Abstract
Nodulation competition is a key factor that limits symbiotic nitrogen fixation between rhizobia and their host legumes. Soybean root exudates (SREs) are thought to act as signals that influence Bradyrhizobium ability to colonize roots and to survive in the rhizosphere, and thus they act as a key determinant of nodulation competitiveness. In order to find the competitiveness-related genes in B. diazoefficiens, the transcriptome of two SREs treated B. diazoefficiens with completely different nodulation abilities (B. diazoefficiens 4534 and B. diazoefficiens 4222) were sequenced and compared. In SREs treated strain 4534 (SREs-4534), 253 unigenes were up-regulated and 204 unigenes were down-regulated. In SREs treated strain 4534 (SREs-4222), the numbers of up- and down-regulated unigenes were 108 and 185, respectively. There were considerable differences between the SREs-4534 and SREs-4222 gene expression profiles. Some differentially expressed genes are associated with a two-component system (i.g., nodW, phyR-σEcfG), bacterial chemotaxis (i.g., cheA, unigene04832), ABC transport proteins (i.g., unigene02212), IAA (indole-3-acetic acid) metabolism (i.g., nthA, nthB), and metabolic fitness (i.g., put.), which may explain the higher nodulation competitiveness of B. diazoefficiens in the rhizosphere. Our results provide a comprehensive transcriptomic resource for SREs treated B. diazoefficiens and will facilitate further studies on competitiveness-related genes in B. diazoefficiens.
Similar content being viewed by others
Introduction
Rhizobia could infect legumes roots where they form nitrogen-fixing root nodules and differentiate into intracellular nitrogen-fixing bacteroids. Bacteroids convert molecular nitrogeninto ammonium, which improves host plant growth, and the plant provides dicarboxylic acids, which are photosynthetically fixed carbon molecules, to rhizobia as a source of carbon, energy, and reductants1. This microbe-plant interaction is the most efficient and productive source of nitrogen fixation, and has a critical potential for use in sustainable agricultural programs, agriculture, especially if it can be optimized2, 3. Due to its higher capacity for N2 fixation and increased adaptation to host rhizospheres compared to other rhizobial strains, it has been broadly used in commercial inoculants since 19924,5,6,7. However, more than 90% of the rhizobial inoculants introduced into field soils have had minimal effects on nodulation of the developing plants because of their low competitiveness or failure to compete with native rhizobial strains8.
For this reason, studies on the mechanism underlying competitiveness may play a key role in the development of commercial inoculants with highly efficient N fixation rates that can be used to improve crop productivity. Rhizobium-legume symbiosis is highly host-specific and is determined by the exchange of diffusible species-specific signals between a host plant and its symbiotic rhizobium9,10,11,12. More than100,000 low-molecular mass natural products, commonly known as secondary metabolites, are secreted by plants and they change the chemistry and biology of the rhizosphere. These changes have a positive effect on the microbial colonization of the root and microbial activity in the rhizosphere12,13,14. The distinct responsiveness of rhizobia to signal induction confers specificity on the rhizobia, which initiates the symbiotic nodulation of the legume plant and leads to the selection of a compatible plant host-microsymbiont pair15, 16. In response to legume-secreted flavonoids, a mechanism involving the NodD family of transcription activators, enables rhizobia to synthesize symbiosis-specific signal molecules called Nod factors (modified lipo-chito-oligosaccharidesor LCOs) by the nod gene operon nodYABCSUIJ, and different species of rhizobia generate a diverse range of Nod factors17. In host plants, the secreted Nod factors also could induce signal transduction cascades and lead to nodule formation18, 19. After Nod factors have been specifically recognized by kinase-like receptors on the host root epidermal cells, they induce root hair deformation, and result in nodule morphogenesis20.
In addition to inducing Nod factor production, legume flavonoids (genistein and daidzein) also act as bacterial growth promoters and chemotactic signals21, induce type III secretion machinery22, 23, lead to the rapid proliferation of rhizobia15, and activate rhizobial quorum sensing systems24. These phenotypes could affect the nodulation competitiveness of the rhizobia. Interestingly, several non-flavonoid compounds, such as betaines, xanthones, jasmonates, and phenolic compounds, have been shown to act as nodulation gene inducers in rhizobia, including B. japonicum and B. diazoefficiens 25,26,27,28. However, some flavonoids act as anti-inducers (antagonists) of nod gene transcriptional activation15, which demonstrates that the responses to soybean root exudates are complex. It is well known that mixtures of legume-derived extracellular compounds are thought to act as signals that affect rhizobial competitiveness towards legume plants29. However, the mechanisms underlying the corresponding recognition and compatibility responses of rhizobia to these root exudates signals are not well understood, and it has not been economically feasible to apply these rhizobia in the field to increase legume yield. The genome sequences of several rhizobia are now readily available6, 30, 31. Therefore, it is now possible to use RNA-seq to investigate gene regulation complexity in host-microbe interactions and obtain a better picture of bacterial behavior under agricultural or natural conditions.
Recently, combined transcriptomic studies on the regulation of symbiotic nitrogen-fixation in rhizobial strains were undertaken for the rhizobial model organism B. diazoefficiens. The main aims of these studies were (i) to detect transcriptional-level changes and identify genes that were specifically up/down-regulated between free-living conditions and the symbiotic lifestyle within root nodules32,33,34; (ii) to analyze the global gene expression changes and identify the differentially expressed genes in B. diazoefficiens in response to a given compound or environmental stresses35,36,37,38; (iii) to investigate the genes needed for symbiosis and identify their transcriptional regulators by comprehensively comparing the transcriptional profiles of mutants to that of the wild-type7, 39,40,41; and (iv) to analyze the functional mechanism underlying the specific adaptation of B. diazoefficiens to different host plant species42. However, only a few studies have investigated the gene expression patterns of rhizobial strains with different nodulation efficiencies, the effects of root exudates, and the role of the genes with altered expression in the specific adaptation to a legume host.
The aim of this study was to investigate and compare the impact of soybean root exudates on the transcriptome profiles of two B. diazoefficiens strains, which are characterized by contrasting nodulation competitiveness capacities: B. diazoefficiens 4534 occupied 90% of the nodules and B. diazoefficiens 4222 only 10% when equal amounts of cells were applied to soybean roots43. As a result, we have increased our biological knowledge of the mechanisms that underlying the host-specific mechanisms controlling rhizobial competitiveness in response to root exudate induction, and what physiological changes occur during the initiation of rhizosphere colonization. This study has also provided a useful resource for further detailed analysis of specific genes, and for systems biology investigations of other rhizobium-legume symbiosis systems.
