Journal of Experimental Botany, Vol. 70, No. 1 pp.

1049–1061, 2019
doi:10.1093/jxb/ery417 Advance Access Publication 21 November 2018
This paper is available online free of all access charges (see https://academic.oup.com/jxb/pages/openaccess for further details)

RESEARCH PAPER

Uncovering Bax inhibitor-1 dual role in the legume–rhizobia

symbiosis in common bean roots
Alejandrina Hernández-López, Mauricio Díaz, Jonathan Rodríguez-López, Gabriel Guillén,
Federico Sánchez* and Claudia Díaz-Camino†,

Downloaded from https://academic.oup.com/jxb/article-abstract/70/3/1049/5194635 by Pfizer Inc., user on 31 March 2020

Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida
Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, Mexico
†
Correspondence: claudia@ibt.unam.mx
* In loving memory.

Received 20 June 2018; Editorial decision 13 November 2018; Accepted 13 November 2018

Editor: Peter Bozhkov, Swedish University of Agricultural Sciences, Sweden

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 symbi-
otic 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 morphologi-
cal 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 pro-
grammed cell death during symbiosis, extending our understanding of its critical function in the modulation of host
immunity while responding to beneficial microbes.

Keywords: Bax inhibitor-1, hypersensitive response, legume–rhizobia symbiosis, Phaseolus vulgaris, plant immune response,
plant programmed cell death, plant autophagy, rhizobial endosymbiosis.

Introduction
Common bean (Phaseolus vulgaris L.) seeds are a major source
of carbohydrates, protein and essential micronutrients for pop-
ulations in eastern Africa and Latin America (Namugwanya
et al., 2014). As a legume, common bean can enter into a
mutualistic relationship with nitrogen-fixing rhizobacteria.
This interaction results in the formation of a new organ in the
root, the nodule, where rhizobia convert atmospheric nitrogen
into ammonia to provide organic nitrogenous compounds to
the plant.
Legume–rhizobia symbiosis is initiated under soil nitrogen-
limiting conditions, when legumes attract host-specific rhizo-
bial strains by the production of phenolic compounds and their
exudation to the rhizosphere.These compounds prompt rhizo-
bia to release nodulation factors (NFs) and attach to the surface
of root hairs in active growth. After contact, the symbiotic sig-
nalling pathway is activated, inducing developmental responses
such as division of pericycle and cortical cells to form the
nodule primordium as well as major deformations of root hair

