Uncovering Bax inhibitor-1 dual role in the legume-rhizobia symbiosis in common bean roots

Abstract

Bax-inhibitor 1 (BI-1) is a cell death suppressor conserved in all eukaryotes that modulates cell death in response to abiotic stress and pathogen attack in plants. However, little is known about its role in the establishment of symbiotic interactions. Here, we demonstrate the functional relevance of an Arabidopsis thaliana BI-1 homolog (PvBI-1a) to symbiosis between the common bean (Phaseolus vulgaris) and Rhizobium tropici. We show that the changes in expression of PvBI-1a observed during early symbiosis resemble those of some defence response-related proteins. By using gain- and loss-of-function approaches, we demonstrate that the overexpression of PvBI-1a in the roots of common bean increases the number of rhizobial infection events (and therefore the final number of nodules per root), but induces the premature death of nodule cells, affecting their nitrogen fixation efficiency. Nodule morphological alterations are known to be associated with changes in the expression of genes tied to defence, autophagy, and vesicular trafficking. Results obtained in the present work suggest that BI-1 has a dual role in the regulation of programmed cell death during symbiosis, extending our understanding of its critical function in the modulation of host immunity while responding to beneficial microbes.

Fig. 1.

PvBaxI-1a expression is spatiotemporally modulated in common bean–R. tropici early symbiosis. (A) PvBaxI-1a (black line) and PvBaxI-1b (grey line) expression levels during nodulation determined using qPCR and normalized against PvEf1-α expression. The fold change in gene expression was obtained by comparing the expression ratio of each gene with its expression in uninoculated common bean roots. Plotted data are the mean values of transcript accumulation ±SD. The statistical significance was determined using two-way ANOVA (*P<0.005). (B–E) Representative images of the PvBI-1a promoter activity in GUS-stained common bean composite roots. (B, C) Uninoculated (B) or R. tropici inoculated (C) hairy-roots transformed with the pBGWFS7 vector. (D, E) Uninoculated (D) or R. tropici inoculated (E) pPvBI1a:pBGWFS7-transformed hairy-roots. Photographs were taken 2 h after treatment. Root hairs are enclosed in squares. Scale bars, 200 μm.

Fig. 2.

PvBI-1a promoter expression in symbiosis is restricted to ITs, symbiotic cells and vascular bundles of root nodules. (A) R. tropici CIAT899 DsRed migrating through infection threads at 3 dpi. (B) The activity of the PvBI-1a promoter is shown by the expression of GFP. (C) Merged image. (D) R. tropici CIAT899 DsRed in infected cells in an 18 dpi nodule. (E) Activity of the PvBI-1a promoter in this tissue. (F) Merged image. Representative images are shown. Transgenic roots or root nodules were analysed by confocal microscopy. Sixteen 1.89 μm optical sections were taken for each experimental condition. IC, infected cell; IT, infection thread; UC, uninfected cell; VB, vascular bundle.

Fig. 3.

Changes in the expression level of PvBI-1a induce deleterious effects in nitrogen-fixing root nodules. (A) PvBI-1a transcript levels in 18 dpi P. vulgaris root nodules of pEarleyGate103 (negative control), 35S:PvBI-1a, pTdT-Sac-RNAi (negative control), and PvBI-1a-RNAi transformed roots determined by qPCR from three independent (n=3) biological replicates. Three technical repeats were used, and the data were normalized to the expression of PvEf1-α. Plotted data are the mean values of PvBI-1a transcript accumulation ±SD. (B) Total number of nodules formed per root (black columns) and their related nitrogenase activity (grey columns) in P. vulgaris transformed roots. Data are means ±SD from independent composite roots (n=20). Statistical significance in (A, B) was determined using an unpaired t-test followed by Welch’s correction (P<0.0001). (C–F) Optical microscopy of pEarleyGate103 (C), 35S:PvBI-1a (D), pTdT-Sac-RNAi (E), or PvBI-1a-RNAi (F) nodules. Representative images of six independent nodules are shown. C, cortex; IC, infected cells; UC, uninfected cells; VB, vascular bundle. Arrows in (D) indicate areas densely stained with toluidine blue. Insets in (C–F): R. tropici survival determined by colony-forming units (CFU) re-isolated from nodules. Values are means ±SD from nine (n=9) nodules of roots of composite plants, and statistical significance was determined with an unpaired t-test. (*P<0.0001). In all cases, 35S:PvBI-1a and PvBI-1a-RNAi data were normalized to associate values obtained from pEarleyGate103- and pTdT-Sac-RNAi-transformed roots, respectively.

Fig. 4.

The overexpression of PvBI-1a in roots of composite common bean plants increases the frequency of R. tropici infection events through time. (A–D) Curled root hairs of transgenic roots inoculated with R. tropici migrating through ITs at 3 dpi. (A, B) pEarleyGate103- (A) or 35S:PvBI-1a- (B) transformed roots inoculated with R. tropici-DsRed. (C, D) pTdT-Sac-RNAi- (C) or PvBI-1a-RNAi- (D) transformed roots inoculated with R. tropici-GFP. Images were taken using a confocal microscope (LSM510; Carl Zeiss, Oberkochen, Germany). Sixteen 1.89 µm optical sections were taken for each experimental condition. Z-Projected confocal images were generated using Fluoview Viewer (Olympus Corporation, Shinjuku, Tokyo, Japan) and ZEN Black (Carl Zeiss). IT, infection thread. Bars, 20 μm. A′–D′ graphs depicting the number of curled root hairs with R. tropici migrating through ITs at 3, 6, or 9 dpi. Values are means ±SD from five (n=5) independent transgenic roots obtained from distinct plants, and statistical significance was determined with a two-way ANOVA followed by Tukey’s test (*P<0.05).

Fig. 5.

High expression levels of PvBI-1a induce premature senescence in young nodules of common bean. High magnification transmission electron micrographs of 18 dpi 35S:PvBI-1a root nodules inoculated with R. tropici at two magnifications. (A, B) Common bean root nodules transformed with pEarleyGate103. (C, D) 35S:PvBI-1a P. vulgaris root nodules. White arrows indicate the symbiosome frontier. Highlighted in yellow, a symbiotic nodule cell undergoing degradation. B, bacteroid; IC, infected cell; PHB, poly-3-hydroxybutyrate; S, starch grain; UC, uninfected cell.

Fig. 6.

PvBI-1a overexpression induces the expression of defence, autophagy, and membrane trafficking genes during P. vulgaris–R. tropici symbiosis. mRNA levels of defence (A, B), autophagy (C, D), and membrane-trafficking (E, F) genes in 35S:PvBI-1a nodules (B, D, F) and in control nodules (A, C, E) of P. vulgaris composite plants 14–26 dpi with R. tropici, quantified using qPCR. Data from three independent (n=3) biological replicates (each with three technical replicates) were normalized to the expression of PvEf1-α. Plotted data are the mean log₂ values of transcript accumulation ±SD. The statistical significance was determined using two-way ANOVA followed by Sidak’s multiple comparison test (*P<0.05; ***P<0.01; ****P<0.001).