as a younger symbiont (probably via acquisition of symbiotic genes from a Burkholderia ancestor) and RM had a typical response of Alphaproteobacterial rhizospheric bacteria. Interestingly, the upregulation of nodulation genes was the only common response among the three strains; the exception was an up-regulated gene encoding a putative fatty acid hydroxylase, which appears to be a novel symbiotic gene specific to Mimosa symbionts.ConclusionThe transcriptional response to root exudates was correlated to each strain nodulation competitiveness, with Burkholderia phymatum appearing as the best specialised symbiont of Mimosa pudica.

Multiple Nodulation Genes Are Up-Regulated During Establishment of Reniform Nematode Feeding Sites in Soybean

The semi-endoparastic reniform nematode (Rotylenchulus reniformis) infects over 300 plant species. Females penetrate host roots and induce formation of complex, multinucleate feeding sites called syncytia. While anatomical changes associated with reniform nematode infection are well documented, little is known about their molecular basis. We grew soybean (Glycine max) in a split-root growth system, inoculated half of each root system with R. reniformis, and quantified gene expression in infected and control root tissue at four dates after inoculation. Over 6,000 genes were differentially expressed between inoculated and control roots on at least one date (false discovery rate [FDR] = 0.01, |log2FC| ≥ 1), and 507 gene sets were significantly enriched or depleted in inoculated roots (FDR = 0.05). Numerous genes up-regulated during syncytium formation had previously been associated with rhizobia nodulation. These included the nodule-initiating transcription factors CYCLOPS, NSP1, NSP2, and NIN, as well as multiple nodulins associated with the plant-derived peribacteroid membrane. Nodulation-related NIP aquaporins and SWEET sugar transporters were induced, as were plant CLAVATA3/ESR-related (CLE) signaling proteins and cell cycle regulators such as CCS52A and E2F. Nodulins and nodule-associated genes may have ancestral functions in normal root development and mycorrhization that have been co-opted by both parasitic nematodes and rhizobial bacteria to promote feeding site and nodule formation.

Phylogeny and genetic diversity assessment of Ensifer strains nodulating Senegalia (Acacia) senegal (L.) Britton. in arid regions of Western Rajasthan, India

The tree Senegalia (Acacia) senegal is well adapted to soils of arid regions having a vital role in reforestation and increasing soil fertility through biological nitrogen fixation in addition to its utility as edible seeds and gum. Fifty-nine root nodule bacterial (RNB) strains were isolated; characterized at phenotypic and genotypic level; and host range was studied. The Senegalia-RNB strains were able to grow up to 3% NaCl and on a wide range of pH (5–11) with optimum growth at alkaline pH showing their adaptation to alkaline desert soil. The carbon utilization pattern of Senegalia-RNB strains was significantly different from existing closely related type strains of Ensifer. The PCR-RFLP (ARDRA) as well as RAPD patterns revealed considerable genetic diversity among the strains forming distinct genetic groups. The 16S rRNA phylogeny of selected six (AS11, AS18, AS27, AS34, AS50 and AS53) strains showed that they belong to fast growing species of Ensifer with few being similar to the previously described Ensifer strains from Vachellia jacquemontii and Vachellia leucophloea from Thar Desert of India. The symbiotic (nifH and nodA) gene phylogeny showed incongruence with that of 16S rRNA gene (species) phylogeny suggesting HGT incidences under hot-arid and alkaline environment. The Senegalia-Ensifer strains possessing novel symbiotic genes could effectively cross-nodulate species of Vachellia (V. jacquemontii, V. leucophloea and V. nilotica) but failed to nodulate the crop Vigna radiata. Multi locus sequence analysis (MLSA) is required to determine novelty of these Ensifer strains and more cross-inoculation studies will help in understanding their potential use as inoculums in nursery practices for reforestation program in arid and semi-arid regions.