Abbreviations: BI-1, Bax inhibitor-1; dpi, days post-inoculation; ER, endoplasmic reticulum; hpi, hours post-inoculation; IT, infection thread; NF, rhizobial nod factor;
PCD, programmed cell death.
© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
1050 | Hernández-López et al.
extension, leading to bacterial invasion (Ferguson et al., 2010;
Oldroyd, 2013; Udvardi and Poole, 2013; Downie, 2014). The
root rhizobial infection process occurs by the formation of a
host-derived inwardly growing tubular compartment, known
as the infection thread (IT), which guides the bacteria toward
nodule primordium cells (Fournier et al., 2015). Rhizobia are
released from the ITs into the cytoplasm of developing nodule
cells by an exocytosis-like pathway (Ivanov et al., 2012), where
they remain surrounded by a membrane called the symbio-
some membrane. Within this membrane, rhizobia differentiate
into bacteroids able to fix nitrogen. Bacterial differentiation is
concomitant with host cell enlargement coupled to repeated
endoreduplication cycles, which result in large polyploid
cells housing thousands of bacteroids (Cermola et al., 2000;
Kondorosi et al., 2000; Jones et al., 2007).
A growing body of evidence suggests that a balanced regu-
lation of the plant’s innate immunity is required through-
out rhizobial infection, symbiotic accommodation into the
nodule cell, and maintenance (Kouchi et al., 2004; Lohar
et al., 2006; Brechenmacher et al., 2008; Libault et al., 2010;
Berrabah et al., 2015; Gourion et al., 2015; Cao et al., 2017;
Zipfel and Oldroyd, 2017). Therefore, investigating the func-
tion of genes traditionally related to defence responses in
legume–rhizobia symbiosis could improve our knowledge of
legume biology.
Bax inhibitor I (BI-1) is an evolutionarily conserved trans-
membrane protein predominantly localized in the endo-
plasmic reticulum (ER) (Ishikawa et al., 2011). BI-1 activity
has been associated to the suppression of the hypersensitive
response (HR), a well-characterized form of cell death that
occurs during the plant immune response (Iwata and Koizumi,
2005;Watanabe and Lam, 2008, 2009). By adjusting the steady-
state level of Ca2+ in the ER during stressful conditions (Kim
et al., 2008), BI-1 also promotes autophagy, a self-degrading
cellular process with adaptive functions (Castillo et al., 2011;
Xu et al., 2017).
Several plant transgenic approaches have shown that the
loss of BI-1 function results in a severe programmed cell
death (PCD) phenotype under abiotic stress (Watanabe and
Lam, 2008, 2009; Duan et al., 2010), whereas BI-1 overexpres-
sion attenuates plant PCD caused by the attack of pathogens
(Matsumura et al., 2003; Kawai-Yamada et al., 2004; Babaeizad
et al., 2008; Watanabe and Lam, 2008, 2009; Isbat et al., 2009;
Ishikawa et al., 2010). However, little is known about its role in
symbiotic interactions. In the present study, we explore the bio-
logical function of a BI-1 homolog (PvBI-1a) in the symbiosis
between the common bean and Rhizobium tropici. PvBI-1a is
transiently induced in the plant root after contact with rhizo-
bia. Overexpression of PvBI-1a in roots of composite common
bean plants promotes rhizobial infection during early symbio-
sis, but induces the premature death of symbiotic nodule cells,
thus diminishing the nitrogen-fixing ability of young nodules.
At the molecular level, the overexpression of PvBI-1a affects
the expression of defence, autophagy, and vesicular traffick-
ing machinery genes; these cellular processes are known to be
essential for the success of diverse plant–microbe interactions
(Yuk et al., 2012; Lamb et al., 2013; Stolz et al., 2014; Estrada-
Navarrete et al., 2016).
Materials and methods
Bacteria and plant material
Escherichia coli DH5α and Agrobacterium rhizogenes K599 (Bond and
Gresshoff, 1993) were grown at 37 °C and 30 °C, respectively, in Luria–
Bertani (LB) medium (Thermo Fisher Scientific, Waltham, MA, USA)
supplemented with the appropriate antibiotics. Rhizobium tropici CIAT899
(Martínez-Romero et al., 1991), R. tropici CIAT899–Discosoma red fluo-
rescent protein (DsRed), and R. tropici–green fluorescent protein (GFP)
were grown at 30 °C for 2 d in peptone-yeast extract (PY) medium, as
previously described (Martínez-Romero et al., 1991).
Surface-sterilized seeds of common bean (Phaseolus vulgaris cv. Negro
Jamapa) were germinated on water-saturated paper towels in the dark at
28 °C for 2 d. The bean seedlings were then transferred to pots contain-
ing vermiculite and were inoculated with 1 ml of R. tropici diluted to
Downloaded from https://academic.oup.com/jxb/article-abstract/70/3/1049/5194635 by Pfizer Inc., user on 31 March 2020
an OD600 of 0.05 (equivalent to 7 × 107) in 10 mM MgSO4 (Martínez-
Romero et al., 1991). The plants were grown in a glasshouse with a con-
trolled environment (26–28 °C, 16 h photoperiod) and watered with
Fahraeus nutrient solution (Fahraeus, 1957). Control plants (non-inoc-
ulated with rhizobia) were irrigated with Fahraeus nutrient solution
supplemented with 8 mM KNO3 and grown in the same conditions).
Alternatively, bean composite plants with transgenic roots were generated
as described (Estrada-Navarrete et al., 2006) and R. tropici inoculated as
described. Wild-type or transgenic roots of common bean plants inocu-
lated or non-inoculated with R. tropici were individually collected at spe-
cific time points, frozen in liquid nitrogen, and ground to a fine powder.
Samples were stored at −80 °C until use.
Plasmid construction and plant transformation
Two BI-1 genes (PvBI-1a and PvBI-1b) with high sequence similarity to
Arabidopsis BI-1 (Sanchez et al., 2000) were identified in the genome
of common bean (Goodstein et al., 2012). Using PCR, both genes were
successfully amplified from a collection of full-length cDNAs gener-
ated from common bean. In addition, 1 kb of each of the PvBI-1a and
PvBI-1b promoters was amplified from the common bean genomic
DNA (see Supplementary Table S1 at JXB online). All DNA frag-
ments were cloned into pENTR/D/TOPO cloning vectors (Thermo
Fisher Scientific). The PvBI-1a overexpression construct was generated
by combining the pENTR/D/TOPO::PvBI-1a entry vector with the
Gateway-compatible plant destination vector pEarleyGate103. In the
resulting construct (35S:PvBI-1a), PvBI-1a was fused in-frame to GFP
(Earley et al., 2006). To produce the PvBI-1a silencing construct, 155 nt
from the PvBI-1a 3′-untranslated region (UTR) was PCR-amplified and
cloned into pENTR/D/TOPO.This vector was then combined with the
pTdT-RNAi plant destination vector (Valdés-López et al., 2008), which
includes the ‘Tomato’ fluorescent protein (tdTomato protein) as a reporter,
to generate the construct PvBI-1a-RNAi. Finally, 1 kb of the PvBI-1a
or PvBI-1b promoter region was cloned into the pENTR/D/TOPO
cloning vector and transferred into pBGWFS7 (pPvBI-1a:pBGWFS7 or
pPvBI-1-1b:pBGWFS7), to produce GFP and β-glucuronidase (GUS)
reporter fusions (Karimi et al., 2002). Empty destination vectors (with
the exception of pTdT-RNAi, which includes a nucleotide-scrambled
sequence, Sac), served as negative controls in all experiments. The result-
ing plasmids were introduced by electroporation into A. rhizogenes K599
and used for plant transformation.
Acetylene reduction assays
The nitrogen fixation rate was determined using the acetylene reduc-
tion method (Vessey, 1994). At 18 d post-inoculation (dpi), the transgenic
roots of the composite bean plants were transferred into glass bottles and
sealed with rubber stoppers. Air was immediately withdrawn from the
closed vial and replaced with acetylene to a final concentration of 10% of
