Physiologia Plantarum 2012 Copyright © Physiologia Plantarum 2012, ISSN 0031-9317

Characterization of the early response of the orchid,

Phalaenopsis amabilis, to Erwinia chrysanthemi infection
using expression profiling
Shih-Feng Fua , Tsung-Mu Tsaib , Ying-Ru Chenb , Ching-Pei Liub , Lin-June Haisob , Li-Hsin Syueb ,
Hsin-Hung Yehc and Hao-Jen Huangb,∗
a
Department of Biology, National Changhua University of Education, Changhua, Taiwan
b
Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan
c
Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan

Correspondence Erwinia chrysanthemi is a devastating bacterial pathogen in Phalaenopsis

*Corresponding author,
e-mail: haojen@mail.ncku.edu.tw
Received 16 September 2011;
revised 28 October 2011
doi:10.1111/j.1399-3054.2012.01582.x
amabilis and causes soft-rotting disease by secretion of cell wall-degrading
enzymes. However, the molecular mechanisms underlying the interaction of
P. amabilis with E. chrysanthemi remain elusive. In this study, early molecular
events of the plant in response to the pathogen attack were investigated. The
alteration in reactive oxygen species accumulation and peroxidase activity
occurred at the site of infection. Subsequently, a systematic sequencing of
expressed sequence tags (ESTs) using suppression subtractive hybridization
(SSH) was performed to obtain the first global picture of the assembly of
genes involved in the pathogenesis. The majority of the SSH clones showed
a high identity with genes coding for proteins that have known roles in
redox homeostasis, responses to pathogens and metabolism. A notable num-
ber of the SSH clones were those encoding WRKY, MYB and basic leucine
zipper transcription factors, indicating the stimulation of intracellular signal
transduction. An orchid gene encoding trans-2-enoyl-CoA reductase (ECR)
was the most abundant transcripts in the EST library. ECR is an enzyme
catalyzing the very long chain fatty acids (VLCFAs) biosynthesis, and the full-
length cDNA of the ECR gene (PaECR1) was obtained. Functional analysis
of PaECR1 was conducted by virus-induced gene silencing to knock down
the gene expression in P. amabilis. The PaECR1-silenced plants were more
susceptible to E. chrysanthemi infection, implying potential roles for VLCFAs
in the pathogenesis. In summary, the pathogen-responsive gene expression
profiles facilitated a more comprehensive view of the molecular events that
underlie this economically important plant–pathogen interaction.

Abbreviations – ACC, acetyl-coenzyme A carboxylase; ADH, short chain alcohol dehydrogenase protein; AGI, Arabidopsis
genome initiative; bZIP, basic leucine zipper transcription factors; CAT, catalase; CaMBP, calmodulin-binding family protein;
CDPK, calcium-dependent protein kinase; CF, culture filtrates; CFU, colony-forming units; CHS, chalcone synthase; CymMV,
cymbidium mosaic virus; DAPI, 4 ,6-diamidino-2-phenylindole; ECR, trans-2-enoyl-CoA reductase; EST, expressed sequence
tags; FDR, false discovery rates; GFP, green fluorescent protein; GR, glutathione reductase; GST, glutathione S-transferase;
HR, hypersensitive response; JA, jasmonic acid; MS, Murashige and Skoog; ORF, open-reading frame; PAMPs, pathogen-
associated molecular patterns; PE, Phalaenopsis-Erwinia; POX, peroxidase; PR10c, pathogenesis-related protein 10c; RACE,
5 and 3 -rapid amplification of cDNA ends; ROS, reactive oxygen species; RT-PCR, reverse transcription-polymerase chain
reaction; SSH, suppression subtractive hybridization; VIGS, virus-induced gene silencing; VLCFA, very long chain fatty acid.