Results and Discussion
The establishment of standard conditions
Mixtures of flavonoids and other compounds that are exuded by plant roots are thought to act as signals that affect the competitiveness of rhizobia and symbiotic interactions with legumes29. It is known that root exudates are primarily responsible for attracting compatible rhizobia and for the expression of a wide range of genes involved in symbiosis15. However, there have been few studies on gene expression patterns in response to root exudates. We performed preliminary experiments to establish a standard technique for sampling. The expression levels of nod genes (nodD1 and nodC) were chosen as criteria to evaluate the effective induction of SREs.
At the beginning of this study, three variables were compared: (i) nutrient solution concentrations, i.e., soybean plants were grown in different concentrations of nutrient solution; (ii) time intervals between SRE collection, i.e., SREs were collected from 0, 6, 12, and 16-day-old soybean plants; and (iii) optimal rhizobia exposure to SREs for maximal expression of nod genes, i.e., the bacterial cells were harvested after exposure to SREs for 0.25-, 1-, 3-, and 6-h. The results were then used to choose the optimal incubation conditions for this study on the effects of SREs on nod gene expression and the rhizobium transcriptome. Both B. diazoefficiens strains were grown to the exponential phase and then incubated with 200 mL SREs.
Treating B. diazoefficiens with adventitious root cultures led to an increase in nod gene expressions, which means they are effective elicitors that are involved in rhizobia nodulation competitiveness and that they are able to trigger rhizobium nodule primordia in legume plants. Therefore, the SREs obtained by this method could be used to induce the expression of bacterial genes involved in the early establishment of microbe-plant interactions. qPCR showed that the nod genes of both strains were all up-regulated in response to root exudates, although there were differences in the rate of gene expression up-regulation. The up-regulation of nod genes was greater in B. diazoefficiens 4534 than in B. diazoefficiens 4222. This result enabled us to determine the optimal conditions for nod gene expression. These were that the root exudates were collected from soybean grown in one-third strength modified N-free Rigaud-Puppo solution for 7 days, and that the gene induction period was 1-h. This resulted in the greatest up-regulation of nodD1 and nodC genes (Supplementary Figs S1–S3).
De novo transcriptome assembly
cDNA libraries from B. diazoefficiens that had been subjected to the different treatments were separately sequenced on the Illumina HiSeqTM 2000 platform and we obtained a total of 69,082,196 raw RNA-seq reads. After filtering out low quality reads, 65,747,612 of clean reads were produced, which represented a majority of the data and the Q20 score was >98%. Each library contained at least 15,244,134 reads, representing a coverage of 67X when compared to the full transcriptome, which is a density regarded as adequate to perform gene expression analysis44. The transcript abundance in the sample was quantified on the basis of the number of sequence reads mapped on the genome of B. diazoefficiens 4534 and B. diazoefficiens 4222 compared to the total number of mapped reads per sample (Table 1). The all unigenes set was used as a reference transcriptome to annotate and analyze the DEGs (differentially expressed ungenes) between the SRE- and control-treated samples.
In order to gain a better understanding of the transcriptome, functional annotation and classification of all the unigenes was performed. In this study, the unigenes with sequence orientations were aligned against public databases, among which total of 8,072 and 8,233 unigenes in stain 4534 and 4222 were mapped in one or more databases, respectively (Table 2).
The functions of all the unigenes were further predicted using GO assignments, a standard system for gene function classification. The unigenes in B. diazoefficiens 4534 treated with SREs were mainly assigned to the “cellular process” (3,017) and the “metabolic process” (3,735) subcategories of the “biological process” category, to the “cell” (1,708) and “cell part” (1,708) subcategories of the “cellular component” category, and to the “binding” (2,486) and “catalytic activity” (3,365) subcategories of the “molecular function” category (Fig. 1a). The unigenes in B. diazoefficiens 4222 treated with SREs were also assigned to the “cellular process” (2,834), “metabolic process” (3,491), “cell” (1,592), “cell part” (1,592), “binding” (2,322), and “catalytic activity” (3,206) subcategories (Fig. 1b).
Pathway assignment for all transcripts was based on the KEGG database. In response to SREs, a total of 8,072 unigenes in B. diazoefficiens 4534 were assigned to 158 KEGG pathways. The analysis indicated that “Metabolic pathways” (486), “Microbial metabolism in diverse environments” (262), “Biosynthesis of secondary metabolites” (243), “ABC transporters” (203), and “Two-component system” (95) were the dominant pathways. Similarly, for SREs treated B. diazoefficiens 4222, a total of 8,233 unigenes were annotated and were assigned to 160 KEGG pathways. The dominant pathways were “Metabolic pathways” (390), “Microbial metabolism in diverse environments” (225), “Biosynthesis of secondary metabolites” (207), “ABC transporters” (171) and “Two-component system” (77).
The analysis of the GO and KEGG categories for the unigenes under SREs induction showed similar enrichment terms in each category for SREs-4534 and SREs-4222, which meant that the symbiosis mechanism was very conservative. However, under the same conditions, the number of unigenes in these categories was greater in SREs-4534 than in SREs-4222.We suggest that the differences in B. diazoefficiens responses to SREs may result in the differences in symbiotic matching ability. Analyzing the DEGs expressed in B. diazoefficiens represents an efficient and simple way to further identify and explore the genes related to nodulation competitiveness.
Differential transcriptomes of two strains treated with soybean root exudates
RNA-seq and a comparative analysis of B. diazoefficiens induced by SREs were performed to explore nodulation competitiveness-related genes. The DEGs was assessed between the SREs and control treatments were obtained from a transcriptome data comparison of the Control-4534 vs. SREs-4534 in B. diazoefficiens 4534 and Control-4222 vs. SREs-4222 in B. diazoefficiens 4222, many genes exhibiting significantly differential expressions were identified. A total of 457 DEGs were identified in SREs-4534(Supplementary Table S1). These consisted of 253 up-regulated and 204 down-regulated genes (Fig. 2a). In SREs-4222, 293 DEGs were identified consisting of 108 up-regulated and 185 down-regulated genes (Fig. 2b) (Supplementary Table S2).
