Journal of Experimental Botany, Vol. 66, No. 11 pp. 3367–3380, 2015
doi:10.1093/jxb/erv147 Advance Access publication 6 April 2015
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Pepper aldehyde dehydrogenase CaALDH1 interacts with
Xanthomonas effector AvrBsT and promotes effectortriggered cell death and defence responses
Nak Hyun Kim* and Byung Kook Hwang†
Laboratory of Molecular Plant Pathology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of
Korea
* Present address: Department of Biology, The University of North Carolina at Chapel Hill, NC27599-3280, USA
† To whom correspondence should be addressed. E-mail: [email protected]
Received 24 December 2014; Revised 5 March 2015; Accepted 5 March 2015
Abstract
Xanthomonas type III effector AvrBsT induces hypersensitive cell death and defence responses in pepper (Capsicum
annuum) and Nicotiana benthamiana. Little is known about the host factors that interact with AvrBsT. Here, we
identified pepper aldehyde dehydrogenase 1 (CaALDH1) as an AvrBsT-interacting protein. Bimolecular fluorescence
complementation and co-immunoprecipitation assays confirmed the interaction between CaALDH1 and AvrBsT in
planta. CaALDH1:smGFP fluorescence was detected in the cytoplasm. CaALDH1 expression in pepper was rapidly
and strongly induced by avirulent Xanthomonas campestris pv. vesicatoria (Xcv) Ds1 (avrBsT) infection. Transient coexpression of CaALDH1 with avrBsT significantly enhanced avrBsT-triggered cell death in N. benthamiana leaves.
Aldehyde dehydrogenase activity was higher in leaves transiently expressing CaALDH1, suggesting that CaALDH1
acts as a cell death enhancer, independently of AvrBsT. CaALDH1 silencing disrupted phenolic compound accumulation, H2O2 production, defence response gene expression, and cell death during avirulent Xcv Ds1 (avrBsT) infection.
Transgenic Arabidopsis thaliana overexpressing CaALDH1 exhibited enhanced defence response to Pseudomonas
syringae pv. tomato and Hyaloperonospora arabidopsidis infection. These results indicate that cytoplasmic CaALDH1
interacts with AvrBsT and promotes plant cell death and defence responses.
Key words: Aldehyde dehydrogenase, cell death, effector AvrBsT, pepper, plant defence, Xanthomonas campestris pv.
vesicatoria.
Introduction
Microbial pathogens attack plants to acquire nutrients for
growth, development, and reproduction (Dangl et al., 2013).
Pathogens that breach the protective waxy cuticular leaf surface encounter an immune system that specifically recognizes
selected pathogens and altered-self molecules generated during pathogen invasion (Eulgem, 2005; Choi et al., 2012; Dangl
et al., 2013). The first tier of the plant immune system contains pattern-recognition receptors (PRRs) localized on the
cell surface that bind to evolutionarily conserved pathogenassociated molecular patterns (PAMPs) (Jones and Dangl,
2006; Dodds and Rathjen, 2010; Monaghan and Zipfel,
2012). PRRs bind to the pathogen-derived ligand, become
Abbreviations: ABA, abscisic acid; ALDH, aldehyde dehydrogenase; Bax, Bcl-2-associated X protein; BiFC, bimolecular fluorescence complementation; Co-IP,
co-immunoprecipitation; DAB, 3,3’-diaminobenzidine; ETI, effector-triggered immunity; HR, hypersensitive response; Hpa, Hyaloperonospora arabidopsidis; LB,
Luria-Bertani; NLR, nucleotide-binding leucine-rich repeat;OX, over-expression; PAMP, pathogen-associated molecular pattern; PCD, programmed cell death; PCR,
polymerase chain reaction; PR, pathogenesis-related; PRRs, pattern-recognition receptors; Pst, Pseudomonas syringae pv. tomato; R, resistance; ROS, reactive
oxygen species; SA, salicylic acid; SGT1, suppressor of the G2 allele of skp1; smGFP, soluble-modified green fluorescent protein; TRV, tobacco rattle virus; VIGS,
virus-induced gene silencing; Xcv, Xanthomonas campestris pv. vesicatoria.
© The Author 2015. 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/3.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
3368 | Kim and Hwang
activated, and trigger an intracellular signalling cascade that
drives transcriptional reprogramming and defence molecule
biosynthesis to inhibit pathogen colonization (Monaghan
and Zipfel, 2012). Plant bacterial pathogens utilize the type
III secretion system to deliver effector proteins that subvert
PRR-triggered defence responses (Kim et al., 2010; Block
and Alfano, 2011). The second tier of the plant immune system contains intracellular nucleotide-binding leucine-rich
repeat (NLR) receptors (Maekawa et al., 2011). NLR proteins recognize specific pathogen effectors either via direct
interaction with the effector protein (Dodds et al., 2006) or
via recognition of pathogen effector-mediated modifications
(van der Hoorn and Kamoun, 2008).
The hypersensitive response (HR)-like cell death that
occurs during pathogen attack is an integral part of plant
immune systems, and is one of the most dramatic displays
of programmed cell death (PCD) in plants (Bozhkov and
Lam, 2011). The hallmarks of HR include rapid oxidative
burst, production of reactive oxygen species (ROS) such as
H2O2, and ion fluxes across the plasma membrane before
cell death (Lamb and Dixon, 1997; Morel and Dangl, 1997).
ROS is intimately related to plant cell death and defence signalling (Torres et al., 2006; Van Breusegem and Dat, 2006).
ROS directly inhibits pathogen growth, stimulates cell wall
cross-linking, and triggers defence- and stress-response gene
expression (Lamb and Dixon, 1997; Skopelitis et al., 2006).
Xanthomonas campestris pv. vesicatoria (Xcv) strain Bv5-4a
secretes type III effector protein AvrBsT, which induces hypersensitive cell death and strong defence responses in pepper
(Capsicum annuum) and Nicotiana benthamiana (Orth et al.,
2000; Escolar et al., 2001; Kim et al., 2010). AvrBsT is a member of the YopJ/AvrRxv family in Xcv (Lewis et al., 2011).
AvrBsT possesses acetyltransferase activity and acetylates
ACIP1 (for ACETYLATED INTERACTING PROTEIN1),
an unknown protein from Arabidopsis (Cheong et al., 2014).
ACIP1 is proposed to function in the defence machinery
required for anti-bacterial immunity. However, the molecular
mechanisms and host factors involved in AvrBsT-triggered
cell death are not completely elucidated.
Aldehyde dehydrogenases (ALDHs; EC 1.2.1.3) catalyse the
conversion of aldehydes to the corresponding carboxylic acids
and reduce NAD+ or NADP+. Human ALDHs are extensively characterized and categorized as cytosolic ALDH1 and
mitochondrial ALDH2 that are involved primarily in ethanol
metabolism (Hsu et al., 1988; Hsu et al., 1989). More than 550
ALDH genes have been identified in mammals, insects, bacteria,
yeast, and plants (Sophos and Vasiliou, 2003). Some ALDHs
oxidize specific substrates, whereas others accept a broad range
of substrates (Yoshida et al., 1998). Comprehensive genetic
information has led to the establishment of the ALDH Gene
Nomenclature Committee that defines specific characteristic
criteria for ALDH proteins (Vasiliou et al., 1999). Family 1
ALDHs include the original Class 1 ALDHs that are targeted
to the cytosol. Family 2 ALDHs include the mitochondrial
Class 2 ALDHs. Family 1 and 2 ALDHs play a major role in
detoxification of ethanol-derived acetaldehyde (Wang et al.,
1998). Aldehydes are produced from lipid peroxidation and are
toxic because of their chemical reactivity. Family 3 ALDHs
detoxify aldehydes formed during lipid peroxidation (Lindahl
and Petersen, 1991).