Physiol. Plant. 2012

Introduction
As plants are confined to the place where they grow,
they have to develop a broad range of defense responses
to cope with microbial pathogen attack such as bacteria,
fungi and viruses. Activation of the defense response is
controlled by a complex transcriptional regulation and
signal transduction network. One of the best character-
ized resistance responses is the gene-for-gene type of
incompatible interactions (Baker et al. 1997, Jones and
Dangl 2006, Collier and Moffett 2009). The response
is triggered by a race/cultivar-specific recognition event
where resistance depends on the recognition of an aviru-
lence (avr ) gene from the pathogen and a corresponding
resistance (R) gene from the plant host (Hammond-
Kosack and Parker 2003). In plant cultivars carrying
R genes, the hypersensitive response (HR) is often the ini-
tial defense response to infection by pathogens. The HR
results in localized cell and tissue death at the site of
infection, which constrains further spread of the infec-
tion. Subsequently, the local HR can induce a long
lasting systemic acquired resistance that primes the plant
for resistance against a broad spectrum of pathogens
(Gozzo 2003, McDowell and Woffenden 2003). In the
absence of an appropriate avr – R gene combination,
the pathogen is able to spread systemically and the
host–pathogen interaction is compatible. The pathogen-
esis is not detected efficiently by the host in a compatible
interaction (Nomura et al. 2005). Therefore, the activa-
tion of defense mechanisms is weak or even suppressed
by the pathogens, granting successful infection and caus-
ing disease symptoms (Nomura et al. 2005, Tyler 2009).
In addition to the cultivar-specific resistance medi-
ated by R genes, plants exhibit broader and more basal
defense responses, which can be activated by a range
of unspecific biotic and abiotic elicitors (Nicaise et al.
2009). Plant bacterial pathogens, and in general other
pathogens, reveal themselves to the host defense system
through molecules called pathogen-associated molecu-
lar patterns (PAMPs). PAMPs that act as general elicitors
in plants include flagellin and lipopolysaccharides from
bacteria, and chitin and ergosterol from fungi. Plants
have evolved specialized cell-surface pattern recogni-
tion receptors to detect conserved features of PAMPs
and activate basal defense (Hou et al. 2009). These basal
defense responses include production of reactive oxygen
species (ROS) and ethylene, transcriptional induction of
a large suite of defense genes, including pathogenesis-
related (PR) genes and post-transcriptional suppression
of the auxin-signaling pathway (Navarro et al. 2006,
Hou et al. 2009). The basal defense systems are gen-
erally sufficient to halt the colonization of pathogens,
and minimize their ability to damage the plant’s normal
growth, development and dispersal. As the activation of
basal defense depends on the perception of PAMPs, it is
also called PAMP-triggered immunity (Jones and Dangl
2006, Nicaise et al. 2009).
The oxidative burst, a rapid production of ROS is
described as one of the earliest responses to pathogen
infection and is generally associated with HR (Torres
2010). Apoplastic generation of superoxide (O2 − ), or
its dismutation product hydrogen peroxide (H2 O2 ), has
been documented following recognition of a variety of
pathogens at the site of attempted invasion (Torres et al.
2006). ROS have direct antimicrobial activities and can
therefore reduce pathogen viability. In addition, ROS
are responsible for strengthening of host cell walls via
cross-linking of glycoproteins, or lipid peroxidation and
membrane damage (Montillet et al. 2005, Torres 2010).
It is also apparent that plant cells generate such reactive
species as signaling molecules, produced at controlled
levels and leading to defense responses (Asai and Yosh-
ioka 2008). Along with the production of ROS, plant cells
deploy enzymatic and non-enzymatic antioxidative sys-
tems to facilitate the removal of such reactive molecules.
The formation of ROS can be removed through the action
of enzymes such as catalase (CAT), peroxidase (POX),
superoxide dismutase, glutathione S-transferase (GST)
and glutathione reductase (GR) (Hancock et al. 2002).
The induction of an oxidative burst and the antioxida-
tive system may work in concert, not only to adjust the
level of ROS formation but also to mediate redox signal
transduction during pathogenesis (Averyanov 2009).
The soft rot Erwinia, Erwinia carotovora subsp.
atroseptica, E. carotovora subsp. carotovora and
E. Erwinia chrysanthemi are destructive to a wide variety
of plants world wide. These highly virulent necrotrophic
pathogens attack a variety of host tissues, including
blossoms, actively growing shoots and fruits. These
pathogens are also capable of rapid systemic movement
downward through stem tissue, resulting in infection and
killing of rootstock crowns (Barras et al. 1994, Toth et al.