Many assembled unigenes were not significantly matched with available databases due to their short sequences or because they represented significantly novel genes. Analysis of the transcriptome profiles showed that the expression of a number of genes was significantly altered (P < 0.05) in both stains in response to SREs, which confirmed the effectiveness of the SRE s collection method. To our knowledge, this is the first report on transcriptome data that shows the response of B. diazoefficiens to SREs treatment. Therefore, this study will help reveal the molecular mechanisms underlying the different nodulation abilities in B. diazoefficiens, and specific adaptations by the legume host. The DEGs were identified under SREs treatment accounts for just a fraction of the total full-length sequences that had been annotated in B. diazoefficiens. One of the reasons is that SREs concentrations are very low and only the genes that are responsive to low concentrations of bioactive compounds have been induced20. Furthermore, symbiotic interaction in the rhizosphere involves a far more complex series of interactions, such as attachment to roots hairs, growth rate on rhizosphere substrates, contact with complicated plant macromolecules, biofilm formation, and cell-cell competition20, 45.
GO and KEGG enrichment analysis of DEGs
We performed GO and KEGG enrichment analyses to assign functional categories to the DEGs in the two strains treated with soybean root exudates. The DEGs in SREs-4534 were enriched in 12 GO terms while the DEGs in SREs-4222 were enriched in 7 GO terms (Supplementary Fig. S4a,b). The enriched 12 GO terms were “ciliary or flagellar motility”(20), “cell motility”(20), “cellular component movement” (20), “locomotion”(36), “chemotaxis” (18), “taxis”(18), “response to external stimulus”(19), “response to chemical stimulus” (20), “oxygen transport” (11), “gas transport” (11), “cell projection organization”(6), and “flagellum organization” (6) in the “biological processes” category; and “motor activity”(13), “oxygen binding”(11),“structural molecule activity”(17),“oxidoreductase activity”, and “acting on other nitrogenous”(3) in the “processes” category. The seven enriched B. diazoefficiens 4222 GO terms were “locomotion” (23), “ciliary or flagellar motility” (23), “cell motility” (23), “cellular component movement”(23), “cell projection organization”(8), “flagellum organization”(8), and “bacterial-type flagellum part” (18) in the “biological processes” category. These results suggest that the GO terms associated with “flagellum motility”, “cell motility”, and “flagellum organization and locomotion” were highly enriched in both of stains treated with SREs. Interestingly, GO terms related to “chemotaxis” and “response to external/chemical stimulus” were highly enriched in SREs-4534, but did not significantly change in SREs-4222. This suggested that a series of activities involved in chemotaxis and stimulus response are triggered in B. diazoefficiens 4534 following SREs treatment, and that these could play a role in nodulation efficiency35. This is in agreement with a previous report about the proteomic analysis of B. japonicumin response to genistein46.
In the KEGG pathway enrichment analysis, the genes that encoded enzymes involved in “flagellar assembly” (29), “bacterial chemotaxis” (37), and the “two-component system” (25) were highly enriched in SREs-4534 (Supplementary Fig. S5a). However, pathways corresponding to “flagellar assembly” (30) and “nitrogen metabolism” (11) were found to be highly enriched in SREs-4222 (Supplementary Fig. S5b). Based on analyses of the pathways, most gene expressions, especially genes related to “bacterial chemotaxis” and the “two-component system”, could have roles in the competitiveness of specific B. diazoefficiens strains. This is consistent with the results for the GO enrichment analysis and the proteomic analysis of B. japonicumin response to genistein.
Differential gene expression patterns between the two strains treated with SREs
Genes associated with the “two-component system”
Many studies have shown that the “two-component system” regulates the specific gene expression of rhizobia by recognizing additional plant and environmental signals, which affects rhizobial ability to colonize roots. It also affects the competitive performance of rhizobia19, 47, 48. This study showed that a total of 19 genes were related to the “two-component system”, which was found to be significantly enriched in the KEGG analysis (Supplementary Table S3). They considerably changed their expression in SREs-4534 where 18 genes were up-regulated and one was down-regulated. Interestingly, three genes were involved in associated with the “two-component” regulatory family, and the genes encoding NodW, PhyR and σEcfG were up-regulated in strain 4534 after SREs treatment, but they did not significantly change in SREs-4222.
Nod W is a two-component response regulator that is phosphorylated at the Asp70 residue by its cognate sensor protein Nod V, which controls the expression of the nodulation genes35. In B. japonicum, Nod VW is thought to recognize different plant flavonoids and environmental signals, or, when combined with Nod D, it may plays a key role in the nodulation of a broader range of specific host plants by mediating Nod factor synthesis19. These results are comparable to our GO enrichment analysis, where the GO term related to the “external/chemical stimulus” response was only highly enriched in SREs treated strain 4534. Furthermore, the Nod VW “two-component system” provides an alternative pathway for nod gene activation In NodD1 mutants (Faruque) and similar systems have been found in many rhizobia, including, Sinorhizobium meliloti (ExoS/ChvI), Mesorhizobiumloti (VirA⁄VirG) and Agrobacterium tumefaciens (ChvG/ChvI)49. Another, PhyR is a “two-component response” regulator that bridges two component signal transduction and is an alternative σ factor regulatory paradigm. It contains an N-terminal σ-like domain and a C-terminal receiver domain50. Previous studies have demonstrated that these PhyR and σEcfG regulons regulators act in the same cascade. Upon sensing a particular stimulus, the PhyR activity is controlled by phosphorylation of a conserved Asp residue and binds NepR, releasing σEcfG that can then associate with the RNA polymerase core enzyme to transcribe stress-related genes51. Subsequently, PhyR/σEcfG has been found to be involved in the responses to a number of stress and cellular growth signals, including nutrient deficiency, low pH, and heat shock, as well as oxidative, UV, ethanol, and hyperosmotic stresses52. PhyR/σEcfG-lacking B. japonicum mutants had impaired nodulation abilities. Further research needs to uncover the signal(s) and transduction mechanisms involved in the control of novel symbiotic genes46.
When B. diazoefficiens was induced by the SREs, a series of “two-component response” regulator (NodW, PhyR and σEcfG) encoding genes were considerably up-regulated, which is consistent with what was previously reported for the comparative proteomic study of B. diazoefficiens 4534 and 4222 by incubation with SREs. Thus, these regulators had positive effects on the higher nodulation competitiveness of B. diazoefficiens 453453.
Genes associated with bacterial chemotaxis
“Flagella and motility related functional” genes were enriched categories shared by both strains. The DEGs associated with chemotaxis were significantly enriched in SREs-4534 compared to SREs-4222. A total of 17 DEGs were observed in this pathway, all of which were up-regulated and were related to flagella and tropism (Supplementary Table S4). For example, cheA was only expressed in SREs-4534, and Donati et al. validated the effect of stimulating factors on the tropism and nodulation of rhizobia by knocking out cheA, which is consistent with the proteomics study37. This indicated that the expression of genes associated with tropism plays important roles in environmental adaptation and selective nodulation by rhizobia54.