ALDHs are involved in plant growth, development, and
stress responses (Sunkar et al., 2003; Kotchoni et al., 2006,
2010; Shin et al., 2009). Maize (Zea mays) mitochondrial
ALDH2B2 (also known as rf2a) is essential for normal
anther development and male fertility (Liu et al., 2001; Liu
and Schnable, 2002). Rice (Oryza sativa) OsALDH7 function
is crucial for seed maturation and viability (Shin et al., 2009).
A well-characterized stress-responsive ALDH is the osmotic
stress-inducible betaine aldehyde dehydrogenase, which
catalyses synthesis of the osmolyte glycine betaine using
betaine aldehyde as the substrate (Chen and Murata, 2002).
ALDH3I1 and ALDH7B4 overexpression in Arabidopsis
significantly enhances tolerance to drought, salinity, and
oxidative stress (Kotchoni et al., 2006). The steady-state
ALDH21A1 transcript level in Tortula ruralis is elevated in
response to dehydration, NaCl, abscisic acid (ABA), and UV
irradiation (Chen et al., 2002). Two Arabidopsis ALDH genes
are upregulated by dehydration, high salinity, and cold (Seki
et al., 2002). Two barley ALDHs are upregulated by drought
stress (Ozturk et al., 2002). ALDH protein accumulates to
high levels in the rice lesion mimic mutant cdr2, suggesting
a role for ALDH in plant programmed cell death (PCD) and
defence signalling (Tsunezuka et al., 2005).
In this study, we isolated and identified pepper aldehyde
dehydrogenase CaALDH1 as an AvrBsT-interacting protein using yeast two-hybrid screening. CaALDH1:smGFP
(soluble-modified green fluorescent protein) fluorescence
was detected in the cytoplasm. Heterologous transient coexpression of CaALDH1 and avrBsT in N. benthamiana
leaves significantly enhanced avrBsT-triggered cell death.
Cell death promotion by CaALDH1 expression depended
on CaALDH1 aldehyde dehydrogenase activity. CaALDH1
expression in pepper was rapidly and strongly induced by
avirulent Xcv Ds1 (avrBsT) infection. CaALDH1 silencing in
pepper disrupted Xcv-induced aldehyde dehydrogenase activity, H2O2 accumulation, and cell death response, and reduced
resistance to avirulent Xcv infection. Defence response
gene expression also was reduced by CaALDH1 silencing.
CaALDH1 overexpression (OX) in transgenic Arabidopsis
plants reduced susceptibility to Pseudomonas syringae pv.
tomato and Hyaloperonospora arabidopsidis Noco2 infection. These results demonstrate the functional importance of
pepper aldehyde dehydrogenase CaALDH1 for regulation of
AvrBsT-triggered cell death and defence responses in plants.
Materials and methods
Plant materials and growth conditions
Pepper (Capsicum annuum L. cv. Nockwang) and Nicotiana
benthamiana were planted in plastic pots containing a soil mix
(loam:perlite:vermiculite, 3:1:1, v/v/v) at 28°C with a long-day photoperiod (16 h light/8 h dark) with a light intensity of 100 μmol
photons m-2 s-1. Arabidopsis thaliana ecotype Col-0 were grown in
pots containing vermiculite, peat moss, and perlite (1:1:0.5, v/v/v)
at 24°C, 60% relative humidity and 130 μmol photons m-2 s-1 with a
16 h light photoperiod in a growth chamber.
CaALDH1 in cell death and defence | 3369
Yeast two-hybrid assay
The avrBsT open reading frame (ORF) was cloned into
BamHI/HindIII sites of the pGBKT7 vector. The yeast prey library
was generated from a pepper cDNA library by ligating cDNA inserts
into the pGADT7 vector. The constructs were co-transformed into
yeast strain AH109 and plated onto synthetic dropout (SD) –histidine,
↕leucine, ↕tryptophan (↕HLT) media (Ito et al., 1983). Approximately
50 000 colonies from the SD-HLT media were transferred onto selection media (SD)↕adenine, ↕histidine, ↕leucine, ↕tryptophan (↕AHLT).
Plasmids were extracted from the surviving yeast colonies and used
to transform E. coli. Colonies carrying pGADT7 were selected on
Luria-Bertani (LB) media containing 100 mg l-1 ampicillin. Isolated
plasmids were sequenced, and sequence homology was analysed using
Genbank BLAST tools (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Bimolecular fluorescence complementation assay
Bimolecular fluorescence complementation (BiFC) analyses were
conducted as described previously (Waadt et al., 2008). To generate
the BiFC constructs, the CaALDH1 coding region without termination codons was PCR-amplified and subcloned into the binary vectors pSPYNE (XbaI/XhoI) and pSCYCE (XbaI/XhoI) under control
of the cauliflower mosaic virus 35S promoter (see Supplementary
Table S1 for the oligonucleotide sequences). pSPYNE:avrBsT and
pSPYCE:avrBsT were generated as described previously (Kim et al.,
2014). AvrBsT and CaALDH1 fusion constructs were co-expressed
in N. benthamiana leaves by infiltrating A. tumefaciens strain GV3101
carrying each construct (OD600=0.5). BiFC signals from the AvrBsT
and CaALDH1 interaction were visualized 40 h after infiltration using
a confocal laser scanning microscope (LSM 5 Exciter, Carl-Zeiss,
Oberkochen, Germany) operated with LSM Imager. pSPYCE:bZIP63
and pSPYNE:bZIP63 were used as positive controls.
Immunoblot analysis
Total soluble proteins were extracted from N. benthamiana leaves
with 1 ml denaturing buffer [50 mM Tris-HCl (pH 8.8), 4 M urea,
10 mM sodium phosphate (pH 7.8), 250 mM NaCl, 0.1% Nonidet
P40, 1 mM EDTA, and 0.5% SDS] per 0.5 g leaf tissue. Insoluble
debris was pelleted by centrifuging leaf extracts at 13 000 ×g for
20 min at 4°C. For co-immunoprecipitation analyses, proteins were
extracted in soluble buffer [50 mM Tris-HCl (pH 8.8), 50 mM NaCl,
10 mM EDTA, 0.1% Triton X-100 and 2× protease inhibitor cocktail (Roche, Mannheim, Germany)]. Proteins in the supernatant
were incubated with HA-agarose or cMyc-agarose (Sigma-Aldrich,
St. Louis, USA) 4°C overnight. Proteins were resolved on 8% SDSPAGE gels and transferred to PVDF membrane (GE Healthcare,
Little Chalfont, United Kingdom). Proteins tagged with HA or
cMyc epitopes were detected with anti-HA-peroxidase or anticMyc-peroxidase antibodies (Sigma), respectively.
in pepper plants. The non-conserved, C-terminal untranslated
region of CaALDH1 cDNA was PCR-amplified and cloned into
the pCR2.1-TOPO vector. The gene-specific primers are listed in
Supplementary Table S1. The cloned fragment was digested with
EcoRI and inserted into pTRV2. The pTRV1, pTRV2:00, and
pTRV2:CaALDH1 constructs were transformed into Agrobacterium
strain GV3101. An equal volume of pTRV1 Agrobacterium culture was mixed with one of the pTRV2 cultures before infiltration
(OD600=0.2). The mixed cultures were infiltrated into cotyledons
of pepper seedlings (Choi and Hwang, 2011). Four to five weeks
after VIGS, CaALDH1-silenced plant leaves were used for quantitative RT-PCR and disease assays. The two other pepper ALDH genes
(accession numbers Capana09g000318 and Capana09g000319) that
share high sequence identities of 95.19 and 86.14% with CaALDH1,
respectively, were identified from a BLASTn search of ALDHs from
the pepper genome sequence (http://peppersequence.genomics.cn)
to use for the specific silencing test of CaALDH1.