2003). Pathogenicity results from the secretion of extra-
cellular enzymes responsible for the disorganization of
the plant cell wall. These include pectin methyl esterases,
pectin acetyl esterases, exo- and endopectate lyases and
exopolygalacturonases (Kazemi-Pour et al. 2004). These
enzymes cause maceration of plant tissues, cell separa-
tion and cell death, indicating that the pectinases are
important virulence determinants (Bell et al. 2004, Zhao
et al. 2005). However, no genetically defined resistance
to Erwinia has been described, and the pathogen does
not appear to contain typical avr genes nor does it nor-
mally cause HR in plants. Resistance to this pathogen
has been observed in Solanum brevidens (Austin et al.
1988, Bains et al. 1999, Tek et al. 2004), a wild and
Physiol. Plant. 2012
non-tuber bearing potato species, but the genetic basis
for this resistance remains elusive. Therefore, develop-
ment of resistance to this pathogen appears to rely on a
generally induced defense mechanism.
The cell wall-degrading enzymes secreted by the soft
rot Erwinia have been proposed to have dual roles: in
addition to being the main virulence determinants of the
pathogen, they also display elicitor properties that trig-
ger basal defense responses (Norman-Setterblad et al.
2000, Hammerschmidt 2004). For example, treatment
of tobacco plants with the cell wall-degrading enzymes
has been shown to induce both local and systemic
expression of several genes encoding PR proteins (Palva
et al. 1993, Vidal et al. 1997, 1998). Moreover, these
enzymes are able to induce defense responses both
locally and systemically to decrease the susceptibility to
E. carotovora in tobacco plants (Palva et al. 1993, Vidal
et al. 1998). Similarly, E. carotovora culture filtrates (CF)
containing cell wall-degrading enzymes and oligogalac-
turonides, which are the breakdown products of pectin,
mediate expression of defense-related genes in non-host
Arabidopsis plants (Norman et al. 1999, Norman-
Setterblad et al. 2000). Several lines of evidence suggest
that ethylene and jasmonic acid (JA) pathways may play
a central role in regulating defense gene induction and
resistance triggered by cell wall-degrading enzymes from
E. carotovora (Norman et al. 1999, Norman-Setterblad
et al. 2000, Brader et al. 2001, Fagard et al. 2007). In
addition, a gene coding for calcium-dependent protein
kinase (CDPK) from orchids was found to be induced
after infection with E. chrysanthemi and the correspond-
ing CF (Tsai et al. 2007). Recent study indicated that
microbial siderophores secreted by E. chrysanthemi can
modulate plant defenses through an antagonistic mech-
anism between SA and JA signaling cascades (Dellagi
et al. 2009). Thus, the soft-rotting Erwinia species pro-
duce enzymes that appear to function both as factors
needed for successful infection as well as indirect induc-
ers of defenses that should stop further development of
the bacteria in host tissue (Wegener and Olsen 2004).
Phalaenopsis spp., a monocotyledon plant species
belonging to the Orchidaceae, is a commercially valu-
able ornamental crop for cut flowers or potted plants.
Soft-rotting disease caused by E. chrysanthemi is one
of the most devastating diseases encountered among
economically important orchids (Liau et al. 2003, Sjahril
et al. 2006). The outbreak of soft-rotting disease in orchid
floriculture is favored by high relative humidity and tem-
perature during cultivation or transportation to markets.
The use of phytochemical has harmful effects on envi-
ronment, and therefore it is essential to control disease
by having extensive information of the plant–pathogen
interactions. Although the knowledge of the molecular
Physiol. Plant. 2012
processes underlying pathogenesis in plants is increas-
ing, very little is known about the early response of
the Orchidaceae to soft rot Erwinia infection. In this
study, a comprehensive analysis of Phalaenopsis orchid
gene expression in response to E. chrysanthemi infec-
tion was performed by combined suppression subtractive
hybridization (SSH) and expression profiling strategies. A
total of 102 expressed sequence tags (ESTs) with different
expression profiles were isolated and subjected to gene
ontology analysis. Noticeably, a Phalaenopsis amabilis
gene encoding trans-2-enoyl-CoA reductase (referred to
as PaECR1) was one of the most abundant transcripts in
the pathogen-induced EST library. The full-length cDNA
sequence of PaECR1 gene was cloned, and the gene
expression in response to Erwinia infection was charac-
terized. Its function in host responses to soft rot Erwinia
was investigated by knocking down the gene expression
with virus-induced gene silencing (VIGS). Furthermore,
the potential roles of PaECR1-mediated pathways lead-
ing to defense responses against Erwinia in Phalaenopsis
orchid are discussed.