the gas phase. The samples were incubated for 60 min at room tempera-
ture, and ethylene production was determined using a Varian 3300 chro-
matograph (Agilent Technologies, Santa Clara, CA, USA), as previously
described (Fuentes-Ramírez et al., 1999). Specific activity was expressed
as µmol–1 C2H2–1 g–1 nodule dry weight (DW) h–1. In these experiments,
 Bax inhibitor-1 dual role in legume–rhizobia symbiosis | 1051
the nitrogen-fixation capacity of untransformed A. rhizogenes K599 root
nodules was considered as 100%.
Quantitative evaluation of root nodule bacteria
Common bean plants were grown and inoculated with R. tropici as
described above. Nine root nodules were harvested from each 18-dpi
plant, surface-sterilized by immersion in ethanol (95%, v/v) for 10 s, and
then in sodium hypochlorite (10%, v/v) for 10 min, rinsed with sterile
water five times and homogenized in 1.5 ml of PY medium containing
20 µg ml–1 nalidixic acid and 10 µg ml–1 tetracycline. Bacterial cultures
were serially diluted and plated on PY medium plates with the appro-
priate antibiotics. Plates were incubated at 30 °C for 3 d and colony-
forming units (CFU) were counted; 200 µl of the wash water used in the
final rinse was plated in the same growth medium as negative control.
RNA extraction and PCR assays
Total RNA was isolated from frozen plant material using the ZR Plant
RNA Miniprep kit (Zymo Research, Irvine, CA, USA), following the
manufacturer’s instructions. RNA quantity was measured spectrophoto-
metrically and only high-quality RNA samples with a 260/280 ratio
between 1.9 and 2.1 and a 260/230 ratio greater than 2.0 were used for
the analysis. For reverse transcription, 3 µg total RNA was treated with
DNaseI (DNaseI (RNase-free) Thermo Fisher Scientific), then 1.5 μg of
the RNA was reverse-transcribed using the RevertAid H Minus First
Strand cDNA Synthesis Kit (Thermo Fisher Scientific), according to the
manufacturer’s instructions. The cDNA was quantified in 15 μl qPCR
reactions using Maxima SYBR Green qPCR Master Mix (Thermo
Fisher Scientific) containing 1 μl cDNA, performed on an iCycler iQ5
(Bio-Rad, Hercules, CA, USA). The cycling conditions were: 3 min at
95 °C; followed by 35 cycles of 20 s at 95 °C, 15 s at 55 °C, and data
acquisition at 81 °C. A negative control reaction without a template was
included for each primer combination (see Supplementary Table S1).The
melting curve protocol began immediately after amplification and con-
sisted of 1 min at 55 °C followed by 80 steps of 10 s each with a 0.5 °C
increase in temperature at each step. The relative numbers for Ct of each
gene were normalized to the Ct of the reference gene, Elongation factor
1-α (PvEf1-α) (Nicot et al., 2005). PvEf1-α has been tested in our labora-
tory and is a stable gene reference for nodulation studies in common bean
(data not shown). Data were analysed using iQ5 Optical System software
(version 2.1, Bio-Rad).
Statistical analyses
In all experiments, at least three biological samples were analysed and
three technical repeats were performed for each biological sample.
Obtained data were subjected to an unpaired Student’s t-test or ANOVA
to identify significant changes in gene expression among the various plant
conditions. The change in gene expression was the dependent variable.
A P value of ≤0.05 or lower was considered as significant. ANOVA was
followed by post hoc multiple comparison tests (Tukey, Welch or Sidak)
depending on the data.
Microscopy analysis
Transgenic roots and 18 dpi root nodules were hand-sectioned using
double-edged razor blades. Sections were mounted on microscope
slides in 0.1 M phosphate buffer, pH 7.4. All fluorescence images were
taken using a confocal microscope (LSM510; Carl Zeiss, Oberkochen,
Germany). Z-Projected confocal images were generated using Fluoview
Viewer (Olympus Corporation, Shinjuku, Tokyo, Japan) and ZEN Black
(Carl Zeiss).
For optical microscopy, control and 18 dpi PvBI-1a overexpression or
RNAi nodules (six nodules per experiment) were fixed in a mixture
of 2% formaldehyde and 0.4% glutaraldehyde in 0.1 M Na-cacodylate
buffer, pH 7.2, at 4 °C for 16 h. The post-fixation was done in 1%
osmium tetroxide for 2 h, and the samples were then dehydrated in a
graded ethanol series. The samples were embedded in LR-White resin
(London Resin Company, Reading, UK) and polymerized under ultra-
violet light at –20 °C for 48 h. Semi-thin sectioning (0.5–1.0 mm) was
performed using an ultramicrotome (Ultracut R; Leica Microsystems,
Wetzlar, Germany), and the sections were stained with 0.1% toluidine
blue. For transmission electron microscopy, samples were stained with
uranyl acetate. Thin (60 nm) sections were prepared with an ultrami-
crotome (Ultracut R). The electron microscopy analyses were performed
using an EM900 transmission electron microscope (Carl Zeiss) dual
vision coupled camera system (Gatan, Inc., Pleasanton, CA, USA).
Results
PvBI-1a expression changes during the common
bean–R. tropici symbiosis
Downloaded from https://academic.oup.com/jxb/article-abstract/70/3/1049/5194635 by Pfizer Inc., user on 31 March 2020
We identified two homologs of Arabidopsis BI-1, PvBI-1a
(Phvul.003G224400) and PvBI-1b (Phvul.002G001400), in
the common bean genome (Goodstein et al., 2012). Using
PCR, we amplified these genes and their respective transcripts
from common bean genomic DNA and full-length cDNA and
sequenced the PCR products. The PvBI-1a sequence had an
open reading frame of 738 nucleotides, preceded by a 251-
bp 5′-UTR and followed by a 221-bp 3′-UTR; analysis of
PvBI-1b revealed a 735-nt open reading frame, with a 5′-UTR
of 152 bp and a 158-bp 3′-UTR (see Supplementary Fig. S1).
The predicted PvBI-1a and PvBI-1b proteins exhibited 72%
and 68% identity and 86% and 87% similarity, respectively, to
Arabidopsis BI-1 (NP_199523.1). An analysis of membrane-
spanning domains by Phyre2 (Kelley et al., 2015) revealed seven
transmembrane domains in both common bean BI-1 predicted
proteins (Supplementary Fig. S1).
We analysed the expression pattern of these two PvBax-I
genes by qPCR throughout the common bean–R. tropici sym-
biosis. In these experiments, five plant roots were pooled and
considered as one biological sample. The fold change in gene
expression was obtained by comparing the expression ratio
of each gene with its expression in uninoculated common
bean roots. As shown in Fig. 1A, both genes were induced in
response to R. tropici, although the amplitude of the PvBI-1a
response was higher (16.3 ± 0.24-fold increase compared
with uninoculated common bean roots) than that of PvBI-1b
(2.2 ± 0.08-fold increase compared with uninoculated roots).
At 6 hours post-rhizobia inoculation (hpi), the expression level
of both genes declined for up to 22 days post-rhizobia inocu-
lation (dpi) (Fig. 1A). Rhizobial colonization of the root and
differentiation into nitrogen-fixing bacteroids are known to
occur during this time (Cermola et al., 2000). In wild-type
common bean nodules, PvBI-1a reached its maximum expres-
sion level at 22–26 dpi (Fig. 1A), coinciding with the high-
est nitrogen-fixing activity of common bean nodules (Olivares
et al., 2011).
To evaluate PvBI-1a and PvBI-1b promoter activity dur-
ing nodulation, we cloned 1 kb of both promoters upstream
of the translation initiation codon and fused them with
GFP–GUS to yield the constructs pPvBI-1a:pBGWFS7 and
pPvBI-1b:pBGWFS7. These constructs were used to generate
transgenic roots in common bean plants (Estrada-Navarrete
et al., 2006). Transgenic roots harbouring the empty vec-
tor pBGWFS7 were used as a negative control. Five (n=5)
1052 | Hernández-López et al.