Arabidopsis transformation
Transgenic Arabidopsis plants expressing CaALDH1 were generated
by the floral-dip method (Clough and Bent, 1998). The CaALDH1
coding region was inserted between the cauliflower mosaic virus
(CaMV) 35S promoter and the nos terminator region in the pBIN35S
binary vector. This construct was introduced into Agrobacterium
tumefaciens GV3101 and used to transform Arabidopsis thaliana
ecotype Columbia 0 (Col-0). Transformed seed stock was selected
for kanamycin resistance by planting seeds on Murashige and Skoog
(Duchefa, Haarlem, The Netherlands) agar plates containing 50 mg
l-1 kanamycin (Duchefa).
Pathogen inoculation
Xanthomonas campestris pv. vesicatoria (Xcv) virulent Ds1 (EV, empty
vector) and avirulent Ds1 (avrBsT) strains, and Pseudomonas syringae
pv. tomato (Pst) DC3000 and DC3000 (avrRpm1) strains, were grown
in YN (5 g of yeast extract and 8 g of nutrient broth l-1) or King’s B
broth (10 g of peptone, 1.5 g of K2HPO4, 15 g of glycerol, and 1 mM
MgSO4 l-1), respectively. The cultured bacteria were harvested and
resuspended in 10 mM MgCl2 solution. Bacterial growth in leaves was
monitored at different time points after infiltration with Xcv (5 × 104
cfu ml-1) and Pst (105 cfu ml-1) using a needleless syringe.
Hyaloperonospora arabidopsidis (Hpa) Noco2 was grown on cotyledons of Arabidopsis seedlings at 16°C, 60% relative humidity, and
a 14 h photoperiod. Hpa conidia (5 × 104 conidia ml-1) were suspended in distilled tap water containing 0.05% Tween 20, and the
inoculum suspension was sprayed onto cotyledons of seven-day-old
Arabidopsis seedlings. The infected plants were incubated at 17°C in
an environmentally controlled chamber.
Agrobacterium-mediated transient expression
For subcellular localization analyses, A. tumefaciens strain GV3101
carrying pBIN35S:GFP or pBIN35S:CaALDH1:GFP was infiltrated
into six-week-old N. benthamiana leaves. To confirm mitochondrial localization, leaf cells were counterstained with MitoTracker
(Invitrogen). The lower epidermal cells were analysed 36↕48 h after
infiltration using a confocal laser scanning microscope (LSM 5 Exciter,
Carl-Zeiss) operated with LSM Imager. For Agrobacterium-mediated
transient expression, the A. tumefaciens strain GV3101 carrying
pBIN35S:avrBsT, pBIN35S:CaALDH1, pBIN35S:CaALDH1E267A, or pBIN35S:CaALDH1-C301A were infiltrated into sixweek-old N. benthamiana leaves (Kim and Hwang, 2015).
RNA gel-blot and real-time RT-PCR analyses
Total RNA was extracted from pepper plants using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was resolved by agarose gel electrophoresis, transferred onto Hybond N+ membranes
(GE Healthcare), and hybridized overnight with 32P-labelled
CaALDH1 cDNA. For real-time RT-PCR, 2 μg of RNA was used
in a reverse-transcription reaction with MMLV reverse transcriptase
(Enzynomics, Daejeon, Korea). Real-time RT-PCR was performed
using iQ SYBR Green Supermix and iCycler iQ (Bio-Rad, Hercules,
CA, USA). Gene-specific primer pairs used for real-time RT-PCR
analysis are listed in Supplementary Table S1. The C. annuum
CaACTIN transcript level was used for normalization of gene transcript levels. Relative expression levels were determined by comparing the calculated values with that of the uninoculated control.
Virus-induced gene silencing
Tobacco rattle virus (TRV)-based virus-induced gene silencing (Liu
et al., 2002) was used to investigate CaALDH1 loss-of-function
Aldehyde dehydrogenase activity assay
Aldehyde dehydrogenase (ALDH) activity was measured from crude
plant extracts (Liu et al., 2001). Leaf tissue was ground in liquid
3370 | Kim and Hwang
nitrogen and extracted with extraction buffer [0.1 M HEPES buffer
(pH 7.4), 1 mM EDTA, 2 mM DTT, and 0.1% Triton X-100]. The
extract was centrifuged at 14 000 ×g for 20 min, and the resulting
supernatant was used as a crude enzyme extract. For each ALDH
assay, 200 mg of crude enzyme extract was added to a reaction mixture containing 1.5 mM NAD (Sigma-Aldrich) and 0.1 M sodium
pyrophosphate buffer (pH 8.5) and the total volume was adjusted to
1.0 ml with distilled water. The mixture was pre-incubated for 30 s
before adding 17 mM acetaldehyde to the mixture. The reaction was
excited at 360 nm, and NADH fluorescence emission was recorded
at 460 nm after 1 min using a model Victor3 fluorescence spectrophotometer (Perkin Elmer, Massachusetts, USA). ALDH activity
was expressed as nanomoles of NADH produced per minute per
milligram of protein.
Electrolyte leakage assay
Leaves of pepper or N. benthamiana plants were harvested at various time points after infiltration with Xcv or Agrobacterium, respectively. Leaf discs (0.5 cm diameter) were excised with a cork borer
and washed in 10 ml of sterile double-distilled water for 30 min with
gentle agitation. Washed leaf discs were transferred to 20 ml of sterile, double-distilled water and incubated for 2 h at room temperature
with gentle agitation. The ion conductivity of the leaf samples was
measured using a Sension 7 conductivity meter (HACH, Loveland,
CO, USA).
Histochemistry
H2O2 accumulation was visualized by placing healthy or inoculated
leaves in 1 mg ml-1 3,3’-diaminobenzidine (DAB) (Sigma-Aldrich)
solution overnight (Thordal-Christensen et al., 1997). Chlorophyll
was cleared from the stained leaves by boiling in 95% ethanol. Cell
death was monitored by trypan-blue staining of healthy or inoculated leaves (Koch and Slusarenko, 1990). Leaves were stained with
lactophenol-trypan blue solution (10 ml of lactic acid, 10 ml of glycerol, 10 g of phenol, and 10 mg of trypan blue, dissolved in 10 ml of
distilled water), and destained in 2.5 g ml-1 chloral hydrate solution.
The samples were photographed using a digital camera (Olympus,
Japan) mounted on a light microscope. Chlorosis, cell death, or phenolic compound accumulation in leaves was visualized using a handheld UV lamp (UVP, CA, USA).
H2O2 measurement
H2O2 accumulation in pepper and Arabidopsis leaves was quantified using xylenol orange (Gay et al., 1999; Choi and Hwang, 2011).
Xylenol orange assay reagent was freshly prepared by adding 500 μl
of solution [25 mM FeSO4 and 25 mM (NH4)SO4 in 2.5 M H2SO4] to
50 ml of 125 μM xylenol orange in 100 mM sorbitol. Eight leaf discs
(0.5 cm2) were floated on 1 ml of distilled water in a microtube for 1 h
and centrifuged for 1 min at 12 000 ×g, and 100 μl of supernatant
was immediately added to 1 ml of xylenol orange assay reagent. The
mixture was incubated for 30 min at room temperature. H2O2 was
quantified by measuring the absorbance at 560 nm using a DU 650
spectrophotometer (Beckman, Urbana, IL, USA) and compared
with a standard curve for H2O2, which was generated by measuring
a serial dilution of 100 nmol to 100 μmol of H2O2.
Results
CaALDH1 interacts with AvrBsT in vitro and in planta
AvrBsT is an Xcv type III effector protein that triggers HR
in pepper and N. benthamiana leaves (Kim et al., 2010).