Vicia faba L. in the Bejaia region of Algeria is nodulated by Rhizobium leguminosarum sv. viciae, Rhizobium laguerreae and two new genospecies

Fifty-eight rhizobial strains were isolated from root nodules of Vicia faba cv. Equina and Vicia faba cv. Minor by the host-trapping method in soils collected from eleven sites in Bejaia, Eastern Algeria. Eleven genotypic groups were distinguished based on the combined PCR/RFLP of 16S rRNA, 16S–23S rRNA intergenic spacer and symbiotic (nodC and nodD-F) genes and further confirmed by multilocus sequence analysis (MLSA) of three housekeeping genes (recA, atpD and rpoB), the 16S rRNA gene and the nodulation genes nodC and nodD. Of the 11 genotypes, 5 were dominant and 2 were the most represented. Most of the strains shared high nodD gene sequence similarity with Rhizobium leguminosarum sv. viciae; their nodC sequences were similar to both Rhizobium leguminosarum and Rhizobium laguerreae. Sequence analyses of the 16S–23S rRNA intergenic spacer showed that all the new strains were phylogenetically related to those described from Vicia sativa and V. faba in several African, European, American and Asian countries, with which they form a group related to Rhizobium leguminosarum. Phylogenetic analysis based on MLSA of 16S rRNA, recA, atpD and rpoB genes allowed the affiliations of strain AM11R to Rhizobium leguminosarum sv. viciae and of strains EB1 and ES8 to Rhizobium laguerreae. In addition, two separate clades with <97% similarity may represent two novel genospecies within the genus Rhizobium.

Strigolactones promote rhizobia interaction and increase nodulation in soybean (Glycine max)

Strigolactones (SLs) play an important role in controlling root growth, shoot branching, and plant-symbionts interaction. Despite the importance, the components of SL biosynthesis and signaling have not been unequivocally explored in soybean. Here we identified the putative components of SL synthesis enzymes GmMAX1a and GmMAX4a with tissue expression patterns and were apparently regulated by rhizobia infection and changed during nodule development. GmMAX1a and GmMAX4a were further characterized in soybean nodulation with knockdown transgenic hairy roots. GmMAX1a and GmMAX4a knockdown lines exhibit decreased nodule number and expression levels of several nodulation genes required for nodule development. Hormone analysis showed that GmMAX1a and GmMAX4a knockdown hairy roots had increased physiological level of ABA and JA but significantly decreased auxin content. This study not only revealed the conservation of SL biosynthesis but also showed close interactions between SL and other hormone signaling in controlling plant development and legume-rhizobia interaction.

Functional characterization of soybean strigolactone biosynthesis and signaling genes in Arabidopsis MAX mutants and GmMAX3 in soybean nodulation

BackgroundStrigolactones (SLs) play important roles in controlling root growth, shoot branching, and plant-symbionts interaction. Despite the importance, the components of SL biosynthesis and signaling have not been unequivocally explored in soybean.ResultsHere we identified the putative components of SL synthetic enzymes and signaling proteins in soybean genome. Soybean genome contains conserved MORE AXILLARY BRANCHING (MAX) orthologs, GmMAX1s, GmMAX2s, GmMAX3s, and GmMAX4s. The tissue expression patterns are coincident with SL synthesis in roots and signaling in other tissues under normal conditions. GmMAX1a, GmMAX2a, GmMAX3b, and GmMAX4a expression in their Arabidopsis orthologs’ mutants not only restored most characteristic phenotypes, such as shoot branching and shoot height, leaf shape, primary root length, and root hair growth, but also restored the significantly changed hormone contents, such as reduced JA and ABA contents in all mutant leaves, but increased auxin levels in atmax1, atmax3 and atmax4 mutants. Overexpression of these GmMAXs also altered the hormone contents in wild-type Arabidopsis. GmMAX3b was further characterized in soybean nodulation with overexpression and knockdown transgenic hairy roots. GmMAX3b overexpression (GmMAX3b-OE) lines exhibited increased nodule number while GmMAX3b knockdown (GmMAX3b-KD) decreased the nodule number in transgenic hairy roots. The expression levels of several key nodulation genes were also altered in GmMAX3b transgenic hairy roots. GmMAX3b overexpression hairy roots had reduced ABA, but increased JA levels, with no significantly changed auxin content, while the contrast changes were observed in GmMAX3b-KD lines. Global gene expression in GmMAX3b-OE or GmMAX3b-KD hairy roots also revealed that altered expression of GmMAX3b in soybean hairy roots changed several subsets of genes involved in hormone biosynthesis and signaling and transcriptional regulation of nodulation processes.ConclusionsThis study not only revealed the conservation of SL biosynthesis and signaling in soybean, but also showed possible interactions between SL and other hormone synthesis and signaling during controlling plant development and soybean nodulation. GmMAX3b-mediated SL biosynthesis and signaling may be involved in soybean nodulation by affecting both root hair formation and its interaction with rhizobia.

