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.

SummaryThe 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.

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: P. coccineus, P. lunatus, and P. acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and beta-glucuronidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root-microbe interactions.

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.

The NodL and NodJ nodulation proteins have been described in different Rhizobium and Bradyrhizobium species. The nodLJ genes belong to the nod regulon. Other genes from this regulon are involved in the biosynthesis and modification of lipo-oligosaccharide molecule(s) which are morphogenic signals when acting on legume roots. It has been proposed that the NodL and NodJ proteins belong to a bacterial inner membrane transport system of small molecules. Nucleotide sequencing of Mudll PR13 insertions in the nodulation region of the symbiotic plasmid from a Rhizobium leguminosarum bv. phaseoli strain CE3 has revealed the presence of nodL and nodJ-related sequences downstream of nodC. Computer nucleotide sequence analysis of the entire NodL and NodJ sequences from R. leguminosarum bv. viciae and Bradyrhizobium japonicum strains show that both proteins are similar to the KpsT and KpsM proteins from Escherichia coli K1 and K5 strains, to the BexB and BexA proteins from Haemophilis influenzae and to the CtrD and CtrC proteins from Neisseria meningitidis, respectively. Except for the NodL and NodJ proteins, all of them have been involved in the mechanism of secretion of polysaccharides in each of their harbouring species. On the basis of the similarity found, we propose that the NodL and the NodJ proteins could be involved in the export of a lipo-oligosaccharide.