Downloaded from https://academic.oup.com/jxb/article-abstract/70/3/1049/5194635 by Pfizer Inc., user on 31 March 2020

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. (This figure is available in colour at JXB online.)

independent GUS-stained roots of composite common bean Fig. 1B–E. The spatiotemporal expression of the PvBaxI-1a
plants uninoculated or 2 hpi with R. tropici were analysed. promoter was detected along the root hair in response to
Representative images of these experiments are shown in R. tropici, as early as 2 hpi (Fig. 1E). The PvBI-1a promoter
 Bax inhibitor-1 dual role in legume–rhizobia symbiosis | 1053

activity was also monitored by GFP fluorescence in early
symbiosis. At 3 dpi, GFP fluorescence was clearly detected in
curled-hair roots and along ITs (Fig. 2A–C), while in nitro-
gen-fixing nodules at 18 dpi this promoter was active in the
infected cells and in vascular bundles (Fig. 2D–F). However,
no fluorescence signal could be detected during nodulation in
pPvBI-1b:pBGWFS7 transgenic roots (data not shown). Given
these results, we decided to focus our study on the functional
characterization of PvBI-1a.
PvBI-1a participates during R. tropici infection and its
establishment in the root nodules of common bean
generated, the transgenic roots of composite P. vulgaris plants
were inoculated with equivalent R. tropici concentrations and
the PvBI-1a transcript levels were determined in 18-dpi nod-
ules using qPCR. In 35S:PvBI-1a nodules, PvBI-1a transcript
levels or PvBI-1a-GFP protein chimera levels were higher than
in the negative control (pEarleyGate103-transformed nodules)
(Fig. 3A and Supplementary Fig. S2, respectively), whereas
expression levels in nodules harbouring PvBI-1a-RNAi were
2.4-fold lower than in the negative control (pTdT-Sac-RNAi,
Fig. 3A).
The PvBI-1a loss-of-function nodules were similar in size
and morphology (i.e. cortex and central tissue) to those of
their corresponding negative control (empty vector pTdT-

Downloaded from https://academic.oup.com/jxb/article-abstract/70/3/1049/5194635 by Pfizer Inc., user on 31 March 2020

To analyse the role of PvBI-1a in the common bean–R. tropici
symbiosis, we altered PvBI-1a expression in roots by express-
ing it under the control of the 35S promoter (35S:PvBI-1a),
or by decreasing its expression using RNA interference
(PvBI-1a-RNAi). To this end, we produced common bean
plants with transgenic roots using A. rhizogenes-mediated root
transformation (Estrada-Navarrete et al., 2006), either with
the 35S:PvBI-1a or with PvBI-1a-RNAi constructs. Once
Sac-RNAi) (Fig. 3E–F), although the number of nodules
formed on the PvBI-1a-RNAi roots (83.3 ± 4.2%) was slightly
lower than the number of nodules found on the negative con-
trol (98.38 ± 2.5%; Fig. 3B). In contrast, R. tropici inocula-
tion elicited 2.2-fold more nodules on the 35S:PvBI-1a roots
(217.2 ± 4.4%) than on the empty vector pEarlyGate103 roots
(94.5 ± 2.5%) (Fig. 3B). Compared with the negative control,
35S:PvBI-1a nodules were smaller in size, and had a diminished

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. (This figure is available in colour at JXB online.)
1054 | Hernández-López et al.

