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Background Rhizobium–legume symbiosis is a specific, coordinated interaction that results in the formation of a root nodule, where biological nitrogen fixation occurs. NADPH oxidases, or Respiratory Burst Oxidase Homologs (RBOHs) in plants, are enzymes that generate superoxide (O2•−). Superoxide produces other reactive oxygen species (ROS); these ROS regulate different stages of mutualistic interactions. For example, changes in ROS levels are thought to induce ROS scavenging, cell wall remodeling, and changes in phytohormone homeostasis during symbiotic interactions. In common bean (Phaseolus vulgaris), PvRbohB plays a key role in the early stages of nodulation. Results In this study, to explore the role of PvRbohB in root nodule symbiosis, we analyzed transcriptomic data from the roots of common bean under control conditions (transgenic roots without construction) and roots with downregulated expression of PvRbohB (by RNA interference) non-inoculated and inoculated with R. tropici. Our results suggest that ROS produced by PvRBOHB play a central role in infection thread formation and nodule organogenesis through crosstalk with flavonoids, carbon metabolism, cell cycle regulation, and the plant hormones auxin and cytokinin during the early stages of this process. Conclusions Our findings provide important insight into the multiple roles of ROS in regulating rhizobia–legume symbiosis.

Metallothioneins (MTs) constitute a heterogeneous family of ubiquitous metal ion-binding proteins. In plants, MTs participate in the regulation of cell growth and proliferation, protection against heavy metal stress, oxidative stress responses, and responses to pathogen attack. Despite their wide variety of functions, the role of MTs in symbiotic associations, specifically nodule-fabacean symbiosis, is poorly understood. Here, we analyzed the role of the

The plant receptor-like-kinase subfamily CrRLK1L has been widely studied, and CrRLK1Ls have been described as crucial regulators in many processes in Arabidopsis thaliana (L.), Heynh. Little is known, however, about the functions of these proteins in other plant species, including potential roles in symbiotic nodulation. We performed a phylogenetic analysis of CrRLK1L subfamily receptors of 57 different plant species and identified 1050 CrRLK1L proteins, clustered into 11 clades. This analysis revealed that the CrRLK1L subfamily probably arose in plants during the transition from chlorophytes to embryophytes and has undergone several duplication events during its evolution. Among the CrRLK1Ls of legumes and A. thaliana, protein structure, gene structure, and expression patterns were highly conserved. Some legume CrRLK1L genes were active in nodules. A detailed analysis of eight nodule-expressed genes in Phaseolus vulgaris L. showed that these genes were differentially expressed in roots at different stages of the symbiotic process. These data suggest that CrRLK1Ls are both conserved and underwent diversification in a wide group of plants, and shed light on the roles of these genes in legume&ndash, rhizobia symbiosis.

Legumes establish a symbiotic relationship with N2-fixing soil bacteria known as rhizobia. This process is initiated by a molecular dialog between roots and bacteria. Rhizobia secrete lipochitooligosaccharide signals, known as Nod factors, into the rhizosphere that are perceived by root hairs and induce an increase in Ca2+ levels, ROS production, and transcriptional activation of nodulin genes. Plant annexins are a family of calcium- and membrane-binding proteins involved in growth and development and in several abiotic and biotic responses. In this study, we identified thirteen annexin genes in the Phaseolus vulgaris (common bean) genome. We show that PvAnn1 transcript abundance was higher in root hairs than in root apices and stripped roots, and that during symbiosis, PvAnn1 transcript abundance was substantially higher in nodules than in nodule-stripped roots. RNAi knock-down of PvAnn1 resulted in reduced root hair lengths and ROS production. Furthermore, PvAnn1-RNAi transgenic roots that were inoculated with rhizobia showed a decrease in the progression and number of infection threads, possibly due to changes in Ca2+-ROS homeostasis and nodulin signaling. Furthermore, a reduced number of nodules and less nitrogen fixation was observed in the transgenic plants at 21 days post infection. Together, these data suggest that PvAnn1 is involved in rhizobial infection and nodule development and function.

