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–rhizobia 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 PvMT1A gene in Phaseolus vulgaris-Rhizobium tropici symbiosis using bioinformatics and reverse genetics approaches. Using in silico analysis, we identified six genes encoding MTs in P. vulgaris, which were clustered into three of the four classes described in plants. PvMT1A transcript levels were significantly higher in roots inoculated with R. tropici at 7 and 30 days post inoculation (dpi) than in non-inoculated roots. Functional analysis showed that downregulating PvMT1A by RNA interference (RNAi) reduced the number of infection events at 7 and 10 dpi and the number of nodules at 14 and 21 dpi. In addition, nodule development was negatively affected in PvMT1A:RNAi transgenic roots, and these nodules displayed a reduced nitrogen fixation rate at 21 dpi. These results strongly suggest that PvMT1A plays an important role in the infection process and nodule development in P. vulgaris during rhizobial symbiosis.

The common bean (Phaseolus vulgaris L.) is one of the legumes used to study the molecular mechanisms that regulate mycorrhizal and rhizobial symbioses. To study these mechanisms, the generation of transgenic hairy roots is a very advantageous method for applying genetic approaches. However, the generation of hairy roots is a difficult task that requires a lot of skill and experience. Here we show a new version of an optimized protocol for the generation of hairy roots in common bean. This version includes videos showing the procedure for preparing the Agrobacterium rhizogenes culture and the infection procedure, which were not included in the original version.

The common bean (Phaseolus vulgaris L.) is one of the legumes used to study the molecular mechanisms that regulate mycorrhizal and rhizobial symbioses. To study these mechanisms, the generation of transgenic hairy roots is a very advantageous method for applying genetic approaches. However, the generation of hairy roots is a difficult task that requires a lot of skill and experience. Here we show a new version of an optimized protocol for the generation of hairy roots in common bean. This version includes photos and videos showing the procedure for preparing the Agrobacterium rhizogenes culture and the infection procedure, which were not included in the original version. Additionally, we included a step for growing hairy roots under hydroponic conditions.