A yeast two-hybrid screen was used to isolate molecular components that interact with AvrBsT (Fig. 1A). A prey library
was generated by fusing the activation domain (AD) to pepper cDNAs synthesized from transcripts of pepper leaves
undergoing hypersensitive response (HR), and AvrBsT was
fused to the binding domain (BD) and used as bait to screen
the library. One of the AvrBsT-interacting cDNAs encoded
an aldehyde dehydrogenase protein (Supplementary Figs
S1, S2). This clone was designated pepper aldehyde dehydrogenase 1 (CaALDH1) and was subjected to further
characterization. In silico CaALDH1 analyses determined
that it encoded a C4-like member of aldehyde dehydrogenase family 2 (Supplementary Fig. S1). We compared the
amino acid sequences of pepper CaALDH1 protein with
those of other plant species. CaALDH1 shared 66–88%
sequence identities with other ALDHs from N. tomentosiformis (88%, NtALDH; XP_009627665), N. sylvestris
(88%, NsALDH; XP_009803645), grass (68%, LcALDH;
ABO93608), Arabidopsis (67%, AtALDH; NP_566749),
rice (66%, OsALDH; NP_001043453), and maize (66%,
ZmALDH; AAL99608) (Supplementary Fig. S2A).
However, any ALDH homologs of N. benthamiana were not
searched from the NCBI’s GenBank database. Residues 267
and 301, glutamic acid and cysteine, respectively, are predicted to be essential for catalytic activity (Supplementary
Figs S1, S2).
To investigate whether CaALDH1 interacts with AvrBsT
in yeast, we swapped vectors and generated a DNA-binding
domain (BD) fused with CaALDH1 and an activation
domain (AD) fused with AvrBsT. We transformed these
constructs into yeast with positive-control and negativecontrol vector pairs. AD-AvrBsT and BD- CaALDH1
interacted with each other and grew on selection media, as
did BD-AvrBsT and AD-CaALDH1 (Fig. 1A). To investigate whether CaALDH1 and AvrBsT interact in planta,
we performed co-immunoprecipitation (co-IP) analysis in N. benthamiana leaves using transiently expressed
CaALDH1:HA and AvrBsT:cMyc (Fig. 1B). Extracted
proteins were immunoprecipitated with α-cMyc or α-HA
beads, separated by SDS-PAGE, and immunoblotted with
α-cMyc and α-HA antibodies. Figure 1B shows that immunoblotting with α-Myc and α-HA antibodies detected
AvrBsT:cMyc and CaALDH1:HA, respectively. Therefore,
the co-IP analysis indicated that CaALDH1:HA physically
interacted with AvrBsT:cMyc in planta.
Bimolecular fluorescence complementation (BiFC) assays
were used to further investigate CaALDH1 and AvrBsT interaction in planta (Fig. 1C). CaALDH1 and AvrBsT were fused
to the yellow fluorescent protein (YFP) N- and C-termini
(Waadt et al., 2008). Agrobacterium cells harbouring the
corresponding constructs were mixed and co-infiltrated into
N. benthamiana leaves. Confocal images of N. benthamiana
epidermal cells detected YFP fluorescence in the cytoplasm
but not in the nucleus (Fig. 1C), suggesting that CaALDH1
and AvrBsT interact with each other in the plant cytoplasm.
As a positive control, the bZIP63:YFP transcription-factor
construct exhibited fluorescence in the nucleus. Immunoblot
assay results indicate that all YFP fusion proteins were transiently expressed with appropriate molecular weights in
N. benthamiana leaves (Fig. 1D).
CaALDH1 in cell death and defence | 3371
CaALDH1 expression is specifically induced in pepper
leaves by avirulent Xcv infection
RNA gel blot analyses were used to investigate CaALDH1
expression profiles during pepper plant interactions with
Xcv. CaALDH1 expression was strongly induced in pepper
leaves during avirulent Xcv Ds1 (avrBsT) infection, compared with that of the mock or virulent Xcv Ds1 (EV) infection (Fig. 2). During incompatible interactions, CaALDH1
expression distinctly increased 5 h after inoculation, and the
high expression level was maintained up to 25 h after inoculation with avirulent Xcv Ds1 (avrBsT), indicating that avrBsT
is required for induction of CaALDH1 expression. In contrast, CaALDH1 expression was not detected at any time
point during mock or compatible interactions. These results
indicate that CaALDH1 expression is strongly and specifically induced during incompatible interactions with avirulent
Xcv Ds1 (avrBsT).
CaALDH1 is localized to the cytoplasm
It has been proposed that human ALDHs be categorized
as cytosolic ALDH1 and mitochondrial ALDH2 that are
involved primarily in ethanol metabolism (Hsu et al., 1988;
Hsu et al., 1989; Wang et al., 1998; Vasiliou et al., 1999).
Family 1 ALDHs include the Class 1 ALDHs that are localized to the cytosol. Family 2 ALDHs are classified as the
mitochondrial Class 2 ALDHs. To investigate CaALDH1
subcellular localization, a CaALDH1 fusion with solublemodified green fluorescent protein (smGFP) (Davis and
Vierstra, 1998) was transiently expressed in N. benthamiana
leaves using agroinfiltration (Fig. 3). The control smGFP
demonstrated that GFP fluorescence was ubiquitously distributed throughout the cell including the nucleus (Fig. 3).
The CaALDH1:GFP fusion protein primarily localized to
the cytoplasm (Fig. 3). The CaALDH1:GFP fusion proteins
were also observed as small dots in the cytoplasm, but did
not co-localize to the mitochondria (Supplementary Fig. S3).
These results suggest that CaALDH1 belongs to the cytosolic
Class1 ALDHs.
Transient CaALDH1 expression promotes avrBsTtriggered cell death, but not Bax-triggered cell death
Fig. 1. AvrBsT interacts with CaALDH1 in yeast and in planta. (A) Yeast
two-hybrid assay. CaALDH1 and AvrBsT fused with GAL4 activation domain
(AD) or binding domain (BD) were co-introduced into Saccharomyces
cerevisiae strain AH109, and reporter gene activation was monitored
on synthetic dropout (SD)-AHLT (minus adenine, histidine, leucine, and
tryptophan) medium containing X-α-gal. Combinations of Lam and p53
with SV40-T were used as negative and positive controls, respectively. (B)
Co-immunoprecipitation analyses of transiently expressed CaALDH1:HA
and AvrBsT:cMyc in N. benthamiana leaves. Extracted proteins were
immunoprecipitated with α-cMyc or α-HA beads, and immunoblotted
with α-cMyc and α-HA antibodies. (C) Bimolecular fluorescence
complementation images of interactions between AvrBsT and CaALDH1
in N. benthamiana leaves. YFP fluorescence was visualized 30 h after
agroinfiltration using a confocal laser scanning microscope. bZIP63:YFPN
and bZIP63:YFPC constructs were used as positive controls. Bars=50 µm.
(D) Immunoblot analyses of YFP fusion proteins transiently expressed in
N. benthamiana leaves. Protein loading was visualized by Coomassie brilliant
blue (CBB) staining. (This figure is available in colour at JXB online.)
Co-infiltration with low titers (OD600=0.05) of
Agrobacterium harbouring CaALDH1 and avrBsT accelerated cell death in N. benthamiana leaves (Fig. 4). Figure 4A
shows that transient CaALDH1 expression (OD600=0.2)
did not induce any cell death response, and transient avrBsT
expression (OD600=0.05) did not induce typical HR cell
death. However, coexpression of CaALDH1 with avrBsT
(OD600=0.05) induced a severe cell death response, similar
to that induced by avrBsT agroinfiltration at OD600=0.2
(Fig. 4A). The Bcl-2-associated X protein (Bax) that induces
hypersensitive cell death in N. benathamiana (Lacomme and
Santa Cruz, 1999) was used as a positive control. As in avrBsT
expression (OD600=0.2), Bax expression (OD600=0.2)
induced typical HR cell death (Fig. 4A). By contrast, coexpression of Bax with CaALDH1 resulting from infiltration
3372 | Kim and Hwang
Xanthomonas campestris pv. vesicatoria
Mock
H
1
5
10 15 20 25 1
Ds 1 (EV)
5
10 15 20 25
Ds1 (avrBsT)
1
5
10 15 20 25 h
CaALDH1
rRNA
Fig. 2. RNA gel blot analysis of CaALDH1 expression in pepper leaves infected with virulent Ds1 (EV) or avirulent Ds1 (avrBsT) Xcv. RNA extracted from
pepper plants was blotted to nylon membrane and hybridized with 32P-labelled CaALDH1 probes. rRNA is shown as a loading control. H, healthy leaves;
Mock, treated with 10 mM MgCl2; EV, empty vector.