Nonspecific Symbiosis Between Sophora flavescens and Different Rhizobia.

We explored the genetic basis of the promiscuous symbiosis of Sophora flavescens with diverse rhizobia. To determine the impact of Nod factors (NFs) on the symbiosis of S. flavescens, nodulation-related gene mutants of representative rhizobial strains were generated. Strains with mutations in common nodulation genes (nodC, nodM, and nodE) failed to nodulate S. flavescens, indicating that the promiscuous nodulation of this plant is strictly dependent on the basic NF structure. Mutations of the NF decoration genes nodH, nodS, nodZ, and noeI did not affect the nodulation of S. flavescens, but these mutations affected the nitrogen-fixation efficiency of nodules. Wild-type Bradyrhizobium diazoefficiens USDA110 cannot nodulate S. flavescens, but we obtained 14 Tn5 mutants of B. diazoefficiens that nodulated S. flavescens. This suggested that the mutations had disrupted a negative regulator that prevents nodulation of S. flavescens, leading to nonspecific nodulation. For Ensifer fredii CCBAU 45436 mutants, the minimal NF structure was sufficient for nodulation of soybean and S. flavescens. In summary, the mechanism of promiscuous symbiosis of S. flavescens with rhizobia might be related to its nonspecific recognition of NF structures, and the host specificity of rhizobia may also be controlled by currently unknown nodulation-related genes.

Genetic diversity and symbiotic effectiveness of Bradyrhizobium strains nodulating selected annual grain legumes growing in Ethiopia.

Vigna unguiculata, Vigna radiata and Arachis hypogaea growing in Ethiopia are nodulated by a genetically diverse group of Bradyrhizobium strains. To determine the genetic identity and symbiotic effectiveness of these bacteria, a collection of 36 test strains originating from the root nodules of the three hosts was investigated using multilocus sequence analyses (MLSA) of core genes including 16S rRNA, recA, glnII, gyrB, atpD and dnaK. Sequence analysis of nodA and nifH genes along with tests for symbiotic effectiveness using δ15N analysis were also carried out. The phylogenetic trees derived from the MLSA grouped most test strains into four well-supported distinct positions designated as genospecies I-IV. The maximum likelihood (ML) tree that was constructed based on the nodA gene sequences separated the entire test strains into two lineages, where the majority of the test strains were clustered on one of a well-supported large branch that comprise Bradyrhizobium species from the tropics. This clearly suggested the monophyletic origin of the nodA genes within the bradyrhizobia of tropical origin. The δ15N-based symbiotic effectiveness test of seven selected strains revealed that strains GN100 (δ15N=0.73) and GN102 (δ15N=0.79) were highly effective nitrogen fixers when inoculated to cowpea, thus can be considered as inoculants in cowpea production. It was concluded that Ethiopian soils are a hotspot for rhizobial diversity. This calls for further research to unravel as yet unknown bradyrhizobia nodulating legume host species growing in the country. In this respect, prospective research should also address the mechanisms of symbiotic specificity that could lead to high nitrogen fixation in target legumes.