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.

Keywords: common bean; metallothionein; nodule symbiosis; reactive oxygen species; rhizobial infection.

Figure 1

Evolutionary relationships among MTs. Rooted approximately maximum-likelihood phylogenetic tree inferred from 77 MTs identified in 12 plant species: P. vulgaris (Ph), G. max (Gm), L. japonicus (Lj), M. truncatula (Mt), A. thaliana (At), V. vinifera (Vv), A. hypochondriacus (Ah), P. trichocarpa (Pt), O. sativa (Os), S. bicolor (Sb), Z. mays (Zm), and H. vulgare (Hv). The clades are shown in different colors according to the MT classes: MT1, pink; MT2, green; MT3, yellow; MT4, brown. A sequence from Saccharomyces cerevisiae was used as the outgroup. The phylogenetic tree was constructed using IQ-TREE software with the Dayhoff substitution model with 1000 bootstrap iterations.

Figure 2

Expression profile analysis of PvMT1A. (A) Relative PvMT1A expression in P. vulgaris roots at 5, 7, 14, 21, and 30 dpi with R. tropici CIAT899 evaluated by qPCR. The elongation factor EF1α and IDE genes were used as endogenous reference genes to normalize expression levels. The blue bars represent non-inoculated roots, and the red bars represent roots inoculated with R. tropici at the indicated times. The top and bottom edges of the boxes delineate the first to third quartiles, the horizontal line within the box represents the median, and the whiskers indicate the smallest and largest outlier in the data set (n = 9). A non-parametric Mann–Whitney test was performed to evaluate significant differences, * p ≤ 0.05, *** p ≤ 0.001, and ns = no significant difference. (B–F) Promoter activity of PvMT1A visualized by GUS staining in non-inoculated (B) or inoculated (C–F) roots carrying pPvMT1A::GFP:GUS. (B) non-infected root hair, (C) curled root hair after rhizobial infection at 7 dpi, (D) nodule primordium at 30 dpi, (E) young nodule at 30 dpi, (F) mature nodule at 30 dpi. r, rhizodermis; c, cortex; bt, bacteroid tissue.

Figure 3

Subcellular localization of PvMT1A. The localization of the PvMT1A protein was monitored in N. benthamiana leaves infiltrated with the 35S::YFP construct as a control (A) or with 35S::YFP:PvMT1A (B) by means of confocal microscope.

Figure 4

Analysis of infection events in control and PvMT1A-silenced transgenic roots detected by GUS staining. Representative images of infection events in control (A) and PvMT1A:RNAi C4 (B) transgenic roots inoculated at 7 dpi with R. tropici-GUS. Close-up of the marked area of panels (A,B) of root hair zones showing infection events in control (C) and PvMT1A:RNAi C4 (D) transgenic roots, respectively; IT of control (E) and PvMT1A:RNAi C4 (F). Cell divisions and IT invasion into cortical layers are shown with an asterisk and an arrow, respectively. The average number of total infection events was scored in control, PvMT1A:RNAi C4, and PvMT1A:RNAi C5 roots at 7 dpi (G) and 10 dpi (H) with R. tropici-GUS. The top and bottom edges of the boxes delineate the first to third quartiles, the horizontal line within the box represents the median, and the whiskers represent 10th and 90th percentiles in the data set (n = 15). A non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparisons test was performed to evaluate significant differences *** p ≤ 0.001. Percentage of IT in control and PvMT1A:RNAi observed in root hairs and cortical cells (I). In (C–F), c, cortex; r, rhizodermis, and rs: root stele.

Figure 5

Measurement of nodulation capacity in control and PvMT1A-silenced transgenic roots. Total number of nodules in control, PvMT1A:RNAi C4, and PvMT1A:RNAi C5 hairy roots at 14 and 21 dpi (A). Nitrogenase activity in control, PvMT1A:RNAi C4, and PvMT1A:RNAi C5 transgenic roots inoculated with R. tropici at 21 dpi, as determined by an acetylene reduction assay (B). The top and bottom edges of the boxes delineate the first to third quartiles, the horizontal line within the box represents the median, and the whiskers represent the smallest and largest outlier in the data set (n = 30). A non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparisons test was performed to evaluate significant differences, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 6

Expression levels of ROS gene markers and ROS production analysis in control and PvMT1A:RNAi transgenic roots. Relative expression profiles of PvSOD (A), PvCAT (B), and PvAPX (C) in control and PvMT1A:RNAi transgenic roots at 7 dpi with R. tropici evaluated by qPCR. The elongation factor EF1α and IDE genes were used as endogenous reference genes to normalize expression levels. The top and bottom edges of the boxes delineate the first to the third quartiles, the horizontal line within the box represents the median, and the whiskers represent the smallest and largest outlier in the data set (n = 9). (A) Non-parametric Mann–Whitney test was performed to evaluate significant differences, * p ≤ 0.05, ** p ≤ 0.01, and ns = no significant difference. In panels (D−O), the visualization of O₂⁻ and H₂O₂ in representative roots at 14 dpi with R. tropici CIAT899 by NBT and DAB staining are shown, respectively. The blue color indicates the presence of O₂⁻ in the tissue, and the brown color indicates the presence of H₂O₂ in the tissues. Typical NBT-stained nodules of control transgenic roots (D), PvMT1A:RNAi C4 (E), and PvMT1A:RNAi C5 (F). Representative samples of control transgenic roots (G), PvMT1A:RNAiC4 (H), and PvMT1A:RNAiC5 (I) stained with NBT. DAB staining of representative early nodule primordia of the control (J), PvMT1A:RNAi C4 (K), and PvMT1A:RNAi C5 (L). Arrows in (J–L) indicate the presence of H₂O₂ in the nodule primordia. Typical nodules of control (M), PvMT1A:RNAi C4 (N), and PvMT1A:RNAi C5 (O) transgenic roots stained with DAB.