Noreide Nava

A Phaseolus vulgaris NADPH Oxidase Gene is Required for Root Infection by Rhizobia

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.

RbohB, a Phaseolus vulgaris NADPH oxidase gene, enhances symbiosome number, bacteroid size, and nitrogen fixation in nodules and impairs mycorrhizal colonization

The reactive oxygen species (ROS) generated by respiratory burst oxidative homologs (Rbohs) are involved in numerous plant cell signaling processes, and have critical roles in the symbiosis between legumes and nitrogen-fixing bacteria. Previously, down-regulation of RbohB in Phaseolus vulgaris was shown to suppress ROS production and abolish Rhizobium infection thread (IT) progression, but also to enhance arbuscular mycorrhizal fungal (AMF) colonization. Thus, Rbohs function both as positive and negative regulators. Here, we assessed the effect of enhancing ROS concentrations, by overexpressing PvRbohB, on the P. vulgaris--rhizobia and P. vulgaris--AMF symbioses. We estimated superoxide concentrations in hairy roots overexpressing PvRbohB, determined the status of early and late events of both Rhizobium and AMF interactions in symbiont-inoculated roots, and analyzed the nodule ultrastructure of transgenic plants overexpressing PvRbohB. Overexpression of PvRbohB significantly enhanced ROS production, the formation of ITs, nodule biomass, and nitrogen-fixing activity, and increased the density of symbiosomes in nodules, and the density and size of bacteroides in symbiosomes. Furthermore, PvCAT, early nodulin, PvSS1, and PvGOGAT transcript abundances were elevated in these nodules. By contrast, mycorrhizal colonization was reduced in roots that overexpressed RbohB. Overexpression of PvRbohB augmented nodule efficiency by enhancing nitrogen fixation and delaying nodule senescence, but impaired AMF colonization.

The role of Nod factor substituents in actin cytoskeleton rearrangements in Phaseolus vulgaris

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.

PvRbohB negatively regulates Rhizophagus irregularis colonization in Phaseolus vulgaris

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.

Down‐regulation of SymRK correlates with a deficiency in vascular bundle development in Phaseolus vulgaris nodules

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.

A Rhizobium tropici DNA region carrying the amino-terminal half of a nodD gene and a nod-box-like sequence confers host-range extension

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.

Nucleotide sequence of the Rhizobium etli nodS gene

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.

Early responses to Nod factors and mycorrhizal colonization in a non-nodulating Phaseolus vulgaris mutant

Legumes can acquire nitrogen through a symbiotic interaction with rhizobial bacteria. The initiation of this process is determined by a molecular dialogue between the two partners. Legume roots exude flavonoids that induce the expression of the bacterial nodulation 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 Ca2+ signaling and early nodulin induction. This supports the idea that both microsymbionts are perceived and trigger different downstream pathways in the host plant.

Sulfation of Nod Factors via nodHPQ Is nodD Independent in Rhizobium tropici CIAT899

A cosmid from the Rhizobium tropici CIAT899 symbiotic plasmid, containing most of the nodulation genes described in this strain, has been isolated. Although this cosmid does not carry a nodD gene, it confers ability to heterologous Rhizobium spp. to nodulate R. tropici hosts (Phaseolus vulgaris, Macroptilium atropurpureum, and Leucaena leucocephala). The observed phenotype is due to constitutive expression of the nodABCSUIJ operon, which has lost its regulatory region and is expressed from a promoter present in the cloning vector. Thin-layer chromatography (TLC) analysis of the Nod factors produced by this construction shows that it is still capable of synthesizing sulfated compounds, suggesting that the nodHPQ genes are organized as an operon that is transcribed in a nodD-independent manner and is not regulated by flavonoids.

Diminished Redundancy of Outer Membrane Factor Proteins in Rhizobiales: A nodT Homolog Is Essential for Free-Living Rhizobium etli

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.