It has been shown that genes related to flagella and bacterial tropism play essential roles at the beginning of the selective nodulation process in rhizobia54, 55. Different rhizobia exhibit different chemotactic responses to the root exudates produced by their hosts55, 56. Environmental factors can induce the expression of taxis factors in Rhizobium leguminosarum. These taxis gene clusters may affect comparative nodulation capabilities by regulating motility swimming and chemotaxis in R. leguminosarum 57. Furthermore, proteomic experiments indicated that more abundant flagellar proteins were detected in the strain 4534 than strain 4222 when both were exposed to SREs53. And similar result was recorded by Lang when rhizobia responded to genistein35. Tropism related genes (e.g., unigene04832) in B. diazoefficiens 4534 showed a stronger activity during the response to SREs than strain 4222. We suggest that the differences in the signal transduction and tropism related genes between the strains during the response to root exudates led to the variation in the selective nodulation capabilities of B. diazoefficiens. This is probably caused by the direct regulation of some nodulation signals or by an indirect impact on flagella synthesis.
Genes associated with ABC-transport proteins
The diverse functions of ABC transport proteins in bacterial physiological processes is due to their adaptability to the environment58, but their main active stage is in the early stages of rhizobium symbiotic system establishment46. After SREs induction, a significant enrichment of the ABC transport protein associated with DEGs was observed. A total of 20 DEGs were identified in this pathway, all of which were up-regulated (Supplementary Table S5). They were mainly involved in amino acid and carbohydrate transportation. The conclusions are found to be in line with what was previously reported for the comparative proteomic study of B. diazoefficiens 4534 and 4222 induced by SREs53. For example, biotin metabolism and the folic acid synthesis process were differentially expressed pathways in SREs-4534 according KEGG, and numerous complex intermediate metabolites and other precursors are produced by these processes. Therefore, to ensure the transport of these molecules, a considerable number of transport proteins are encoded by rhizobium species, and the ABC transport protein is the most important one46. The study of B. japonicum type bacteria demonstrated that only certain ABC transport protein types were expressed during the early stage of rhizobium symbiosis59, 60. Transport protein related to amino acid metabolism was one of the important classes, which is consistent with the data for S. meliloti type bacteria61. Based on the mutation analysis of the specific amino acid ABC transport proteins in R. leguminosarum, Lodwig et al. proposed a complex mechanism of amino acid recycling for the symbiotic nitrogen fixation process62.
These data indicated that the carbohydrate ABC transport system in rhizobium promoted the absorption of nutrients that were only presented at low concentrations in the rhizosphere and soil environment, and that this difference in nutrient uptake possess a strong relation to competitiveness for nodulation1.
Genes associated with phytohormone metabolism
All known phytohormones affect the nodulation of rhizobium. Auxin and cytokinin act as positive regulators in the formation and development of nodule and ethylene acts as an inhibitor that prevents root infection by rhizobia and nodule formation6, 63, 64. Indole-3-acetic acid (IAA) is a plant hormone that can regulate the formation of root nodules, and it plays important roles in many cellular processes, such as nodule formation, stimulation of early cell division, and nodule differentiation65. It has been shown that the IAA produced by Bradyrhizobium may affect symbiosis with the host66, 67, but how this occurs is still unclear. Therefore, IAA produced by rhizobium during the symbiosis process might affect the IAA balance inthe host plant, which would influence the establishment of the rhizobia and host symbiont6, 65.
These results demonstrate that two genes related to IAA synthesis, nthA encoding nitrile hydratase subunit alpha protein and nthB encoding nitrile hydratase subunit beta protein, which constitute nitrile hydratase (Nhase), were up-regulated in SREs-4534. Nitrile hydratase is an enzyme that converts indol-3-acetonitrile into indole-3-acetamide (IAM). Most rhizobia can synthesize IAA via different pathways, and at least five of them have been shown to initiate the synthesis of tryptophan6, 68, 69. In this group of synthetic pathways, most is known about the IAM route, where the tryptophan is first converted into IAM in the presence of tryptophan monooxygenase and then converted to IAA by IAM hydrolase66. Recent studies have shown that in Bradyrhizobium, the synthesis of IAM is independent of TMO activity and related to nitrile hydratase activity6, 65, 66. Although the regulation mechanisms for the biosynthesis of IAA from tryptophan in B. diazoefficiens have not been fully characterized, it may play a key role in its competitive colonization of rhizospheres. It also suggests that the regulation of IAA in nodules is very complex.
Genes associated with metabolic fitness
It is well known that the quantity of single carbon sources in soil is limited and that there are various types of carbon sources. Some special carbohydrates around the root, such as stachydrine, myo-inositol, and homoserine, can be used by rhizobia for growth70, 71, which is necessary if symbiotic nodulation and nitrogen fixation are to occur. To a certain extent, the nutrient utilization ability of rhizobium in the soil when faced by limited nutrient resources determines its selectively competitive nodulation capability72, 73.
Stachydrine and homoserine are major components of SREs, and they can lead to the production of large amounts of proline after degradation. In this experiment, putA expression in strain 4534 was significantly higher than in strain 4222. The proline dehydrogenase encoded by the putA gene in S. meliloti oxidizes proline into glutamate. Glutamate is then deaminated to produce α-ketoglutarate, which can participate in the citric acid cycle to produce energy or carbohydrate. Therefore, proline is an essential source of energy at the early stage of rhizobium infection. Mutant analysis showed that putA played a role in the selective nodulation capability and the colonization ability of rhizobia around host roots74, 75. Besides, the results of the proteome analysis showed that under the induction of SREs, more proteins related to metabolic fitness were found up-regulated in B. diazoefficiens 4534 than that of in B. diazoefficiens 422253.
In addition, the gene encoding prolidase (e.g., unigene05621) was up-regulated in both strains, and it effectively decomposes compounds containing proline residues, which increases the amount of proline that can be utilized by rhizobia. Therefore, we believe that the genes related to metabolism fitness are surely playing an important role in nodulation competition. Rhizobia lability to effectively utilize nutrients in soils containing limited nutrient resources determines the selectively competitive nodulation capabilities between different strains and the host.