Downloaded from https://academic.oup.com/jxb/article-abstract/70/3/1049/5194635 by Pfizer Inc., user on 31 March 2020

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. (This figure is available in
colour at JXB online.)

infection zone (Fig. 3C, D). As expected, their nitrogen fixa-
tion efficiency was considerably lower (~50% when consider-
ing total nodule number). However, this drop in the nitrogen
fixation rate was compensated by the high number of nodules
in the root (80 ± 5.5% compared with 97.4 ± 4.3 µmol C2H2
g–1 nodule DW h–1 in control nodules) so that it had no impact
on plant growth (data not shown) (see Supplementary Fig. S3).
We determined the number of viable bacteroids within all
these nodules. The 35S:PvBI-1a and the PvBI-1a-RNAi
root nodules had fewer viable bacteroids (16.8 ± 0.9% and
89.4 ± 1.0% CFU, respectively) compared with the negative
controls (97 ± 0.6% in pEarleyGate103 and 97.6 ± 1.3% in
pTdT-Sac-RNAi nodules) (inset graphs in Fig. 3C–F). While
nitrogen fixation levels were lower in PvBI-1a-overexpressing
nodules compared with the negative control, the drastic reduc-
tion (~80%) in the number of free-living rhizobia recovered
from these organs was unexpected. Presumably, the increased
PvBI-1a levels could be detrimentally affecting bacteroids
 Bax inhibitor-1 dual role in legume–rhizobia symbiosis | 1055
inhabiting symbiotic cells. However, given that these nodules
are morphologically distinct from PvBI-1a-RNAi and nega-
tive control nodules, we cannot rule out a technical bias arising
from the sample preparation required for this analysis. Further
experiments are necessary to investigate the cause of such a
reduced recovery.
Since PvBI-1a and PvBI-1b were both expressed in R. trop-
ici-inoculated common bean roots (Fig. 1A), we wondered
whether the lack of a clear and unequivocal phenotype in
PvBI-1a-RNAi nodules could be due to a functional redun-
dancy with PvBI-1b. Thus, we determined the expression level
of PvBI-1b in 10–26 dpi transgenic nodules and compared it
with the negative control. As shown in Supplementary Fig. S4,
the expression level of PvBI-1b was unaltered in 35S:PvBI-1a
and PvBI-1a-RNAi nodules, indicating that PvBI-1b was not
likely to be compensating the function of PvBI-1a in PvBI-1a-
silenced nodules.
PvBI-1a overexpression increases the number of
rhizobial infection events in roots of composite
common bean plants
The number of nodules formed in a legume root host is locally
controlled by the HR, which prevents IT formation, growth,
and ramification (Vasse et al., 1993). In consequence, the final
number of nodules is programmed in early symbiosis. At this
stage, the number of successful infection events can be deter-
mined by the number of curled root hairs with ramified ITs
harbouring living rhizobia. Since the overexpression of PvBI-1a
in transgenic roots of composite bean plants resulted in a dra-
matic rise in root nodule number (Fig. 3B), we determined the
frequency of R. tropici successful infection events in PvBI-1a
gain- or loss-of-function roots at different times (3, 6, and 9
dpi) (Fig. 4). As expected, the number of successful infection
events in the negative control decreased over time after R. trop-
ici inoculation (Fig. 4A′). The frequency of these events was
sustained in PvBI-1a-overexpressing roots (Fig. 4B′). In con-
trast, silencing of PvBI-1a caused no significant differences over
time compared with the control (Fig. 4C′, D′). Collectively,
our results indicate that the overexpression of PvBI-1a in roots
of common bean plants inoculated with R. tropici resulted in a
higher number of infection events over time, thereby increas-
ing the final number of nodules per root.
Overexpression of PvBI-1a in common bean nodules
induces premature cell death in symbiotic nodule cells
During the microscopic analysis of 18 dpi PvBI-1a-
overexpressing nodules, we detected areas between nodule
cells that were densely stained with toluidine blue (Fig. 3D).
These regions were further analysed using transmission elec-
tron microscopy (Fig. 5), which revealed the presence of old
bacteroids (characterized by the overaccumulation of poly-β-
hydroxybutyrate granules) as well as some degraded bacteroids
(ghost, empty bacteroids) (Fig. 5C, D). No bacterial clusters
were observed in control or PvBI-1a-silenced nodules (see
Supplementary Fig. S5). We assessed the level of cell death in
PvBI-1a-overexpressing nodules by measuring the expression
levels of a vacuolar processing enzyme (γVPE), and comparing
them to those in PvBI-1a-RNAi and in negative control nod-
ules.VPEs, and particularly γVPE, are caspase-like proteins that
have been implicated in the execution of the ER-dependent
PCD (Hatsugai et al., 2004). Supplementary Fig. S6 shows
γVPE induction in 18 dpi PvBI-1a-overexpressing nodules
compared with the control, but not in PvBI-1a-RNAi nodules.
This fact, in conjunction to the previously described morpho-
logical observations, suggests that at least some of the symbi-
otic nodule cells of 18 dpi PvBI-1a-overexpressing nodules are
in PCD.
PvBI-1a overexpression induces the premature death
Downloaded from https://academic.oup.com/jxb/article-abstract/70/3/1049/5194635 by Pfizer Inc., user on 31 March 2020
of symbiotic nodule cells
Defence, autophagy, and membrane trafficking are fundamen-
tal cellular processes in plant–microbe interactions, including
pathogenesis and symbiosis (Yuk et al., 2012; Lamb et al., 2013;
Stolz et al., 2014; Estrada-Navarrete et al., 2016). We analysed
the expression profile of a set of genes related to these inter-
connected cellular processes in control and PvBI-1a gain-of-
function nodules (Fig. 6). Typically, the common bean nodule
primordia may be unequivocally distinguished from lateral root
primordia after 10 dpi in wild-type nodules. Given that the
development of nodule primordia in common bean hairy roots
inoculated with A. rhizogenes was slightly delayed, we decided
to use 14–26 dpi nodules in these experiments. Nitrogen fixa-
tion levels from 22–26 dpi transgenic control nodules were
similar (data not shown), indicating that rhizobia were fully
differentiated to bacteroids after 22 dpi.
Compared with negative control nodules (pEarlyGate103
transformed root nodules), the onset of changes in the expres-
sion of defence-related genes occurred 4 d earlier in PvBI-1a-
overexpressing nodules (Fig. 6A, B). In addition, the induction
of core autophagy genes, including ATG6, which mediates
PCD during the plant immune response in Arabidopsis (Liu
et al., 2005; Fig. 6D compared with control in Fig. 6C), and
of genes tied to membrane trafficking (Fig. 6F compared with
control in Fig. 6E) both support the pro-death role of BI-1
in 35S:PvBI-1a young root nodules. Compared with control
(Supplementary Fig. S7 A, C, D), the silencing of PvBI-1a
in 18 dpi transgenic root nodules induces defence and most
membrane trafficking genes (Supplementary Fig. S7B, F),
but does not change the expression of autophagy-tied genes
(Supplementary Fig. S6C–D).
Discussion
While being mutually beneficial, rhizobial nodulation is ener-
getically costly to the legume host, and it is known to be tightly
controlled. Nodulation in legumes is regulated by local and
systemic mechanisms. Locally, elicitation of the HR (a type of
plant PCD) limits the number of infection events allowed, and
therefore, contributes to determining the final number of active
nitrogen-fixing nodules formed in the root of the legume host
(Vasse et al., 1993; Stacey et al., 2006). Remarkably, the HR is
also one of the earliest responses to pathogen attack in plants.
1056 | Hernández-López et al.