Materials and methods
Plant materials and inoculation
with E. chrysanthemi
P. amabilis orchid at five-leaf stage (24-month-old)
‘TS97K’ (P . amabilis W1–10 × P . amabilis W1–22) was
kindly provided by the Taiwan Sugar Company (Tainan,
Taiwan). The plants were grown in a greenhouse under
natural light and controlled conditions. The temperature
ranged from 23 to 27◦ C, and the relative humidity was
70%. For inoculation of orchid leaves with E. chrysan-
themi, the bacterial cells were grown overnight on
Luria-Bertani liquid medium and harvested by centrifu-
gation at 4000 g for 10 min. The bacterial pellets were
resuspended in 0.9% sodium chloride solution, and then
adjusted to an absorbance (600 nm) of 1.0 containing
approximately 8–9 × 107 colony-forming units (CFU).
A newly emerging leaf with approximately 10 cm in
length in the orchid plant was selected for inoculation.
Subsequently, the 1-cm leaf apex of the orchid plant
was transversally sliced off with a sterile razor blade.
The remaining part of the leaf blade was subjected to
inoculation with E. chrysanthemi. The cross section of
leaf blade was then rubbed gently with 30 μl of the
bacterial suspension. After inoculation, the orchid plants
were incubated for the indicated time periods at 28◦ C
and high humidity to facilitate bacterial growth. Mock
inoculation was performed in parallel by wounding the
leaves without the bacterial suspension. Leaf samples
containing the regions 1 cm from the excised (or inocu-
lated) sites were collected and frozen in liquid nitrogen
immediately. For each treatment, at least three individual
leaf samples were harvested from the orchid plants.
Chemical applications and CF preparation
CF from E. chrysanthemi was prepared as previously
described (Vidal et al. 1997). Supernatant from overnight
cultures, corresponding to 107 CFU ml−1 , was obtained
by centrifugation of the bacterial suspension (10 min at
4000 g ) and was filter sterilized. Treatment of the plants
with chemicals was performed with a method similar to
leaf inoculation. An aliquot (30 μl) of freshly prepared
CF or methyl-JA (50 μM) was spotted onto the cross
section of the leaf blade. Plant samples were collected
at the indicated time after treatment.
Detection of ROS accumulation in orchid plants
Leaves of orchid plants were collected at the indi-
cated times after inoculation with E. chrysanthemi. The
samples were subsequently incubated in 5 μM 5-(and-
6)-chloromethyl-2 ,7 -dichlorodihydrofluorescein diac-
etate, acetylester (CM-H2DCF-DA, Molecular Probes,
Eugene, OR) for 60 min in the dark. The samples
were then washed thoroughly with water and visualized
under a microscope equipped with a green filter (Leica
MPS60 fluorescent microscope, excitation 450–490 nm;
emission 500–530 nm). The period of exposure was
consistent throughout the experiment.
Antioxidative enzyme activity assays
Leaf samples were collected and ground into fine powder
with liquid nitrogen. The protein extract was obtained by
homogenization of leaf samples in 1 M phosphate buffer
pH 7.0 (2:1, fresh weight/volume). After centrifugation at
120 000 g at 4◦ C for 20 min, the supernatant containing
soluble protein was quantified with bovine serum
albumin as the calibration standard by the method of
Bradford (1976). POX activity was determined according
to the method described by Beyer and Fridovich (1987).
The protein samples (6 μg) were loaded onto 10% native-
polyacrylamide gels, and electrophoresis was conducted
at 150 V for 30 min in a cold room (4◦ C). The gels were
stained with 50 mM sodium phosphate buffer pH 7.0
containing 3,3-diaminobenzidine (0.05 ml mg−1 ) and
hydrogen peroxide (35%) for 5 h until the dark brown
bands were visible.