Quantitative real-time PCR (qPCR) verification
To confirm the accuracy and reproducibility of the results of transcriptome analysis, quantitative PCR analysis was performed on 10 selected DEGs identified from the transcriptome profiling experiments, which divided into two groups according to their expression levels. In the first group, containing three genes encoding a sugar transporter (unigene00729), prolidase (unigene05621) and flagellin (unigene01736), the gene expression was up-regulated in both strains after SREs treatement (Fig. 3a). In another group, the DEGs were up-regulated in SREs-4534, but no significantly change in SREs-4222. The genes encoding two-component system (nodW, phyR), bacterial chemotaxis (cheA, unigene04832), ABC transport proteins (unigene02212), IAA (indole-3-acetic acid) metabolism (nthA) and metabolic fitness (putA) fitted this pattern (Fig. 3b). The qPCR results of 10 selected genes were consistent with their transcript abundance changes as determined by RNA-seq, which indicated the reliability of the transcriptome data.
Conclusion
This study is a comparative survey of the impact of SREs on the transcriptome profiles of two B. diazoefficiens strains that have different nodulation abilities. It reveals several previously uncharacterized genes involved in the competitive nodulation process. A majority of the genes led to the differential expression of a diverse array of proteins/enzymes, which suggested that there was a considerable variation in metabolic and physiological activities between the two SREs treated strains. This could affect B. diazoefficiens competitiveness. The genes involved in a variety of metabolic pathways, such as the two-component system, bacterial chemotaxis, ABC transport proteins, IAA metabolism, and metabolic fitness, were highly expressed in SREs-4534 compared to SREs-4222. When exposure to SREs, we observed that the most DEGs in both B. diazoefficiens strain 4534 and 4222 were highly correlated to differentially expressed proteins in the proteomic study53. Differential responses to induction by particular compounds in the root exudates may mediate the selection of specific rhizosphere populations. We found that competitive strains more readily responded to a wide range of synthetic flavonoids and seed exudates in comparison to uncompetitive strains.
Some other important DEGs were also validated in previous comparative proteomics analysis of B. diazoefficiens treatment with different flavonoids (genistein, coumestrol and daidzein, etc.) as well as the proteome profiles of B. diazoefficiens bacteroid during soybean nodule development46, 76,77,78,79,80. The data further revealed that the regulation of signaling transduction, receptor-mediated recognition, and the ability to metabolize various nutrient substances and phytohormones at the transcriptional level were fundamental to successful host colonization by rhizobia. Overall, this study has produced new information about the genetic and functional responses of B. diazoefficiens during competitive nodulation, and has provided a molecular basis for further investigations into the mechanisms underlying the host-specific symbiosis of bacteria with soybean and other legume hosts.
Methods
Bacterial strains, medium, and culture conditions
B. diazoefficiens 4534 and B. diazoefficiens 4222 were originally isolated from the nodules of field grown soybeans in Henan Province and Anhui Province, China, respectively. The soybean cultivar was Zhong-huang 13, which is widely cultivated in the Huang-Huai-Hai region on the North China Plain. All the rhizobia were pre-cultured at 180 rpm and 30 °C to an optical absorbance at 600 nm in TY medium (5 g/L tryptone, 3 g/L yeast extract, and 1.3 g/L CaCl2.6 H2O, [pH 7.0])81. The cultured cells were harvested, immediately flash frozen in liquid nitrogen, and stored at −80 °C until needed for RNA isolation.
Seedling growth and preparation of soybean root exudates (SREs)
Mixtures of flavonoids and other compounds that are exuded by plant roots can be more effective at improving the competitiveness of rhizobia than single compound26. Soybean seeds were surface-sterilized and germinated on a nitrogen-free mediumin the dark at 28 °C for 24–48 h5. Twenty-five pre-germination seeds were transferred to a polypropylene lattice placed in a glass cylinder containing 300 mL of sterilized, modified N-free solution under aseptic conditions82. No seeds were sown in the control treatment. The plant and control microcosms were arranged in a replicated randomized block design and maintained at 30 °C for 16 h and then at 15 °C for 8 h. The root exudates and control eluent (liquid from the blank control) were applied on TY solid medium and no growth indicated that no microbial contamination was present. Each collection had three biological replicates.
Sample preparation for RNA-seq RNA extraction
Total RNA was extracted using the SV Total RNA Isolation System (Promega, Madison, WI, USA) according to the manufacturer’s protocol, and then the genomic DNA was purified with RNase-free DNase I (Promega). The total RNA concentration was assessed using a NanoDrop-100 (Thermo Scientific, Wilmington, USA) and its quality was tested using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). All samples had A260/A280 and A260/A230 ratios of 2.13–2.23, RIN (RNA Integrity Number) value above 8.5, and 23 S/16 S values above 1.6 except for one.
Quantitative real-time PCR (qPCR) certification
qPCR as used to detect the effects of root exudate on nodC, nodD1, and nodD2 gene expressions and verify the reliability of the transcriptome experiments. Total RNAs were isolated as described previously. The first strand cDNAs were synthesized using1.0 μg of RNA and a ProtoScript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA). The RT samples were used for quantitative qPCR with the primers shown in Table 1, which were designed using the Premier 5.0 based on the genomic B. diazoefficiens USDA11083, which targets an amplicon size of 150–200 bp. The specificity of the primers was certified by agarose gel electrophoresis and product dissociation curves84. The expression of 16 S rRNA was used as an internal control for normalization. The equipment used included a 7500 Sequence Detection system (Applied Biosystems, Foster City, CA, USA) and SYBR Green PCR Master Mix (Applied Biosystems).
RNA-Seq experimental design, library construction, and sequencing
An equal amount (at least 5 μg) of total RNA from each sample was pooled for RNA-Seq so that a comprehensive range of transcripts could be obtained. Isolated mRNAs were extracted using Oligo (dT) magnetic beads and broken down to about 200 bp lengths using fragmentation buffer. The fragmented mRNA was reverse-transcribed into cDNA using a Truseq RNA-seq kit (Illumina Inc., CA, USA) and random primers. After end reparation and the addition of a single nucleotide A (adenine) at the 3′-end, the cDNAs were connected with adapters and prepared as libraries. After qualification and quantification using an Agilent 2100 Bioanaylzer and a StepOnePlus Real-Time PCR System (ABI, CA, USA), their sequences were determined using an Illumina HiSeq™ 2000 (Illumina Inc).