Rice is the principle staple food for more than half of humankind. Frequently, productivity of rice is affected by low nitrogen in the soil and hence, for enhanced rice production it heavily relies on synthetic nitrogen fertilizers that beget economic and ecological costs. In this context, the interest in transferring legume-like biological nitrogen fixation capability to rice has increased lately. The rice-arbuscular mycorrhizal (AM) symbiosis is mediated by genes that are orthologous to legume-genes known to be essential constituents of the common symbiotic pathway (CSP) that facilitates the establishment of both rhizobial nitrogen fixation- and AM-symbioses in legumes. Particularly, DMI3 (Ca+2/calmodulin-dependent serine/threonine protein kinase, CCaMK), a component of the CSP, was found to play a paramount role in promoting the development of both types of symbioses. In fact, expression of autoactive version of DMI3 was shown to be sufficient to trigger downstream developmental processes leading to the induction of spontaneous nodulation in the absence of rhizobia. Hence, in the present investigation, we expressed in transgenic rice a gain-of-function Medicago truncatula DMI3 T271D gene (gofMtDMI3) and assessed if legume-like symbiotic responses can be mimicked in rice roots. Ectopic expression of gofMtDMI3 in common bean induced spontaneous nodulation in the roots in the absence of rhizobia, but in rice plants it did not produce any such legume-like nodular manifestations. Conversely, the expression of gofMtDMI3 supported elevated AM colonization in rice roots that could improve plant nutrition/growth. In addition, gofMtDMI3 expression induced higher transcript levels of the CSP orthologues OsDMI3, OsIPD3 and OsNSP1, as well as triggered changes in the expression of several genes involved in biotic and abiotic stress responses. Our results with gofMtDMI3 lay the basis for the potential development of a biotechnological approach towards improvement of rice production.

BackgroundReactive oxygen species (ROS) are generated by NADPH oxidases known as respiratory burst oxidase homologs (RBOHs) in plants. ROS regulate various cellular processes, including the mutualistic interactions between legumes and nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi.Rbohis a multigene family comprising nine members (RbohA–I) in common bean (Phaseolus vulgaris).The RNA interference-mediated silencing ofRbohB(PvRbohB-RNAi) in this species diminished its ROS production and greatly impaired nodulation. By contrast, thePvRbohB-RNAi transgenic roots showed early hyphal root colonization with enlarged fungal hypopodia; therefore, we proposed thatPvRbohBpositively regulates rhizobial infection (Rhizobium tropici) and inhibits AM colonization byRhizophagus irregularisinP. vulgaris.ResultsTo corroborate this hypothesis, an RNA-Seq transcriptomic analysis was performed to identify the differentially expressed genes in thePvRbohB-RNAi roots inoculated withRhizobium tropiciorRhizophagus irregularis. We found that, in the early stages, root nodule symbioses generated larger changes of the transcriptome than did AM symbioses inP. vulgaris. Genes related to ROS homeostasis and cell wall flexibility were markedly upregulated in the early stages of rhizobial colonization, but not during AM colonization. Compared with AM colonization, the rhizobia induced the expression of a greater number of genes encoding enzymes involved in the metabolism of auxins, cytokinins, and ethylene, which were typically repressed in thePvRbohB-RNAi roots.ConclusionsOur research provides substantial insights into the genetic interaction networks in the early stages of rhizobia and AM symbioses withP. vulgaris, as well as the differential roles thatRbohBplays in processes related to ROS scavenging, cell wall remodeling, and phytohormone homeostasis during nodulation and mycorrhization in this legume.

Additional file 11: Figure S8. KEGG pathway map of plant hormone signal transduction for upregulated genes in mycorrhized control roots.

Reactive oxygen species (ROS) produced by respiratory burst oxidase homologs (RBOHs) regulate numerous plant cell processes, including the symbiosis between legumes and nitrogen-fixing bacteria. Rapid and transient ROS production was reported after Phaseolus vulgaris root hairs were treated with Nod factors, indicating the presence of a ROS-associated molecular signature in the symbiosis signaling pathway. Rboh is a multigene family containing nine members (RbohA-I) in P. vulgaris. RNA interference of RbohB suppresses ROS production and attenuates rhizobial infection thread (IT) progression in P. vulgaris root hairs. However, the roles of other Rboh members in symbiotic interactions are largely unknown. In this study, we characterized the role of the NADPH oxidase-encoding gene RbohA (Phvulv091020621) in the P. vulgaris-Rhizobium tropici symbiosis. The spatiotemporal activity of the RbohA promoter colocalized with growing ITs and was associated with vascular bundles in developing nodules. Subcellular localization studies indicated that RBOHA was localized in the plasma membrane of P. vulgaris root hairs. After rhizobial inoculation, PvRBOHA was mainly distributed in the infection pocket and, to a lesser extent, throughout the IT. In PvRbohA RNAi lines, the rhizobial infection events were significantly reduced and, in successful infections, IT progression was arrested within the root hair, but did not impede cortical cell division. PvRbohA-RNAi nodules failed to fix nitrogen, since the infected cells in the few nodules formed were empty. RbohA-dependent ROS production and upregulation of several antioxidant enzymes was attenuated in rhizobia-inoculated PvRbohA-RNAi roots. These combined results indicate that PvRbohA is crucial for effective Rhizobium infection and its release into the nodule cells. This oxidase is partially or indirectly required to promote nodule organogenesis, altering the expression of auxin- and cyclin-related genes and genes involved in cell growth and division.