Bright field
Merged
CaALDH1:smGFP
smGFP
GFP
Fig. 3. Subcellular localization of CaALDH1. Agrobacterium-mediated
transient expression of CaALDH1:smGFP in N. benthamiana epidermal
cells. GFP fluorescence was visualized using a confocal laser scanning
microscope 48 h after agroinfiltration. Bars=50 µm. (This figure is available
in colour at JXB online.)
with Agrobacterium at OD600=0.05 did not produce a
cell death response. Co-expression of avrBsT or Bax with
CaALDH1-E267A or CaALDH1-C301A (inactive mutations
of CaALDH1) did not produce severe cell death in N. benthamiana leaves. These results indicate that CaALDH1 specifically interacts with AvrBsT to promote AvrBsT-triggered
cell death.
The extent of cell death was quantified by measuring electrolyte leakage from N. benthamiana leaf discs transiently
expressing CaALDH1 and/or avrBsT or Bax (Fig. 4B).
Control leaves expressing empty vector and leaves transiently expressing CaALDH1 did not exhibit electrolyte
leakage. These results were consistent with the observed
cell death phenotypes. However, leaf tissues co-expressing
CaALDH1 with avrBsT, but not with Bax, at the low titers
(OD600=0.05) exhibited high levels of electrolyte leakage,
similar to that of leaves transiently expressing high levels of avrBsT or Bax at 24 and 48 h after agroinfiltration
(OD600=0.2). Leaves co-expressing inactive CaALDH1
mutants with avrBsT or Bax did not show high levels of
electrolyte leakage. Immunoblot analyses confirmed that
CaALDH1,
CaALDH1-E267A,
CaALDH1-C301A,
AvrBsT and Bax proteins were transiently expressed in
N. benthamiana leaves (Fig. 4C). Collectively, these results
indicate that CaALDH1 expression promotes avrBsT-triggered hypersensitive cell death response.
CaALDH1 exhibits aldehyde dehydrogenase activity in
planta
Crude extracts prepared from N. benthamiana leaves agroinfiltrated with empty vector control, CaALDH1, CaALDH1E267A, CaALDH1-C301A, and avrBsT were assayed
for aldehyde dehydrogenase activity using acetaldehyde
as substrate (Fig. 5A). Transient CaALDH1 expression
(OD600=0.2) caused a 3-fold increase in ALDH activity
compared to that of the empty vector control 24 h after agroinfiltration. Transient avrBsT expression (OD600=0.05)
caused a slight elevation in ALDH activity 48 h after agroinfiltration. However, avrBsT and CaALDH1 co-expression
(OD600=0.05) caused a 2-fold increase in ALDH activity
24 h after agroinfiltration, similar to that of avrBsT expression (OD600=0.2). Co-expression of inactive CaALDH1
mutants (E267A or C301A) with avrBsT (OD600=0.05) did
not affect ALDH activity. Crude extracts prepared from
N. benthamiana leaves infiltrated with empty vector control,
CaALDH1, inactive CaALDH1 mutants (E267A or C301A),
and Bax were assayed for ALDH activity using acetaldehyde
as the substrate (Fig. 5B). Co-expression of CaALDH1 or
inactive CaALDH1 mutants (E267A or C301A) with Bax
(OD600=0.05) did not affect ALDH activity. These results
indicate that CaALDH1 expression induces ALDH activity,
which is required for avrBsT-triggered plant cell death.
CaALDH1 silencing reduces avrBsT-mediated
resistance, ROS burst, and cell death during Xcv
infection
The tobacco rattle virus (TRV)-based virus-induced gene
silencing (VIGS) technique was used to generate CaALDH1
loss-of-function pepper plants (Liu et al., 2002). To reduce the
nonspecific silencing effect, the non-conserved, C-terminal
untranslated region (UTR) of CaALDH1 cDNA was cloned
into the TRV2 vector to specifically silence CaALDH1.
RT-PCR analysis showed that CaALDH1 was effectively
silenced in pepper plants infected with Xcv (Supplementary
Fig. S4). Leaves of non-silenced (TRV:00) and CaALDH1silenced (TRV:CaALDH1) pepper were inoculated with virulent Xcv Ds1 (EV) or avirulent Ds1 (avrBsT) strains (107 and
108 cfu ml-1). Avirulent Xcv infection (108 cfu ml-1) of nonsilenced (TRV:00) leaves caused HR and complete necrosis
two days after inoculation; however, CaALDH1 silencing
greatly reduced HR to avirulent Xcv infection (Fig. 6A).
Reduced cell death in CaALDH1-silenced leaves was evident
CaALDH1 in cell death and defence | 3373
(A)
600 :
EV
0.2
avrBsT
0.05
CaALDH1
/ avrBsT
0.05/0.05
CaALDH1 CaALDH1
E267A
C301A
/ avrBsT
/ avrBsT
0.05/0.05 0.05/0.05
avrBsT
0.2
Bax
0.05
CaALDH1
/ Bax
0.05/0.05
CaALDH1 CaALDH1
E267A
C301A
/ Bax
/ Bax
0.05/0.05 0.05/0.05
Bax
0.2
CaALDH1
0.2
Cell death
600 :
d
d
3
c
2
b
b
1
a
b
b
b
b
b
a
0
Conductivity (µS cm-1 )
(B)
(C)
25
c
20
15
c
10
5
c
a a a aa a a aa a a a
0
b
b
b
a a
a a a a
aa
0
a a
48
+
-
-
-
-
-
-
-
-
-
-
- +
- +
-
-
-
-
CaALDH1E267A:HA
-
-
-
+ -
-
-
CaALDH1C301A:HA
-
-
-
-
-
+ -
avrBsT:cMyc
-
-
+ + + + +
Bax
-
-
-
-
-
-
-
b b b b
bb
24
-
CaALDH1:HA
b
-
hai
+ - - + -
-
-
+
-
-
-
-
- + + + +
+
-
-
OD600 :
kDa 70
α-HA
50
α cMyc 50
CBB
kDa 70
α HA
50
25
α -Bax
20
CBB
Fig. 4. Transient CaALDH1 expression promotes avrBsT-triggered cell death, but not Bax-triggered cell death. (A) Cell-death phenotypes and
quantification in N. benthamiana leaves 2 days after infiltration with Agrobacterium carrying CaALDH1, CaALDH1-E267A, CaALDH1-C301A, avrBsT
or Bax at different inoculum ratios. Cell death was quantified based on a 0−3 scale: 0, no cell death (<10%); 1, weak cell death (10–30%); 2, partial
cell death (30–80%); and 3, full cell death (80–100%). Data are means ±SD from three independent experiments. Different letters indicate statistically
significant differences (LSD, P<0.05). (B) Electrolyte leakage from leaf discs at different time points after infiltration with Agrobacterium carrying the
indicated constructs at different inoculum ratios. Data are means ±SD from three independent experiments. Different letters indicate statistically significant
differences (LSD, P<0.05). (C) Immunoblot analyses of transient expression of CaALDH1, CaALDH1-E267A, CaALDH1-C301A, BAX and avrBs 2 days
after agroinfiltration. Protein loading was visualized by Coomassie brilliant blue (CBB) staining. (This figure is available in colour at JXB online.)
under UV illumination (Fig. 6A). Avirulent Xcv Ds1 (avrBsT)
growth in CaALDH1-silenced leaves was approximately
7-fold higher than that in non-silenced (TRV:00) leaves three
days after inoculation (Fig. 6B).