Transcript assembly and functional gene annotation
The raw data outputs from the Illumina equipment were filtered to remove adaptor sequences, unknown bases higher than 10%, and low quality bases (bases with a quality value ≤ 5), which left just high quality (clean) reads. The clean reads were assembled using the Trinity software short reads assembly program (http://trinityrnaseq.sourceforge.net/). The high quality reads were assembled into contigs, and then integrated to obtain unigenes. Subsequently, the “all unigenes” was assembled from the unigenes of Control-4534, SREs-4534, Control-4222, and SREs-4222, and used as a reference transcriptome for further annotation and analyses.
To obtain annotation information for the transcriptome in B. diazoefficiens, the sequences of the unigenes were aligned with the following databases: NR (non-redundant database), NT (nucleotide database), Swiss-Prot (Swiss Protein database), KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Clusters of Orthologous Groups) and GO (Gene Ontology).
Analysis of differentially expressed genes (DEGs)
The expression levels of the B. diazoefficiens DEGs in the different treatments were calculated by the RPKM (reads per kilobase per million mapped reads) method. An FDR (false discovery rate) ≤0.001 and a log2 (RPKM-SREs/FPKM-Control) ≥1 were adopted to judge the DEGs between the SREs and the control treatments. To understand the distribution of the DEGs, GO annotations for the DEGs were assigned to three categories (“molecular function”, “biological process”, and “cellular component”) using the Blast2GO software (http://www.geneontology.org). The GO enrichment analysis of the DEGs was undertaken using Goatools software (https://github.com/tanghaibao/GOatools) to find significantly enriched GO terms among the DEGs. Differentially expressed gene enrichment in the KEGG pathways was carried out by KOBAS software (http://kobas.cbi.pku.edu.cn/home.do). The DEGs that were significantly enriched in GO terms and KEGG pathways were identified after hypergeometric testing (Bonferroni-correction P ≤ 0.05)85.
References
Prell, J. & Poole, P. Metabolic changes of rhizobia in legume nodules. Trends Microbiol. 14, 161–168 (2006).
Biswas, B. & Gresshoff, P. M. The role of symbiotic nitrogen fixation in sustainable production of biofuels. Int. J. Mol. Sci. 15, 7380–7397 (2014).
Delamuta, J. R. et al. Polyphasic evidence supporting the reclassification of Bradyrhizobium japonicum group ia strains as Bradyrhizobium diazoefficiens sp. nov. Int. J. Syst. Evol. Microbiol. 63, 3342–3351 (2013).
Hungria, M. et al. Nitrogen fixation capacity and nodule occupancy by Bradyrhizobium japonicum and B. elekanii strains. Biol. Fert. Soils 27, 393–399 (1998).
Albareda, M. et al. Factors affecting the attachment of rhizospheric bacteria to bean and soybean roots. FEMS Microbiol. Lett. 259, 67–73 (2006).
Siqueira, A. F. et al. Comparative genomics of Bradyrhizobium japonicum, CPAC 15 and Bradyrhizobium diazoefficiens, CPAC 7: elite model strains for understanding symbiotic performance with soybean. BMC Genomics 15, 420 (2014).
Čuklina, J. et al. Genome-wide transcription start site mapping of Bradyrhizobium japonicum grown free-living or in symbiosis-a rich resource to identify new transcripts, proteins and to study gene regulation. BMC Genomics 17, 1–19 (2016).
Schumpp, O. & Deakin, W. J. How inefficient rhizobia prolong their existence within nodules. Trends Plant Sci. 15, 189–195 (2010).
Oldroyd, G. E. et al. The rules of engagement in the legume-rhizobial symbiosis. Annu. Rev. Genet 45, 119–44 (2011).
Cesco, S. et al. Plant-borne flavonoids released into the rhizosphere: impact on soil bio-activities related to plant nutrition. a review. Biol. Fert. Soils 48, 123–149 (2012).
Hayashi, S. et al. Transient nod factor-dependent gene expression in the nodulation-competent zone of soybean (glycine max, [L.] merr.) roots. Plant Biotechnol J. 10, 995–1010 (2012).
Palacios, O. A., Bashan, Y. & Debashan, L. E. Proven and potential involvement of vitamins in interactions of plants with plant growth-promoting bacteria-an overview. Biol. Fert. Soils 50, 415–432 (2014).
Mark, G. L. et al. Transcriptome profiling of bacterial responses to root exudates identifies genes involved in microbe-plant interactions. Proc. Natl. Acad. Sci. USA 102, 17454–17459 (2005).
Dardanelli, M. S. et al. Changes in flavonoids secreted by Phaseolus vulgaris roots in the presence of salt and the plant growth-promoting rhizobacterium Chryseobacterium balustinum. Appl. Soil. Ecol. 57, 31–38 (2012).
Moscatiello, R., Squartini, A., Mariani, P. & Navazio, L. Flavonoid-induced calcium signalling in Rhizobium leguminosarum bv. viciae. New. Phytol. 188, 814–823 (2010).
Maj, D. et al. Response to flavonoids as a factor influencing competitiveness and symbiotic activity of Rhizobium leguminosarum. Microbiol. Res. 165, 50–60 (2010).
Lemaire, B. et al. Symbiotic diversity, specificity and distribution of rhizobia in native legumes of the Core Cape Subregion (South Africa). FEMS Microbiol. Ecol. 91, 1–17 (2015).
Kouchi, H. et al. How many peas in a pod? legume genes responsible for mutualistic symbioses underground. Plant Cell Physiol. 51, 1381–1397 (2010).
Okazaki, S. et al. Genome analysis of a novel Bradyrhizobium sp. DOA9 carrying a symbiotic plasmid. Plos One 10, e0117392 (2015).
Ramachandran, V. K. et al. Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet rhizospheres investigated by comparative transcriptomics. Genome Biol. 12, R106 (2011).
Somers, E., Vanderleyden, J. & Srinivasan, M. Rhizosphere bacterial signalling: a love parade beneath our feet. Crit. Rev. Microbiol. 30, 205–240 (2004).
Krause, A., Doerfel, A. & Göttfert, M. Mutational and transcriptional analysis of the type III secretion system of Bradyrhizobium japonicum. Mol. Plant Microbe. In. 15, 1228–1235 (2002).
Lópezbaena, F. J. et al. Regulation and symbiotic significance of nodulation outer proteins secretion in Sinorhizobium fredii HH103. Microbiol. 154, 1825–1836 (2008).
Pérezmontaño, F. et al. Nodulation-gene-inducing flavonoids increase overall production of autoinducers and expression of N-acyl homoserine lactone synthesis genes in rhizobia. Res. Microbiol. 162, 715–723 (2011).