The symbiotic interaction of legumes and rhizobia results in the formation of nitrogen-fixing nodules. Nodulation depends on the finely coordinated expression of a battery of genes involved in the infection and the organogenesis processes. After Nod factor perception, symbiosis receptor kinase (SymRK) receptor triggers a signal transduction cascade essential for nodulation leading to cortical cell divisions, infection thread (IT) formation and final release of rhizobia to the intracellular space, forming the symbiosome. Herein, the participation of SymRK receptor during the nodule organogenesis in Phaseolus vulgaris is addressed. Our findings indicate that besides its expression in the nodule epidermis, in IT, and in uninfected cells of the infection zone, PvSymRK immunolocalizes in the root and nodule vascular system. On the other hand, knockdown expression of PvSymRK led to the formation of scarce and defective nodules, which presented alterations in both IT/symbiosome formation and vascular system.

Rhizobium etli is a gram-negative soil bacterium that induces nitrogen-fixing nodules on common bean roots (Phaseolus vulgaris). R. etli encodes two genes homologous to nodT of Rhizobium leguminosarum. nodTch is chromosomal and forms an operon with new genes resembling a multi-drug efflux pump of the resistance-nodulation-cell division (RND) family. nodTch is the last gene of this operon and can also be independently transcribed; the gene product is located in the bacterial outer membrane. Cell survival requires nodTch under all conditions tested. A second nodT gene, nodTpc, is encoded by plasmid c; it is constitutively transcribed but does not complement the essential function encoded by nodTch. NodT proteins belong to the outer membrane efflux proteins of the TolC superfamily. The number of duplications in the tolC gene family positively correlates with genome size in gram-negative bacteria. Nonetheless, some α-proteobacteria, including R. etli, encode fewer outer membrane factor exporters than expected suggesting further roles in addition to detoxification.

In order to define the symbiotic role of some of the chemical substituents in the Rhizobium etli Nod factors (NFs), we purified Nod metabolites secreted by the SM25 strain, which carries most of the nodulation genes, and SM17 with an insertion in nodS. These NFs were analyzed for their capabilities to induce root hair curling and cytoskeletal rearrangements. The NFs secreted by strain SM17 lack the carbamoyl and methyl substituents on the nonreducing terminal residue and an acetyl moiety on the fucosyl residue on the reducing-terminal residue as determined by mass spectrometry. We have reported previously that the root hair cell actin cytoskeleton from bean responds with a rapid fragmentation of the actin bundles within 5 min of NF exposure, and also is accompanied by increases in the apical influxes and intracellular calcium levels. In this article, we report that methyl-bearing NFs are more active in inducing root hair curling and actin cytoskeleton rearrangements than nonmethylated NFs. However, the carbamoyl residue on the nonreducing terminal residue and the acetyl group at the fucosyl residue on the reducing terminal residue do not seem to have any effect on root hair curling induction or in actin cytoskeleton rearrangement.

Plant NADPH oxidases (RBOHs) regulate the early stages of rhizobial infection in Phaseolus vulgaris and affect nodule function in Medicago truncatula. In contrast, the role of RBOHs in the plant-arbuscular mycorrhizal (AM) symbiosis and in the regulation of reactive oxygen species (ROS) production during the establishment of the AM interaction is largely unknown. In this study, we assessed the role of P. vulgaris Rboh (PvRbohB) during the symbiosis with the AM fungus, Rhizophagus irregularis. Our results indicate that the PvRbohB transcript is significantly up-regulated in the mycorrhized roots of P. vulgaris. Further, the PvRbohB promoter was found to be active during the invasion of R. irregularis. Down-regulation of PvRbohB transcription by RNAi (RNA interference) silencing resulted in diminished ROS levels in the transgenic mycorrhized roots and induced early hyphal root colonization. Interestingly, the size of appressoria increased in PvRbohB-RNAi roots (760 ± 70.1 µm) relative to controls (251 ± 73.2 µm). Finally, the overall level of mycorrhizal colonization significantly increased in PvRbohB-RNAi roots [48.1 ± 3.3% root length colonization (RLC)] compared with controls (29.4 ± 1.9% RLC). We propose that PvRbohB negatively regulates AM colonization in P. vulgaris.