Downloaded from https://academic.oup.com/jxb/article-abstract/70/3/1049/5194635 by Pfizer Inc., user on 31 March 2020

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). (This figure is available in colour at JXB online.)

The onset of the HR is associated with the synthesis of salycilic
acid (SA), which induces extracellular calcium influx, produc-
tion of reactive oxygen species (ROS), and PR gene expres-
sion (Grant et al., 2000; Lam et al., 2001; Hatsugai et al., 2004).
In parallel, SA also activates the expression of BI-1 (Lu et al.,
2018), whose expression is induced in biotic and abiotic types
of cell death in plants (Kawai-Yamada et al., 2001; Watanabe
and Lam, 2008, 2009; Isbat et al., 2009; Ishikawa et al., 2010).
Plant BI-1s have been implicated in ER stress-induced PCD
and calcium homeostasis, and in promoting autophagy during
pathogen attack (Kim et al., 2008; Watanabe and Lam, 2008;
Kawai-Yamada et al., 2009; Duan et al., 2010; Xu et al., 2017).
Considering the relevant role of the HR in the context of
legume nodulation, in this work we characterized the func-
tion of PvBI-1a, a common bean homolog of Arabidopsis BI-1,
throughout the process of symbiosis with R. tropici. As it occurs
with ROS and some PR genes in early legume nodulation
(Kouchi et al., 2004; Lohar et al., 2006, 2007; Brechenmacher
et al., 2008; Libault et al., 2010), PvBI-1a expression transiently
increased in common bean after rhizobial inoculation (Fig. 1A).
A temporary increase of BI-1 expression after rhizobial inocu-
lation has also been observed in soybean (Brechenmacher et al.,
2008), another tropical legume closely related to the common
bean. The spatiotemporal induction of the PvBI-1a promoter
in early symbiosis shows that this gene is being expressed in
the root hair after rhizobial exposure, and during IT elonga-
tion (Figs 1B–E , 2A–C), precisely where the HR induces the
abortion of a number of infection attempts (Vasse et al., 1993).
Given its role as a PCD suppressor in plants, these results lead
us to hypothesize that during the early steps of the common
 Bax inhibitor-1 dual role in legume–rhizobia symbiosis | 1057

Downloaded from https://academic.oup.com/jxb/article-abstract/70/3/1049/5194635 by Pfizer Inc., user on 31 March 2020

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. (This figure is available in
colour at JXB online.)

bean–R. tropici interaction, PvBI-1a could help modulate the
HR, thus providing an additional step in defining the num-
ber of nodules formed in the root host. To test this hypoth-
esis, we produced transgenic roots of composite common bean
plants with either increased or decreased expression of PvBI-1a
(Fig. 3A; Supplementary Fig. S2). Both PvBI-1a variants were
similar to control transgenic roots in aspects of hair root curling
and IT development (Fig. 4A–D). However, the overexpres-
sion of PvBI-1a increased the frequency of R. tropici infec-
tion events over time (Fig. 4A′–D′), and consequently, the final
number of nodules formed in the root (Fig. 3B). These results
support the notion that PvBI-1a promotes rhizobial infection
by attenuating the HR, and is consistent with previous reports
where the susceptibility of diverse barley cultivars to Blumeria
graminis f.sp. hordei (a biotrophic fungus) was found to cor-
relate with higher BI-1 expression levels (Hückelhoven et al.,
2003; Babaeizad et al., 2008), whereas in barley roots colonized
by the mutualistic fungal endophyte Piriformospora indica, the
expression of BI-1 is attenuated (Deshmukh et al., 2006).
Even though the final number of nodules formed in PvBI-
1a-overexpressing roots was twice as large as the number on
control roots (Fig. 3B), 35S:PvBI-1a nodules were smaller
in size and poorly infected (Fig. 3C–D). Given that resident
bacteroids inhabiting these organs were able to fix nitrogen
(Fig. 3B), bacterial release from ITs to symbiotic nodule cells
was likely not affected. A detailed analysis of the infection zone
of PvBI-1a-overexpressing nodules revealed the presence of old
or degraded bacteroids between uninfected or infected nodule
cells (Fig. 5C–D). A similar phenotype has been observed in
nodules of Medicago truncatula activated defence 1 mutant plants
(Wang et al., 2016), where bacteroids and their symbiotic plant
cells become necrotic as a consequence of an active plant
defence. Such bacterial clusters were not observed in control or
PvBI-1a-silenced nodules (see Supplementary Fig. S5).We con-
firmed the premature death of symbiotic cells in 35S:PvBI-1a
nodules by assessing the expression level of γVPE in control
or PvBI-1a-transgenic nodules. Because γVPE transcription
increases during soybean nodule senescence (Roux et al., 2014;
Van Wyk et al., 2014), γVPE induction in 35S:PvBI-1a nod-
ules (Supplementary Fig. S6) suggests that these organs may be
undergoing PCD. This is further supported by the premature
gene activation observed during nodulation in 35S:PvBI-1a
nodules not only of defence-related genes (Fig. 6A–B), but
also of autophagy (Fig. 6C–D) and membrane trafficking genes
(Fig. 6E–F). Induced expression of defence genes such as PR1,
PR2, and phenylalanine ammonia lyase (PAL) have been also
observed in plant senescence (Egli et al., 1989; Quirino et al.,
1999; Morris et al., 2000; Gourion, et al., 2015) and it has been
demonstrated that plant autophagy determines the resistance
level against invading pathogens by controlling the amount of
proteins involved in cell death through membrane trafficking
events (Inada and Ueda, 2014; Leborgne-Castel and Bouhidel,
2014; Gourion, et al., 2015; Ishikawa et al., 2015). Additionally,
Xu et al. (2017) recently demonstrated that BI-1 promotes
1058 | Hernández-López et al.