Mabood, F., Souleimanov, A., Khan, W. & Smith, D. L. Jasmonates induce nod factor production by Bradyrhizobium japonicum. Plant Physiol. Biochem. 44, 759–765 (2006).
Cooper, J. E. Early interactions between legumes and rhizobia: disclosing complexity in a molecular dialogue. J. Appl. Microbiol. 103, 1355–1365 (2007).
Lee, H. I. et al. Effect of soybean coumestrol on Bradyrhizobium japonicum nodulation ability, biofilm formation, and transcriptional profile. Appl. Environ. Microbiol. 78, 2896–2903 (2012).
Gao, T. G. et al. Nodulation characterization and proteomic profiling of Bradyrhizobium liaoningense CCBAU05525 in response to water-soluble humic materials. Sci. Rep. 5, 10836, doi:10.1038/srep10836 (2015).
Cooper, J. E. Multiple responses of rhizobia to flavonoids during legume root infection. Adv. Bot. Res. 41, 1–62 (2004).
Kaneko, T. et al. Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110 (supplement). DNA. Res. 9, 189–197 (2002).
Kaneko, T. et al. Complete genome sequence of the soybean symbiont Bradyrhizobium japonicum strain USDA6T. Genes 2, 763–787 (2011).
Pessi, G. et al. Genome-wide transcript analysis of Bradyrhizobium japonicum bacteroids in soybean root nodules. Mol. Plant Microbe. In. 20, 1353–1363 (2007).
Chang, W. S. et al. An oligonucleotide microarray resource for transcriptional profiling of Bradyrhizobium japonicum. Mol. Plant Microbe. In. 20, 1298–1307 (2007).
Delmotte, N. et al. An integrated proteomics and transcriptomics reference data set provides new insights into the Bradyrhizobium japonicum, bacteroid metabolism in soybean root nodules. Proteomics 10, 1391–1400 (2010).
Lang, K., Lindemann, A., Hauser, F. & Göttfert, M. The genistein stimulon of Bradyrhizobium japonicum. Mol. Genet. Genomics 279, 203–211 (2008).
Jeon, J. M. et al. Whole-genome expression profiling of Bradyrhizobium japonicum in response to hydrogen peroxide. Mol. Plant Microbe. In. 24, 1472–1481 (2011).
Donati, A. J. et al. Effects of indole-3-acetic acid on the transcriptional activities and stress tolerance of Bradyrhizobium japonicum. Plos One 8, e76559, doi:10.1371/journal.pone.0076559 (2013).
Sanzsáez, Á. et al. Inoculation with an enhanced N2 -fixing Bradyrhizobium japonicum strain (USDA110) does not alter soybean (Glycine max merr.) response to elevated [CO2]. Plant Cell Environ. 38, 2589–2602 (2015).
Stockwell, S. B., Reutimann, L. & Guerinot, M. L. A role for Bradyrhizobium japonicum ECF16 sigma factor EcfS in the formation of a functional symbiosis with soybean. Mol. Plant Microbe. Interact 25, 119–128 (2011).
Naoko, O. O. et al. Growth rate of and gene expression inbradyrhizobium diazoefficiens USDA110 due to a mutation in blr7984, a TetR family transcriptional regulator gene. Microbes. Environ. 31, 249–259 (2016).
Lardi, M. et al. Metabolomic profiling of Bradyrhizobium diazoefficiens-induced root nodules reveals both host plant-specific and developmental signatures. Int. J. Mol. Sci. 17, 815, doi:10.3390/ijms17060815 (2016).
Koch, M. et al. Rhizobial adaptation to hosts, a new facet in the legume root-nodule symbiosis. Mol. Plant Microbe. Interact. 23, 784–790 (2010).
Li, J. et al. Proteomic study on two Bradyrhizobium Japonicum strains with different competitivenesses for nodulation. Agr. Sci. China 10, 1072–1079 (2011).
Morin, R. D. et al. Application of massively parallel sequencing to microrna profiling and discovery in human embryonic stem cells. Genome Res. 18, 610–621 (2008).
Edwards, A. et al. The cin and rai quorum-sensing regulatory systems in Rhizobium leguminosarum are coordinated by ExpR and CinS, a small regulatory protein coexpressed with CinI. J. Bacteriol 191, 3059–3067 (2009).
Batista, J. S. D. S. et al. Proteomics reveals differential expression of proteins related to a variety of metabolic pathways by genistein-induced Bradyrhizobium japonicum strains. J. Proteomics 75, 1211–1219 (2012).
Loh, J. et al. Two-component regulator mediates population-density-dependent expression of the Bradyrhizobium japonicum nodulation genes. J. Bacteriol 184, 1759–1766 (2002).
Wenzel, M. et al. Characterization of the flavonoid-responsive regulator FrrA and its binding sites. J. Bacteriol 194, 2363–2370 (2012).
Vanderlinde, E. M. & Yost, C. K. Mutation of the sensor kinase chvG in Rhizobium leguminosarum negatively impacts cellular metabolism, outer membrane stability, and symbiosis. J. Bacteriol 194, 768–777 (2012).
Fiebig, A. et al. General stress signaling in the Alphaproteobacteria. Annu. Rev. Genet 49, 603–625 (2015).
Kaczmarczyk, A. et al. Complex two-component signaling regulates the general stress response in Alphaproteobacteria. Proc. Natl. Acad. Sci. USA 111, 5196–5204 (2014).
Gourion, B. et al. The PhyR-sigma(EcfG) signalling cascade is involved in stress response and symbiotic efficiency in Bradyrhizobium japonicum. Mol. Microbiol. 73, 291–305 (2009).
Liu, Y. et al. Proteins involved in nodulation competitiveness of two Bradyrhizobium diazoefficiens, strains induced by soybean root exudates. Biol Fert Soils. 51, 251–260 (2015).
Covelli, J. M. et al. Swarming motility in Bradyrhizobium japonicum. Res. Microbiol. 164, 136–144 (2013).
Mongiardini, E. J. Overproduction of the rhizobial adhesin RapA1 increases competitiveness for nodulation. Soil Biol. Biochem. 41, 2017–2020 (2009).
Quelas, J. I. et al. Swimming performance of Bradyrhizobium diazoefficiens is an emergent property of its two flagellar systems. Sci. Rep. 6, 23841, doi:10.1038/srep23841 (2016).
Miller, L. D., Yost, C. K., Hynes, M. F. & Alexandre, G. The major chemotaxis gene cluster of Rhizobium leguminosarum, bv. viciae, is essential for competitive nodulation. Mol. Microbiol. 63, 348–362 (2007).