Early defence responses such as ROS (H2O2) accumulation and cell death response were analysed in non-silenced
(TRV:00) and CaALDH1-silenced pepper leaves during
Xcv infection (Fig. 6C, D). H2O2 production and cell death
were visualized by DAB (Fig. 6C) and trypan-blue staining
(Fig. 6E), respectively. Significantly reduced H2O2 accumulation and cell death responses were observed in CaALDH1silenced leaves inoculated with Xcv. Xylenol orange assay and
ion conductivity measurements were used to quantify H2O2
production and cell death, respectively. CaALDH1 silencing
3374 | Kim and Hwang
(A)
5
35S:EV (OD600=0.2)
35S:CaALDH1 E267A / 35S:avrBsT (OD600=0.05)
35S:CaALDH1 (OD600=0.2)
35S:CaALDH1 C301A / 35S:avrBsT (OD600=0.05)
35S:avrBsT (OD600=0.05)
35S:avrBsT (OD600=0.2)
35S:CaALDH1 /
35S:avrBsT (OD600=0.05)
RFU 460 nm
d
c
4
3
2
b
a
aaaa aa
a
a
aa
c
c
b
bb
b
a
1
0
0
24
48
Hours after infiltration
(B)
35S:EV (OD600=0.2)
5
35S:CaALDH1 E267A / 35S:Bax (OD600=0.05)
35S:CaALDH1 (OD600=0.2)
35S:CaALDH1 C301A / 35S:Bax (OD600=0.05)
35S:Bax(OD600=0.05)
35S:Bax (OD600=0.2)
35S:CaALDH1 /
35S:Bax (OD600=0.05)
RFU 460 nm
4
d
c
3
2
c
aaaaaa a
a
a
aa a a
a
a
b
aa
1
0
0
24
48
Hours after infiltration
Fig. 5. Aldehyde dehydrogenase activity assay. (A) Coexpression of CaALDH1, CaALDH1-E267A, or CaALDH1-C301A with avrBsT. (B) Coexpression of
CaALDH1, CaALDH1-E267A, or CaALDH1-C301A with Bax. N. benthamiana leaves were infiltrated with Agrobacterium carrying the indicated constructs
at different inoculum ratios. ALDH activity in crude extracts was assayed at 0, 24 and 48 h after agroinfiltration. RFU 460 nm, relative fluorescence units at
460 nm.
significantly reduced H2O2 production in pepper leaves during virulent Xcv Ds1 and avirulent Xcv Ds1 (avrBsT) infection (Fig. 6D). Cell death triggered by avirulent Xcv Ds1
(avrBsT) infection was significantly reduced, with a corresponding reduction in electrolyte leakage from infected pepper leaf tissue (Fig. 6F). Collectively, these results indicate
that CaALDH1 is required for avrBsT-mediated resistance to
Xcv infection.
CaALDH1 silencing attenuates ALDH activity and
defence-responsive gene expression
ALDH activity was analysed in leaves of non-silenced
(TRV:00) and silenced (TRV:CaALDH1) pepper plants during Xcv infection. Crude extracts prepared from pepper leaves
0, 6, 12, 18 and 24 h after inoculation with Xcv were analysed
for ALDH activity using acetaldehyde as the substrate (Fig. 7).
Avirulent Xcv Ds1 (avrBsT) infection induced substantially
higher ALDH activity in non-silenced (TRV:00) leaves than
that of virulent Xcv Ds1 (EV) infection. CaALDH1 silencing
significantly reduced induction of ALDH activity during virulent Xcv Ds1 (EV) infection. Avirulent Xcv Ds1 (avrBsT) infection induced 2↕4-fold higher ALDH activity in empty-vector
control leaves than in CaALDH1-silenced leaves 6↕24 h after
inoculation.
Real-time RT-PCR analyses were performed using genespecific primer pairs for CaALDH1, CaPR1 (PR1) (Kim
and Hwang, 2000), CaDEF1 (defensin) (Do et al., 2004),
and CaPR10 (PR10) (Choi et al., 2012) during Xcv infection
(Fig. 8) to determine the effects of CaALDH1 silencing on the
expression of defence-related genes at early infection stages
in pepper. CaALDH1 silencing in pepper leaves significantly
CaALDH1 in cell death and defence | 3375
(A)
Visible
UV
(B)
cfu mL-1
6
107
TRV:00
Log cfu cm-2
108
107
TRV:
CaALDH1
TRV:CaALDH1
TRV:00
7
*
5
4
3
2
1
108
0
0
1
3
0
1
3 dai
Xcv Ds1 (avrBsT)
Xcv Ds1 (EV)
Ds1
Ds1
Ds1
Ds1
(EV) (avrBsT) (EV) (avrBsT)
Xcv Ds1 (EV) Xcv Ds1(avrBsT)
(D)
TRV:00
TRV:
CaALDH1
2.5
2.0
1.5
*
1.0
*
*
*
*
*
0.5
0.0
Xcv Ds1 (EV)
Xcv Ds1(avrBsT)
(F)
TRV:00
TRV:
CaALDH1
8
Conductivity (µS cm-1 )
(E)
TRV:CaALDH1
TRV:00
3.0
H2O2 (µmol cm-2 )
(C)
H
6 12 18
Xcv Ds1(EV)
TRV:00
6
12
18 hai
Xcv Ds1(avrBsT)
TRV:CaALDH1
6
*
4
*
2
0
0
12
24
Xcv Ds1(EV)
0
12
24 hai
Xcv Ds1(avrBsT)
Fig. 6. Enhanced susceptibility of CaALDH1-silenced pepper leaves to avirulent Ds1 (avrBsT) or virulent Ds1 (EV) Xcv infection. (A) Enhanced disease symptoms
on CaALDH1-silenced pepper leaves. Yellow line, symptoms visible only under UV illumination; orange line, visible symptoms; red line, complete cell death.
(B) Bacterial growth in leaves at 0, 1 and 3 days after Xcv infiltration. Xcv Ds1 (EV) and Ds1 (avrBsT) (5 × 105 cfu ml-1) were infiltrated into empty-vector control
(TRV:00) and CaALDH1-silenced (TRV:CaALDH1) leaves. (C) DAB staining and (D) quantification of H2O2 at different time points after infiltration with Xcv Ds1 (EV)
and Ds1 (avrBsT) (5 × 107 cfu ml-1). (E) Trypan blue staining and (F) electrolyte leakage from leaves infiltrated with Xcv Ds1 (EV) and Ds1 (avrBsT) (5 × 107 cfu ml-1).
Trypan blue staining was performed 24 h after infiltration. Asterisks indicate statistically significant differences (t-test; P<0.05). Data are mean values ±SD from
three independent experiments with four replicates each. EV, empty vector. (This figure is available in colour at JXB online.)
5
attenuated expression of salicylic acid (SA)-dependent
defence genes CaPR1 and CaPR10, but not the jasmonaterelated gene CaDEF1, during avirulent Xcv infection. These
results indicate that CaALDH1 is involved in SA-dependent
defence signalling during compatible and incompatible interactions of Xcv with pepper.
TRV:CaALDH1
TRV:00
RFU 460 nm
4
3
2
*
1
0
*
*
*
0
6 12 18 24
Xcv Ds1 (EV)
*
*
0 6 12 18 24 hpi
Xcv Ds1 (avrBsT)
Fig. 7. Decreased aldehyde dehydrogenase activity in CaALDH1-silenced
pepper leaves infiltrated with Xcv Ds1 (EV) and Ds1 (avrBsT) (5 × 105
cfu ml-1). ALDH activity in pepper leaf crude extracts was analysed at
different time points after Xcv infiltration. Asterisks indicate statistically
significant differences (t-test; P<0.05). Data are mean values ±SD from
three independent experiments. RFU 460 nm, relative fluorescence units at
460 nm; EV, empty vector.