Downloaded from https://academic.oup.com/jxb/article-abstract/70/3/1049/5194635 by Pfizer Inc., user on 31 March 2020

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).

autophagy during pathogenesis, establishing a direct connection
between autophagy and plant resistance. Thus, the gene expres-
sion pattern we observed in 35S:PvBI-1a nodules also supports
the notion that constitutive expression of PvBI-1a through-
out the common bean–R. tropici symbiosis, induces premature
PCD in symbiotic nodule cells. On the other hand, PvBI-1a
is highly induced in wild-type common bean nodules during
active nitrogen fixation (Fig. 1A, 22–26 dpi nodules), although
in this physiological context, where ROS are being intensively
produced (Chang et al., 2009), PvBI-1a could potentially be
playing a separate role related to antioxidant pathways. Such a
function has been previously reported in mammals (Lee et al.,
2007; Kim et al., 2009).
As we showed, the silencing of PvBI-1a gave no distinctive
phenotype in early or late symbiosis). Since functional redun-
dancy between BI-1 genes in common bean is unlikely (see
Supplementary Fig. S4), these data suggest that other molecu-
lar signals involved in the positive control of nodule number,
not yet identified, could compensate the lack of PvBaxI-1a at
this step of symbiosis. However, in later stages, a strict control
of PvBI-1a expression must take place to avoid the premature
death of young nodules.
Overall, our results suggest that PvBI-1a has a dual role dur-
ing the legume–rhizobia symbiosis. While in early symbiosis
BI-1 promotes rhizobial infection by repressing HR, in the
later nodulation stages its overexpression leads to cell death, as
 Bax inhibitor-1 dual role in legume–rhizobia symbiosis | 1059
occurs in other plant–microbe interactions (Xu et al., 2017).
By demonstrating the functional role of a BI-1 during nodu-
lation in common bean, we have extended our understand-
ing of the function of this protein in beneficial plant–microbe
interactions.
Supplementary data
Supplementary data are available at JXB online.
Fig. S1. PvBI-1a and PvBI-1b genes and sequence-deduced
proteins.
Fig. S2. Overexpression of PvBI-1a in root nodules of com-
posite common bean plants.
Fig. S3. Control (pEarlyGate 103) and PvBI-1a-
overexpressing composite bean plants inoculated with R. trop-
ici CIAT899 (18 d later).
Fig. S4. Overexpression or silencing of PvBI-1a does not
modify the expression level of PvBI-1b in common bean
nodules.
Fig. S5. High magnification transmission electron micro-
graphs of 18 dpi control and PvBI-1a-RNAi root nodules
show no disctintive phenotype.
Fig. S6. γVPE expression in 18 dpi PvBI-1a gain- or loss-of-
function nodules.
Fig. S7. Silencing of PvBI-1a in symbiotic nodules of com-
mon bean affects host defence and vesicular trafficking, but not
autophagy.
Table S1. Oligonucleotides used in this study.
Acknowledgements
We thank Olivia Santana, Alfonso Leija, Xóchitl Alvarado-Affantranger
and Guadalupe Zavala for their technical advice; Pedro Saucedo for the
nodule ontogeny drawing; the Laboratorio Nacional de Microscopía
Avanzada (LNMA) at the Instituto de Biotecnología of the Universidad
Nacional Autónoma de México for the use of their equipment; and
Paul Gaytán, Jorge Yañez, and Eugenio López for primer synthesis and
DNA sequencing (Unidad de Síntesis y Secuenciación de ADN at the
Instituto de Biotecnología). All authors are grateful to Caspar Chater,
Helena Porta, and Marcela Treviño and the team of Plant Editors for
their detailed and professional editing of this manuscript. AH-L was
supported by a PhD scholarship (215023) from Consejo Nacional de
Ciencia y Tecnología (CONACYT)-México. This research was par-
tially supported by diverse research grants from CONACYT-México
to Federico Sánchez, and from the Universidad Nacional Autónoma
de México (Dirección General de Asuntos del Personal Académico
PAPIIT No. IN206815 and No. IN201418) to CD.
References
Babaeizad V, Imani J, Kogel KH, Eichmann R, Hückelhoven R. 2008.
Over-expression of theell death regulator BAX inhibitor-1 in barley confers
reduced or enhanced susceptibility to distinct fungal pathogens. Theoretical
and Applied Genetics 18, 455–463.
Berrabah F, Ratet P, Gourion B. 2015. Multiple steps control immunity
during the intracellular accommodation of rhizobia. Journal of Experimental
Botany 66, 1977–1985.
Bond JE, Gresshoff PM. 1993. Soybean transformation to study molecular
physiology. In: Gresshoff PM, ed. Plant responses to the environment.
London: CRC Press, 25–44.
Brechenmacher L, Kim MY, Benitez M, et al. 2008. Transcription
profiling of soybean nodulation by Bradyrhizobium japonicum. Molecular
Plant-Microbe Interactions 21, 631–645.
Cao Y, Halane MK, Gassmann W, Stacey G. 2017. The role of plant
innate immunity in the legume-rhizobium symbiosis. Annual Review of Plant
Biology 68, 535–561.
Castillo K, Rojas-Rivera D, Lisbona F, et al. 2011. BAX inhibitor-1
regulates autophagy by controlling the IRE1α branch of the unfolded protein
response. The EMBO Journal 30, 4465–4478.
Cermola M, Fedorova E, Taté R, Riccio A, Favre R, Patriarca EJ.
2000. Nodule invasion and symbiosome differentiation during Rhizobium
etli-Phaseolus vulgaris symbiosis. Molecular Plant-Microbe Interactions 13,
733–741.
Chang C, Damiani I, Puppo A, Frendo P. 2009. Redox changes during
the legume-rhizobium symbiosis. Molecular Plant 2, 370–377.
Deshmukh S, Hückelhoven R, Schäfer P, Imani J, Sharma M, Weiss
Downloaded from https://academic.oup.com/jxb/article-abstract/70/3/1049/5194635 by Pfizer Inc., user on 31 March 2020
M, Waller F, Kogel K-H. 2006. Proceedings of the National Academy of
Sciences, USA 103, 18450–18457.
Downie JA. 2014. Legume nodulation. Current Biology 24, R184–R190.
Duan Y, Zhang W, Li B, Wang Y, Li K, Sodmergen, Han C, Zhang
Y, Li X. 2010. An endoplasmic reticulum response pathway mediates
programmed cell death of root tip induced by water stress in Arabidopsis.
New Phytologist 186, 681–695.
Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song K, Pikaard
CS. 2006. Gateway-compatible vectors for plant functional genomics and
proteomics. The Plant Journal 45, 616–629.
Egli MA, Griffith SM, Miller SS, Anderson MP, Vance CP. 1989. Nitrogen
assimilating enzyme activities and enzyme protein during development and
senescence of effective and plant gene-controlled ineffective alfalfa nodules.
Plant Physiology 91, 898–904.
Estrada-Navarrete G, Alvarado-Affantranger X, Olivares JE, et al.
2006. Agrobacterium rhizogenes transformation of the Phaseolus spp.:
a tool for functional genomics. Molecular Plant-Microbe Interactions 19,
1385–1393.
Estrada-Navarrete G, Cruz-Mireles N, Lascano R, et al. 2016. An
autophagy-related kinase is essential for the symbiotic relationship between
Phaseolus vulgaris and both rhizobia and arbuscular mycorrhizal fungi. The
Plant Cell 28, 2326–2341.
Fahraeus G. 1957. The infection of clover root hairs by nodule bacteria
studied by a simple glass slide technique. Journal of General Microbiology
16, 374–381.
Ferguson BJ, Indrasumunar A, Hayashi S, Lin MH, Lin YH, Reid DE,
Gresshoff PM. 2010. Molecular analysis of legume nodule development
and autoregulation. Journal of Integrative Plant Biology 52, 61–76.
Fournier J, Teillet A, Chabaud M, Ivanov S, Genre A, Limpens E,
de Carvalho-Niebel F, Barker DG. 2015. Remodeling of the infection
chamber before infection thread formation reveals a two-step mechanism
for rhizobial entry into the host legume root hair. Plant Physiology 167,
1233–1242.
Fuentes-Ramírez LE, Caballero-Mellado J, Sepúlveda J, Martínez-
Romero E. 1999. Colonization of sugarcane by Acetobacter diazotrophicus
is inhibited by high N-fertilization. FEMS Microbiology Ecology 29, 117–128.
Goodstein DM, Shu S, Howson R, et al. 2012. Phytozome: a
comparative platform for green plant genomics. Nucleic Acids Research
40, D1178–D1186.
Gourion B, Berrabah F, Ratet P, Stacey G. 2015. Rhizobium-legume
symbioses: the crucial role of plant immunity. Trends in Plant Science 20,
186–194.
Grant M, Brown I, Adams S, Knight M, Ainslie A, Mansfield J. 2000.
The RPM1 plant disease resistance gene facilitates a rapid and sustained
increase in cytosolic calcium that is necessary for the oxidative burst and
hypersensitive cell death. The Plant Journal 23, 441–450.
Hatsugai N, Kuroyanagi M, Yamada K, Meshi T, Tsuda S, Kondo M,
Nishimura M, Hara-Nishimura I. 2004. A plant vacuolar protease, VPE,
mediates virus-induced hypersensitive cell death. Science 305, 855–858.
Hückelhoven R, Dechert C, Kogel KH. 2003. Overexpression of barley
BAX inhibitor 1 induces breakdown of mlo-mediated penetration resistance
to Blumeria graminis. Proceedings of the National Academy of Sciences,
USA 100, 5555–5560.
1060 | Hernández-López et al.
Inada N, Ueda T. 2014. Membrane trafficking pathways and their roles in
plant-microbe interactions. Plant & Cell Physiology 55, 672–686.
Isbat M, Zeba N, Kim SR, Hong CB. 2009. A BAX inhibitor-1 gene in
Capsicum annuum is induced under various abiotic stresses and endows
multi-tolerance in transgenic tobacco. Journal of Plant Physiology 166,
1685–1693.
Ishikawa T, Aki T, Yanagisawa S, Uchimiya H, Kawai-Yamada M. 2015.
Overexpression of BAX INHIBITOR-1 links plasma membrane microdomain
proteins to stress. Plant Physiology 169, 1333–1343.
