Metallothionein1A Regulates Rhizobial Infection and Nodulation in Phaseolus vulgaris

Abstract

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.

A comprehensive, improved protocol for generating common bean (Phaseolus vulgaris L.) transgenic hairy roots and their use in reverse-genetics studies

“…Using this protocol, transgenic hairy roots can be generated in common bean in approximately 16 days. In our laboratory, we have successfully used this method to obtain hairy roots using A. rhizogenes strains transformed with specific plasmids for overexpression and RNA interference (RNAi)-mediated gene silencing [5,7]. Fig 1 shows the transcript levels of pPLA-IIγ and PvPLDα2 in common bean transgenic hairy roots transformed with the 35S:pPLA-IIγ-EGFP or PvPLDα2-RNAi plasmid, respectively.…”

“…Composite plants are widely used to study the molecular mechanisms underlying plant developmental programs, particularly those regulating symbiosis between common bean roots and arbuscular mycorrhizal fungi or rhizobia [ 2 , 3 , 5 ]. Here, we present an update of the original protocol for generating transgenic hairy roots in common bean.…”

“…This crop species also has agronomic and nutritional values, making it a widely used experimental model to study the mechanisms that regulate these symbiotic interactions. In particular, to better understand the molecular basis of bean-mycorrhiza and bean-rhizobia symbioses, two reverse-genetics approaches are commonly used, gene overexpression and gene silencing, together with promoter activity analysis using reporter genes [2][3][4][5]. These methods require the generation of transgenic hairy roots in wild-type plants (hereafter referred to as composite plants) via Agrobacterium rhizogenes-mediated transformation.…”

Extracellular Self- and Non-Self DNA Involved in Damage Recognition in the Mistletoe Parasitism of Mesquite Trees

“…The method can quantify O 2 •− radicals that are produced in plant tissues under stress and is based on the selective extraction of formazan produced after reduction with nitroblue tetrazolium (NBT) in histochemical staining [ 119 ]. O 2 •− was measured according to Fonseca-García et al [ 120 ], with some modifications. Briefly, 500 µL of 0.1% NBT (Sigma Aldrich, St. Louis, MO, USA) in 50 mM pH 7.5 NaH 2 PO 4 were added to a tube containing 10 pinnae (leaves).…”

“…•− was measured according to Fonseca-García et al [120], with some modifications. Briefly, 500 µL of 0.1% NBT (Sigma Aldrich, St. Louis, MO, USA) in 50 mM pH 7.5 NaH 2 PO 4 were added to a tube containing 10 pinnae (leaves).…”