Plant NADPH oxidases [respiratory burst oxidase homologs (RBOHs)] have emerged as key players in the regulation of plant-pathogen interactions. Nonetheless, their role in mutualistic associations, such as the rhizobia-legume symbiosis, is poorly understood. In this work, nine members of the Phaseolus vulgaris Rboh gene family were identified. The transcript of one of these, PvRbohB, accumulated abundantly in shoots, roots and nodules. PvRbohB promoter activity was detected in meristematic regions of P. vulgaris roots, as well as during infection thread (IT) progression and nodule development. RNA interference (RNAi)-mediated PvRbohB down-regulation in transgenic roots reduced reactive oxygen species (ROS) production and lateral root density, and greatly impaired nodulation. Microscopy analysis revealed that progression of the ITs was impeded at the base of root hairs in PvRbohB-RNAi roots. Furthermore, the few nodules that formed in PvRbohB-down-regulated roots displayed abnormally wide ITs and reduced nitrogen fixation. These findings indicate that this common bean NADPH oxidase is crucial for successful rhizobial colonization and probably maintains proper IT growth and shape.

The symbiotic interaction of legumes and rhizobia results in the formation of nitrogen-fixing nodules. Nodulation depends on the finely coordinated expression of a battery of genes involved in the infection and the organogenesis processes. After Nod factor perception, symbiosis receptor kinase (SymRK) receptor triggers a signal transduction cascade essential for nodulation leading to cortical cell divisions, infection thread (IT) formation and final release of rhizobia to the intracellular space, forming the symbiosome. Herein, the participation of SymRK receptor during the nodule organogenesis in Phaseolus vulgaris is addressed. Our findings indicate that besides its expression in the nodule epidermis, in IT, and in uninfected cells of the infection zone, PvSymRK immunolocalizes in the root and nodule vascular system. On the other hand, knockdown expression of PvSymRK led to the formation of scarce and defective nodules, which presented alterations in both IT/symbiosome formation and vascular system.

Rhizobium etli is a gram-negative soil bacterium that induces nitrogen-fixing nodules on common bean roots (Phaseolus vulgaris). R. etli encodes two genes homologous to nodT of Rhizobium leguminosarum. nodTch is chromosomal and forms an operon with new genes resembling a multi-drug efflux pump of the resistance-nodulation-cell division (RND) family. nodTch is the last gene of this operon and can also be independently transcribed; the gene product is located in the bacterial outer membrane. Cell survival requires nodTch under all conditions tested. A second nodT gene, nodTpc, is encoded by plasmid c; it is constitutively transcribed but does not complement the essential function encoded by nodTch. NodT proteins belong to the outer membrane efflux proteins of the TolC superfamily. The number of duplications in the tolC gene family positively correlates with genome size in gram-negative bacteria. Nonetheless, some alpha-proteobacteria, including R. etli, encode fewer outer membrane factor exporters than expected suggesting further roles in addition to detoxification.

Legumes can acquire nitrogen through a sym- biotic interaction with rhizobial bacteria. The initiation of this process is determined by a molecular dialogue between the two partners. Legume roots exude flavo- noids that induce the expression of the bacterial nodu- lation genes, which encode proteins involved in the synthesis and secretion of signals called Nod factors (NFs). NFs signal back to the plant root and trigger several responses, leading to bacterial invasion and nodule formation. Here, we describe the molecular and cellular characterization of a Phaseolus vulgaris non- nodulating mutant (NN-mutant). Root hair cells of the NN-mutant plant respond with swelling and branching when inoculated with Rhizobium etli, albeit without curling induction. Furthermore, neither initiation of cell division in the outer cortex, nor entrapment of bacteria nor infection thread formation was observed. Both the bean wild-type and the NN-mutant responded with elevated intracellular calcium changes in the root hairs. Although the NN-mutant is deficient in early nodulin gene expression when inoculated with R. etli, it can be effectively colonized by arbuscular mycorrhizal fungi (Glomus intraradices). Our data indicate that the P. vulgaris NN-mutant is not blocked at the NFs early perception stage, but at later downstream stages between Ca 2+ signaling and early nodulin induction. This sup- ports the idea that both microsymbionts are perceived and trigger different downstream pathways in the host plant.

Summary Rhizobium tropici CIAT899 is a broad-host-range strain that, in addition to Phaseolus, nodulates other plant legumes such as Leucaena and Macroptilium. The narrow-host-range of Rhizobium leguminosarum biovars phaseoli (strain CE3) and trifolii (strain RS1051) can be extended to Leucaena esculents and Phaseolus vulgaris plants, respectively, by the introduction of a DNA fragment 521 bp long, which carries 128 amino acids of the amino-terminal region of a nodD gene from R. tropici, as well as a putative nod-box-like sequence, divergently oriented. The 521 bp fragment, in the presence of L. esculenta or P. vulgaris root exudates, induced a R. leguminosarum bv. viciae nodA-lacZ fusion in either a CE3 or RS1051 background, respectively.

The complete nucleotide sequence of the nodS gene from the bean-nodulating Rhizobium etli, presumably encoding a methyltransferase, was determined. A phylogenetic analysis of five different NodS proteins from three genera of Gram ~ soil bacteria, Azorhizobium, Bradyrhizobium and Rhizobium, was performed.