Ishikawa T, Takahara K, Hirabayashi T, Matsumura H, Fujisawa S,
Terauchi R, Uchimiya H, Kawai-Yamada M. 2010. Metabolome analysis
of response to oxidative stress in rice suspension cells overexpressing cell
death suppressor Bax inhibitor-1. Plant & Cell Physiology 51, 9–20.
Ishikawa T, Watanabe N, Nagano M, Kawai-Yamada M, Lam E. 2011.
Bax inhibitor-1: a highly conserved endoplasmic reticulum-resident cell
death suppressor. Cell Death and Differentiation 18, 1271–1278.
Ivanov S, Fedorova EE, Limpens E, et al. 2012. Rhizobium–legume
symbiosis shares an exocytotic pathway required for arbuscule formation.
Proceedings of the National Academy of Sciences, USA 109, 8316–8321.
Iwata Y, Koizumi N. 2005. Unfolded protein response followed by
induction of cell death in cultured tobacco cells treated with tunicamycin.
Planta 220, 804–807.
Jones KM, Kobayashi H, Davies BW, Taga ME, Walker GC. 2007. How
rhizobial symbionts invade plants: the Sinorhizobium–Medicago model.
Nature Reviews. Microbiology 5, 619–633.
Karimi M, Inzé D, Depicker A. 2002. GATEWAY vectors for Agrobacterium-
mediated plant transformation. Trends in Plant Science 7, 193–195.
Kawai-Yamada M, Hori Z, Ogawa T, Ihara-Ohori Y, Tamura K,
Nagano M, Ishikawa T, Uchimiya H. 2009. Loss of calmodulin binding
to Bax inhibitor-1 affects Pseudomonas-mediated hypersensitive response-
associated cell death in Arabidopsis thaliana. The Journal of Biological
Chemistry 284, 27998–28003.
Kawai-Yamada M, Jin U, Yoshinaga K, Hirata A, Uchimiya H. 2001.
Mammalian Bax-induced plant cell death can be down-regulated by
overexpression of Arabidopsis Bax inhibitor-1 (AtBI-1). Proceedings of the
National Academy of Sciences, USA 98, 12295–12300.
Kawai-Yamada M, Ohori Y, Uchimiya H. 2004. Dissection of Arabidopsis
Bax inhibitor-1 suppressing Bax-, hydrogen peroxide-, and salicylic acid-
induced cell death. The Plant Cell 16, 21–32.
Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. 2015. The
Phyre2 web portal for protein modeling, prediction and analysis. Nature
Protocols 10, 845–858.
Kim HR, Lee GH, Cho EY, Chae SW, Ahn T, Chae HJ. 2009. Bax inhibitor
1 regulates ER-stress-induced ROS accumulation through the regulation of
cytochrome P450 2E1. Journal of Cell Science 122, 1126–1133.
Kim HR, Lee GH, Ha KC, et al. 2008. Bax inhibitor-1 is a pH-dependent
regulator of Ca2+ channel activity in the endoplasmic reticulum. The Journal
of Biological Chemistry 283, 15946–15955.
Kondorosi E, Roudier F, Gendreau E. 2000. Plant cell-size control:
growing by ploidy? Current Opinion in Plant Biology 3, 488–492.
Kouchi H, Shimomura K, Hata S, et al. 2004. Large-scale analysis of
gene expression profiles during early stages of root nodule formation in a
model legume, Lotus japonicus. DNA Research 11, 263–274.
Lam E, Kato N, Lawton M. 2001. Programmed cell death, mitochondria
and the plant hypersensitive response. Nature 411, 848–853.
Lamb CA, Yoshimori T, Tooze SA. 2013. The autophagosome: origins
unknown, biogenesis complex. Nature Reviews. Molecular Cell Biology 14,
759–774.
Leborgne-Castel N, Bouhidel K. 2014. Plasma membrane protein
trafficking in plant-microbe interactions: a plant cell point of view. Frontiers
in Plant Science 5, 735.
Lee GH, Kim HK, Chae SW, Kim DS, Ha KC, Cuddy M, Kress C,
Reed JC, Kim HR, Chae HJ. 2007. Bax inhibitor-1 regulates endoplasmic
reticulum stress-associated reactive oxygen species and heme oxygenase-1
expression. The Journal of Biological Chemistry 282, 21618–21628.
Libault M, Farmer A, Brechenmacher L, et al. 2010. Complete
transcriptome of the soybean root hair cell, a single-cell model, and
its alteration in response to Bradyrhizobium japonicum infection. Plant
Physiology 152, 541–552.
Liu Y, Schiff M, Czymmek K, Tallóczy Z, Levine B, Dinesh-Kumar SP.
2005. Autophagy regulates programmed cell death during the plant innate
immune response. Cell 121, 567–577.
Lohar DP, Haridas S, Gantt JS, VandenBosch KA. 2007. A transient
decrease in reactive oxygen species in roots leads to root hair deformation
in the legume–rhizobia symbiosis. New Phytologist 173, 39–49.
Lohar DP, Sharopova N, Endre G, Peñuela S, Samac D, Town C,
Silverstein KA, VandenBosch KA. 2006. Transcript analysis of early
nodulation events in Medicago truncatula. Plant Physiology 140, 221–234.
Lu PP, Yu TF, Zheng WJ, Chen M, Zhou YB, Chen J, Ma YZ, Xi YJ,
Xu ZS. 2018. The wheat bax inhibitor-1 protein interacts with an aquaporin
tapip1 and enhances disease resistance in Arabidopsis. Frontiers in Plant
Science 9, 20.
Martínez-Romero E, Segovia L, Mercante FM, Franco AA, Graham P,
Pardo MA. 1991. Rhizobium tropici, a novel species nodulating Phaseolus
vulgaris L. beans and Leucaena sp. trees. International Journal of Systematic
Downloaded from https://academic.oup.com/jxb/article-abstract/70/3/1049/5194635 by Pfizer Inc., user on 31 March 2020
Bacteriology 41, 417–426.
Matsumura H, Nirasawa S, Kiba A, Urasaki N, Saitoh H, Ito M,
Kawai-Yamada M, Uchimiya H, Terauchi R. 2003. Overexpression of
Bax inhibitor suppresses the fungal elicitor-induced cell death in rice (Oryza
sativa L) cells. The Plant Journal 33, 425–434.
Morris K, MacKerness SA, Page T, John CF, Murphy AM, Carr JP,
Buchanan-Wollaston V. 2000. Salicylic acid has a role in regulating gene
expression during leaf senescence. The Plant Journal 23, 677–685.
Namugwanya M, Tenywa JS, Otabbong E, Mubiru D, Masamba T.
2014. Development of common bean (Phaseolus vulgaris L.) production
under low soil phosphorus and drought in Sub-Saharan Africa: a review.
Journal of Sustainable Development 7, 128–139.
Nicot N, Hausman JF, Hoffmann L, Evers D. 2005. Housekeeping gene
selection for real-time RT-PCR normalization in potato during biotic and
abiotic stress. Journal of Experimental Botany 56, 2907–2914.
Oldroyd GE. 2013. Speak, friend, and enter: signalling systems that
promote beneficial symbiotic associations in plants. Nature Reviews.
Microbiology 11, 252–263.
Olivares JE, Díaz-Camino C, Estrada-Navarrete G, Alvarado-
Affantranger X, Rodríguez-Kessler M, Zamudio FZ, Olamendi-
Portugal T, Márquez Y, Servín LE, Sánchez F. 2011. Nodulin 41, a novel
late nodulin of common bean with peptidase activity. BMC Plant Biology
11, 134.
Quirino BF, Normanly J, Amasino RM. 1999. Diverse range of gene
activity during Arabidopsis thaliana leaf senescence includes pathogen-
independent induction of defense-related genes. Plant Molecular Biology
40, 267–278.
Roux B, Rodde N, Jardinaud MF, et al. 2014. An integrated analysis of
plant and bacterial gene expression in symbiotic root nodules using laser-
capture microdissection coupled to RNA sequencing. The Plant Journal 77,
817–837.
Sanchez P, de Torres Zabala M, Grant M. 2000. AtBI-1, a plant
homologue of Bax inhibitor-1, suppresses Bax-induced cell death in yeast
and is rapidly upregulated during wounding and pathogen challenge. The
Plant Journal 21, 393–399.
Stacey G, McAlvin CB, Kim SY, Olivares J, Soto MJ. 2006. Effects
of endogenous salicylic acid on nodulation in the model legumes Lotus
japonicus and Medicago truncatula. Plant Physiology 141, 1473–1481.
Stolz A, Ernst A, Dikic I. 2014. Cargo recognition and trafficking in
selective autophagy. Nature Cell Biology 16, 495–501.
Udvardi M, Poole PS. 2013. Transport and metabolism in legume-rhizobia
symbioses. Annual Review of Plant Biology 64, 781–805.
Valdés-López O, Arenas-Huertero C, Ramírez M, Girard L, Sánchez
F, Vance CP, Luis Reyes J, Hernández G. 2008. Essential role of MYB
transcription factor: PvPHR1 and microRNA: PvmiR399 in phosphorus-
deficiency signalling in common bean roots. Plant, Cell & Environment 31,
1834–1843.
Van Wyk SG, Du Plessis M, Cullis CA, Kunert KJ, Vorster BJ. 2014.
Cysteine protease and cystatin expression and activity during soybean
nodule development and senescence. BMC Plant Biology 14, 294.
Vasse J, de Billy F, Truchet G. 1993. Abortion of infection during the
Rhizobium meliloti-alfalfa symbiotic interaction is accompanied by a
hypersensitive reaction. The Plant Journal 4, 555–566.
 Bax inhibitor-1 dual role in legume–rhizobia symbiosis | 1061
Vessey JK. 1994. Measurement of nitrogenase activity in legume root nodules:
In defense of the acetylene reduction assay. Plant and Soil 158, 151–162.
Wang C, Yu H, Luo L, et al. 2016. NODULES WITH ACTIVATED DEFENSE
1 is required for maintenance of rhizobial endosymbiosis in Medicago
truncatula. New Phytologist 212, 176–191.
Watanabe N, Lam E. 2008. BAX inhibitor-1 modulates endoplasmic
reticulum stress-mediated programmed cell death in Arabidopsis. The
Journal of Biological Chemistry 283, 3200–3210.
Watanabe N, Lam E. 2009. Bax Inhibitor-1, a conserved cell death
suppressor, is a key molecular switch downstream from a variety of biotic
and abiotic stress signals in plants. International Journal of Molecular
Sciences 10, 3149–3167.
Xu G, Wang S, Han S, Xie K, Wang Y, Li J, Liu Y. 2017. Plant Bax
Inhibitor-1 interacts with ATG6 to regulate autophagy and programmed cell
death. Autophagy 13, 1161–1175.
Yuk JM, Yoshimori T, Jo EK. 2012. Autophagy and bacterial infectious
diseases. Experimental & Molecular Medicine 44, 99–108.
Zipfel C, Oldroyd GE. 2017. Plant signalling in symbiosis and immunity.
Nature 543, 328–336.
Downloaded from https://academic.oup.com/jxb/article-abstract/70/3/1049/5194635 by Pfizer Inc., user on 31 March 2020