Enhanced defence response of CaALDH1overexpressing Arabidopsis to P. syringae pv. tomato
and H. arabidopsidis
The effect of ectopic CaALDH1 expression on disease resistance was analysed by generating transgenic
CaALDH1-overexpressing (OX) Arabidopsis plants using the
floral-dip method (Clough and Bent, 1998). Three independent CaALDH1-OX transgenic lines (#5, #6, and #9) were
confirmed by RT-PCR to constitutively express CaALDH1
(Supplementary Fig. S5). Four-week-old wild-type and
3376 | Kim and Hwang
TRV:00
4
CaALDH1
5
Relative gene expression
3
CaPR1
3
*
1
0.8
0.6
0.4
0.2
0.0
TRV:CaALDH1
4
2
0
1.6
1.4
1.2
1.0
6
1
*
*
CaPR10
*
0
12
24
Xcv Ds1(EV)
2
12
24 hai
Xcv Ds1(avrBsT)
0
16
14
12
10
8
6
4
2
0
*
*
*
CaDEF1
0
12
24
Xcv Ds1(EV)
12
24 hai
Xcv Ds1(avrBsT)
Fig. 8. Quantitative real-time RT-PCR analysis of defence-related gene expression in empty-vector control (TRV:00) and CaALDH1-silenced
(TRV:CaALDH1) pepper leaves infiltrated with Xcv Ds1 (EV) and Ds1 (avrBsT) (5 × 107 cfu ml-1). CaPR1, PR1; CaPR10, PR10; CaDEF1, defensin1.
Expression levels of Capsicum annuum CaACTIN were used to normalize defence-related gene expression levels. Asterisks indicate statistically
significant differences (t-test; P<0.05). Data are mean values ±SD from three independent experiments. EV, empty vector.
CaALDH1-OX Arabidopsis plants were inoculated with Pst
DC3000 and DC3000 (avrRpm1) (5 × 105 cfu ml-1) (Fig. 9A).
Viable bacterial counts in leaves increased approximately
10-fold more in wild-type plants than in CaALDH1-OX
plants three days after inoculation of Pst DC3000. The Pst
DC3000 (avrRpm1) bacterial titers in CaALDH1-OX plants
were significantly lower than those in wild-type plants. These
data indicate that CaALDH1 overexpression enhances
Arabidopsis defence responses and attenuates Pst DC3000
and DC3000 (avrRpm1) growth in planta.
CaALDH1 overexpression strengthened early defence
responses such as ROS burst and cell death response in
Arabidopsis leaves during Pst infection. CaALDH1-OX lines
exhibited higher H2O2 accumulation levels in response to Pst
infection compared with that of wild-type (Fig. 9B). H2O2
bursts during 3–12 h after inoculation with Pst were significantly higher in CaALDH1-OX Arabidopsis leaves compared
with those in wild-type leaves. The cell death response was
quantified as the level of electrolyte leakage from discs of
infected leaves (Fig. 9C). CaALDH1 overexpression led to
significantly higher electrolyte leakage from leaf tissues 9–12 h
after Pst infection compared with that in wild-type leaves
(Fig. 9C). In general, infection with avirulent Pst DC3000
(avrRpm1) induced higher ROS burst and electrolyte leakage
from Arabidopsis leaves compared with that of virulent Pst
DC3000 infection.
ALDH activity levels were analysed in leaves of wild-type
and CaALDH1-OX Arabidopsis plants inoculated with Pst
DC3000 (5 × 107 cfu ml-1) (Fig. 10). When challenged with
Pst DC3000 and DC3000 (avrRpm1), CaALDH1-OX plants
exhibited significantly higher ALDH activity compared
with that of wild-type leaves during infection. Pst DC3000
(avrRpm1) infection induced higher ALDH activity compared with that of Pst DC3000 infection in Arabidopsis
leaves.
Seven-day-old wild-type and transgenic Arabidopsis seedlings were inoculated with a conidiospore suspension of
Hpa isolate Noco2 (Fig. 11). CaALDH1-OX lines exhibited
reduced susceptibility to Hpa infection (Fig. 11A). A significant decrease in the number of sporangiophores was observed
in cotyledons of CaALDH1-OX seedlings compared with the
observed sporangiophore proliferation in wild-type plants
five days after inoculation (Fig. 11B).
Discussion
Pepper leaves infected with the virulent Xcv Ds1 develop
susceptible lesions, which appear water-soaked and turn yellow three days after inoculation (Lee and Hwang, 1996). Xcv
type III effector AvrBsT induces hypersensitive cell death and
defence responses in pepper, but not in tomato (Kim et al.,
2010). Introduction of AvrBsT into Xcv Ds1 rendered the
strain avirulent to pepper plants (Kim et al., 2010). Xcv Ds1
(avrBsT) infection induced hypersensitive response (HR) in
pepper leaves. AvrBsT translocates into pepper cells via the
Xcv type III secretion system, and triggers a HR during infection (Escolar et al., 2001). AvrBsT also triggers hypersensitive cell death in N. benthamiana (Orth et al., 2000; Kim et al.,
2010). AvrBsT activates effector-triggered immunity (ETI)
in Arabidopsis thaliana Pi-0 plants (Cunnac et al., 2007). We
recently identified that pepper CaSGT1 (suppressor of the
G2 allele of skp1) interacted with AvrBsT (Kim et al., 2014)
and promoted AvrBsT-triggered hypersensitive cell death in
CaALDH1 in cell death and defence | 3377
(A)
WT
8
Log cfu cm-2
#6
#9
Pst DC3000 (avrRpm1)
b
aa a
b
aaa
a a aa
a aaa
b
b
aa a
aa a
2
0
(B)
H2O2 (µmol cm-2 )
Pst DC3000
6
4
#5
2.0
0
1
1.0
0.0
Conductivity (µS cm-1 )
(C)
60
50
40
30
20
10
0
0
Pst DC3000
aa aa
a
bb b a
0
b bb
3 dai
a
b bb
a
bb b
a
bb b a
bb b a
bb b a
bb b
a aa a
3
6
9
12
a aa a a a a a
0
3
6
9
12 hai
Pst DC3000 (avrRpm1)
Pst DC3000
0
1
Pst DC3000 (avrRpm1)
1.5
0.5
3
a aa a
3
a
b bb a
aaa a
b bb
a
bb b
a
bb b
a aa a
a aa a
6
9
12
0
3
6
9
12 hai
Fig. 9. Enhanced resistance of CaALDH1-overexpressing Arabidopsis plants to Pst DC3000 and DC3000 (avrRpm1). (A) Bacterial growth in leaves of
wild-type and CaALDH1-overexpressing plants at 0, 1 and 3 days after inoculation (5 × 105 cfu ml-1). (B) H2O2 quantification at different time points after
inoculation (5 × 107 cfu ml-1). (C) Electrolyte leakage measurement. Wild-type and CaALDH1-overexpressing leaves were infiltrated with Pst DC3000
and DC3000 (avrRpm1) (5 × 107 cfu ml-1), and electrolyte leakage was monitored at the indicated time points. Data are mean values ±SD from three
independent experiments with four replicates each. Different letters indicate significant differences at different time points (Fisher’s least significant
differences; P<0.05).
WT
RFU 460 nm
5
#5
Pst DC3000
#6
#9
Pst DC3000 (avrRpm1)
4
3
b bb
2
1
0
b bb
a
0
bbb
a
bb
b bb
a
a
b bb
a
bbb
b
bbb
a
a
a
6
12
24
0
6
12
24 hai
Fig. 10. Enhanced aldehyde dehydrogenase activity in leaves of CaALDH1-overexpressing Arabidopsis lines in response to Pst DC3000 and DC3000
(avrRpm1) (5 × 107 cfu ml-1) infection. ALDH activity in pepper leaf crude extracts was analysed at the indicated time points after infiltration with Pst. RFU
460 nm, relative fluorescence units at 460 nm.
a phosphorylation-dependent manner. Our previous findings
support the possibility that AvrBsT may interact with some host
receptors related to plant cell death. Here, we demonstrated
that pepper aldehyde dehydrogenase 1 (CaALDH1) interacted with AvrBsT in planta, and promoted AvrBsT-triggered
cell death and defence responses in plants. The compelling
data prompted an investigation into the role of CaALDH1 in
AvrBsT-triggered cell death and defence response.