Davidson, A. L. Structure, function, and evolution of bacterial ATP-binding cassette systems. Microbiol. Mol. Biol. 72, 317–364 (2008).
Sarma, A. D. & Emerich, D. W. A comparative proteomic evaluation of culture grown vs nodule isolated Bradyrhizobium japonicum. Proteomics 6, 3008–3028 (2006).
Cabrera, J. J. et al. An integrated biochemical system for nitrate assimilation and nitric oxide detoxification in Bradyrhizobium japonicum. Biochem. J. 473, 297–309 (2015).
Djordjevic, M. A. Sinorhizobium meliloti metabolism in the root nodule: a proteomic perspective. Proteomics 4, 1859–1872 (2004).
Lodwig, E. M. et al. Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids. Mol. Plant Microbe. 18, 67–74 (2005). In.
Ding, Y. & Oldroyd, G. E. D. Positioning the nodule, the hormone dictum. Plant Signal Behav. 4, 89–93 (2009).
Ryu, H., Cho, H., Choi, D. & Hwang, I. Plant hormonal regulation of nitrogen-fixing nodule organogenesis. Mol. Cells 34, 117–126 (2012).
Mathesius, U. Auxin: at the root of nodule development? Funct. Plant Biol. 35, 651–668 (2008).
Vegahernández, M. et al. Indole-3-acetic acid production from indole-3-acetonitrile by strains of Bradyrhizobium. Soil Biol. Biochem 34, 665–668 (2002).
Boiero, L. et al. Phytohormone production by three strains of Bradyrhizobium japonicum and possible physiological and technological implications. Appl. Microbiol. Biotechnol 74, 874–880 (2007).
Spaepen, S. & Vanderleyden, J. Auxin and plant-microbe interactions. Csh. Perspect Biol. 3, 704–704 (2011).
Patten, C. L., Blakney, A. J. & Coulson, T. J. Activity, distribution and function of indole-3-acetic acid biosynthetic pathways in bacteria. Crit. Rev. Microbiol. 39, 395–415 (2013).
Jiang, G. et al. A functional myo-inositol dehydrogenase gene is required for efficient nitrogen fixation and competitiveness of Sinorhizobium fredii USDA191 to nodulate soybean (Glycine max [L.] Merr.). Appl. Environ. Microbio. 183, 2595–2604 (2001).
Fry, J. et al. Investigation of myo-inositol catabolism in Rhizobium leguminosarum bv. viciae and its effect on nodulation competitiveness. Mol. Plant Microbe. In. 14, 1016–1025 (2001).
Quelas, J. I. et al. Effects of N-starvation and C-source on Bradyrhizobium japonicum exopolysaccharide production and composition, and bacterial infectivity to soybean roots. Arch. Microbiol. 186, 119–128 (2006).
Gao, S. et al. Genomic analysis of cyclic-di-GMP-related genes in rhizobial type strains and functional analysis in Rhizobium etli. Appl. Microbiol. Biotechnol. 98, 4589–4602 (2014).
Phillips, D. A. et al. A new genetic locus in Sinorhizobium meliloti is involved in stachydrine utilization. Appl. Environ. Microbiol. 64, 3954–3960 (1998).
Van, D. P. et al. Construction and environmental release of a Sinorhizobium meliloti strain genetically modified to be more competitive for alfalfa nodulation. Appl. Environ. Microbiol. 67, 3860–3865 (2001).
Sarma, A. D. & Emerich, D. W. Global protein expression pattern of Bradyrhizobium japonicum bacteroids: a prelude to functional proteomics. Proteomics 5, 4170–4784 (2005).
Sarma, A. D. & Emerich, D. W. A comparative proteomic evaluation of culture grown vs nodul nodule isolated Bradyrhizobium japonicum. Proteomics 6, 3008–3028 (2006).
Süß, C. et al. Identification of genistein-inducible and type III secreted proteins of Bradyrhizobium japonicum. J. Biotechnol 126, 69–77 (2006).
Hempel, J. et al. Analysis of the secretome of the soybean symbiont Bradyrhizobium japonicum. J. Bacteriol 140, 51–58 (2009).
Nomura, M. et al. Differential protein profiles of Bradyrhizobium japonicum USDA110 bacteroid during soybean nodule development. Soil Sci Plant Nutr 56, 579–590 (2010).
Beringer, J. E. R factor transfer in Rhizobium leguminosarum. J. Gen. Microbio. 84, 188–198 (1974).
Rigaud, J. & Puppo, A. Indole-3-acetic acid catabolism by soybean bacteroids. J.Gen.Microbiol. 88, 223–228 (1975).
Kaneko, T. et al. Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110 (supplement). DNA. Res. 9, 225–256 (2002).
Yan, Y. et al. Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated Pseudomonas stutzeri A1501. Proc. Natl. Acad. Sci. USA 105, 7564–7569 (2008).
Wu, S. et al. Transcriptome analysis of the signalling networks in coronatine-induced secondary laticifer differentiation from vascular cambia in rubber trees. Sci. Rep. 6, 36384, doi:10.1038/srep36384 (2016).
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No. 41573066), the National Key Basic Research Program of China (973 Program: 2015CB150506), the Special Fund for China Agriculture Research System (CARS-04), and the National High Technology Research and Development Program of China (2013AA102802–04).
Author information
Authors and Affiliations
Contributions
Y.L., X.J., W.Z. and J.L. conceived and designed the study. Y.L. conducted the experiments and wrote the manuscript. D.G. and F.C. participated in the preparation of experimental material. L.L. helped to performe the q.P.C.R. assay. J.L. and B.Z. reviewed the manuscript. All authors read and approved the final manuscript.
Corresponding authors
Ethics declarations
Competing Interests
The authors declare that they have no competing interests.
Additional information
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Liu, Y., Jiang, X., Guan, D. et al. Transcriptional analysis of genes involved in competitive nodulation in Bradyrhizobium diazoefficiens at the presence of soybean root exudates. Sci Rep 7, 10946 (2017). https://doi.org/10.1038/s41598-017-11372-0
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-017-11372-0
- Springer Nature Limited
This article is cited by
-
Identification and functional analysis of recent IS transposition events in rhizobia
Mobile DNA (2024)
-
Mycelial network-mediated rhizobial dispersal enhances legume nodulation
The ISME Journal (2020)
-
Revealing the roles of y4wF and tidC genes in Rhizobium tropici CIAT 899: biosynthesis of indolic compounds and impact on symbiotic properties
Archives of Microbiology (2019)