NAD(P)+-dependent aldehyde dehydrogenase is involved in
the detoxification of stress-generated aldehydes (Kirch et al.,
3378 | Kim and Hwang
Fig. 11. Enhanced resistance of CaALDH1-overexpressing Arabidopsis
plants to Hpa Noco2 infection. (A) Disease symptoms on cotyledons of
wild-type and CaALDH1-overexpressing plants 7 days after inoculation
with Hpa Noco2. (B) Number of sporangiophores per cotyledon of wildtype and CaALDH1-overexpressing plants at 5 days after inoculation.
Data were quantified based on the number of sporangiophores counted
per cotyledon as follows: 0, no sporulation; 1−9, light sporulation; 10−19,
medium sporulation; ≥20, heavy sporulation. More than 50 cotyledons of
wild-type and CaALDH1-overexpressing plant lines were counted. Average
numbers of sporangiophores are given below the chart. Data represent
mean values ±SD from three independent experiments. Different letters
indicate statistically significant differences (LSD, P<0.05). (This figure is
available in colour at JXB online.)
2004). Aldehyde dehydrogenases are generally localized to
mitochondria or cytosol (Sophos and Vasiliou, 2003; Kirch
et al., 2004). The Arabidopsis ALDH2C4 shares 98% identity
with CaALDH1, and is predicted to accumulate in the cytosol
(Skibbe et al., 2002). We show that CaALDH1:smGFP accumulates primarily in the cytosol. ALDHs scavenge toxic aldehydes
generated from abiotic stresses. ALDH3I1 and ALDH7B4
overexpression enhances tolerance to drought, salinity, and
oxidative stress in Arabidopsis (Sunkar et al., 2003; Kotchoni
et al., 2006). Our transient co-expression analyses indicate that
CaALDH1 promotes AvrBsT-triggered cell death, but not
Bax-triggered cell death, in N. benthamiana. The Bax protein
has been known as a general cell death trigger in N. benthamiana (Lacomme and Santa Cruz, 1999). Bax is reported to
promote apoptosis through its action on mitochondria and
downstream activation of caspases in mammals (Arnoult
et al., 2003). Yeast two-hybrid, bimolecular fluorescence complementation and co-immunoprecipitation assays revealed the
physical interaction between CaALDH1 and AvrBsT in yeast
and in planta. These results support the notion that CaALDH1
may be directly involved in promoting AvrBsT-triggered cell
death. Heterologous CaALDH1 coexpression with avrBsT
in N. benthamiana significantly enhances avrBsT-triggered
cell death. Cell death induction is dependent on aldehyde
dehydrogenase activity of CaALDH1. However, co-expression
of inactive CaALDH1 mutants (E267A and C301A) did not
promote avrBsT-triggered cell death. Collectively, these results
suggest that the CaALDH1-complex formation contributes
positively to the promotion of AvrBsT-triggered cell death.
RNA gel-blot analysis indicates that CaALDH1 is strongly
induced in pepper leaves by avirulent Xcv (avrBsT) infection.
ALDHs are predicted to play an important role for defence
responses by detoxifying stress-generated aldehydes (Kirch
et al., 2004). Loss-of-function analyses of CaALDH1 via virusinduced gene silencing shows that CaALDH1 aldehyde dehydrogenase activity is important for R gene-mediated defence
responses in pepper. CaALDH1-silenced pepper plants exhibit
significantly reduced ALDH activity in leaves. CaALDH1
silencing attenuated ALDH activity and ROS burst, and significantly reduced cell death and defence responses to avirulent
Xcv (avrBsT) infection. CaALDH1-silenced plants also exhibit
significantly reduced expression of defence-related genes such
as CaPR1 (Kim and Hwang, 2000) and CaPR10 (Choi et al.,
2012), which have been identified in pepper as a defenceresponse marker and a cell-death regulator, respectively.
CaALDH1 overexpression in Arabidopsis suppressed Pst
DC3000 and DC3000 (avrRpm1) growth. CaALDH1-OX
plants accumulate significantly higher ROS levels during Pst
infection, ultimately resulting in enhanced cell death. The
ROS burst mediates cell-defence responses in pepper plants
(Choi et al., 2007). These results support a potential functional role of CaALDH1 in plant immunity to Pst infection.
This role is observed during obligate biotrophic downy mildew infection. CaALDH1 overexpression strongly suppresses
Hpa Noco2 growth. These results support the proposal that
CaALDH1 activity effectively enhances defence response to
biotrophic oomycete infection. Together, these data suggest
that CaALDH1 is required to trigger basal defence and R
gene-mediated resistance to microbial pathogens in plants.
We integrate these data to propose a working model for
the role of the CaALDH1 and AvrBsT complex in plant cell
death and defence signalling in response to microbial pathogens (Supplementary Fig. S6). Xcv Ds1 (avrBsT) secretes the
type III effector AvrBsT into host plant cells to modulate
immune signalling during infection. Expression of avrBsT
in Xcv Ds1 rendered the strain avirulent to pepper plants
(Kim et al., 2010). Infection of pepper leaves with Xcv Ds1
(avrBsT) expressing avrBsT triggers hypersensitive response
(HR) accompanied by strong H2O2 generation, callose deposition and defence-marker gene expressions. Rapid induction
of pepper CaALDH1 is triggered by Xcv (avrBsT) challenge.
CaALDH1 expression induces ALDH activity, ROS burst,
cell death response as well as expression of salicylic acid
(SA)-dependent defence genes in plants. CaALDH1 interacts
with AvrBsT in the plant cell cytoplasm to promote AvrBsTtriggered cell death and defence responses. The CaALDH1
and AvrBsT complex promotes ALDH activity, which may
promote ROS burst and induce expression of some PR
genes such as CaPR1 (Kim and Hwang, 2000) and CaPR10
(Choi et al., 2012). The cumulative effect of CaALDH1
activity enhances HR-like cell death and defence responses.
CaALDH1 together with AvrBsT may function upstream
CaALDH1 in cell death and defence | 3379
of ROS-mediated cell-death signalling during Xcv (avrBsT)
infection. Collectively, the results presented in this study suggest that CaALDH1 acts as a positive regulator that promotes
AvrBsT-triggered cell death and defence responses in plants.
Supplementary material
Supplementary data are available at JXB online.
Supplementary Fig. S1. Nucleotide and deduced amino
acid sequences of pepper CaALDH1 cDNA.
Supplementary Fig. S2. Comparison of aldehyde dehydrogenase homologues in plants.
Supplementary Fig. S3. CaALDH1 does not localize to the
mitochondria.
Supplementary Fig. S4. RT-PCR analysis of expression
of CaALDH1, Capana09g000318 and Capana09g000319 in
leaves of empty-vector control (TRV:00) and CaALDH1silenced (TRV:CaALDH1) pepper plants.
Supplementary Fig. S5. RT-PCR analysis of CaALDH1
expression in leaves of four-week-old wild-type and
CaALDH1-overexpressing Arabidopsis lines.
Supplementary Fig. S6. Proposed model for the functional
role of CaALDH1-AvrBsT complex in plant cell death and
defence signalling.
Supplementary Table S1. Gene-specific primers used in
this study.
Acknowledgments
This work was carried out with the support of Cooperative Research
Program for Agriculture Science and Technology (project no. PJ00802701),
Rural Development Administration, Republic of Korea.
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