RNA extraction and construction of the SSH
cDNA library
Total RNA was extracted from the leaf tissues of orchid
plants using the RNeasy Plant Mini Kit (Qiagen, Hilden,
Germany). SSH was performed with a polymerase chain
reaction (PCR)-Select cDNA subtraction kit (Clontech,
CA) according to the manufacturer’s instructions with
some modifications. The cDNA was synthesized from
total RNA with a SMART cDNA synthesis kit (Clontech)
using an oligo dT primer that allowed the amplifica-
tion of the complete mRNA population in each sample.
The cDNA prepared from the leaves 24 h after inocu-
lation with of E. chrysanthemi was used as the ’tester’
and cDNA from the control samples was used as the
’driver’. The tester and driver cDNA populations were
digested with restriction enzyme RsaI and ligated with
the specific adaptors provided in the kit. The tester pool
was then hybridized separately with an excess of driver
cDNA. After two rounds of hybridizations, the fragments
expressed in the tester but not in the driver were specifi-
cally amplified. The products were cloned into pGEM-T
vector (Promega, WI) and transformed into Escherichia
coli strain JM109. The pathogen-induced EST clones
were stored and used for PCR amplification of the cDNA
inserts.
DNA sequencing and gene ontology analysis
The pathogen-induced EST clones were sequenced using
the T7 promoter primer. Sequence similarity searches
were performed against the National Center for Biotech-
nology Information database using the basic local align-
ment search tool (BLASTX), which compared translated
nucleotide sequences with protein sequences (Altschul
et al. 1997). These ESTs were named according to
homologous sequences in the database. Sequences with
a BLASTX e value >10−5 were designated as having no
significant similarity (unknown protein). Sequences of
the unique pathogen-induced ESTs were then imported
into BLAST2GO, a web-based gene ontology annotation
and analysis tool for subsequent analysis (Conesa et al.
2005). This involved automated retrieval of gene ontol-
ogy terms associated with the hits. The ESTs were
manually assigned to functional categories based on
the analysis of BLAST2GO and also with the aid of informa-
tion reported by the scientific literature. The sequences
of ESTs and genes described in this study have all been
submitted to GenBank.
Semi-quantitative RT-PCR analysis
Total RNA was reversed transcribed into cDNA by the
ImProm-II™ Reverse Transcription (RT) System with a
mix of oligo (dT)18 and (dT)20 as primers according to
the manufacturer’s manual (Promega). The sequence-
specific primer pairs and recommended annealing
temperatures (Ta ) corresponding to each gene are
Physiol. Plant. 2012
summarized in a table (Appendix S1). The cDNA was
added to the PCR mixture containing 1 U Taq DNA
polymerase (Promega), 25 mM MgCl2 , 10 mM dNTPs
and 1 μM of each primer pair. The PCR conditions con-
sisted of an initial denaturation step at 94◦ C for 2 min, 30
cycles of amplification (94◦ C for 1 min, primer-specific
annealing temperature for 1 min, and 72◦ C for 1.5 min)
and a final elongation step at 72◦ C for 10 min (Appendix
S1). The PCR products were immediately separated by
electrophoresis in a 1% agarose gel.
5 and 3 -rapid amplification of cDNA ends
The EST fragments that contain the consensus WRKY
and ECR sequences were initially isolated from the
E. chrysanthemi -infected orchid plants by SSH. The
full-length cDNAs of the WRKY and ECR genes were
subsequently obtained by 5 and 3 -rapid amplifica-
tion of cDNA ends (RACE) using the SMARTer RACE
cDNA amplification kit (Clontech) according to the
manufacturer’s instructions. After RT with primers and
SMARTScribe Reverse Transcriptase supplied by Clon-
tech, the first strand cDNA was used directly in 5 - and
3 -RACE PCR. Primary PCR amplification reactions to
obtain individual 5 or 3 sequence were achieved using
a proof-reading enzyme, Advantage 2 Polymerase Mix
(Clontech). The WRKY and ECR gene-specific primers
(Appendix S1) were designed to generate the 5 - and
3 -cDNA fragments, respectively. The obtained cDNA
ends were ligated into pGEM-T vector (Promega) and
sequenced. Secondary PCR amplifications were per-
formed to combine 5 and 3 sequences and generate
full-length cDNAs. The full-length cDNAs of the WRKY
and ECR were amplified by using primers designed from
the extreme 5 and 3 ends. The following PCR amplifi-
cation parameters were used: 94◦ C for 5 min followed
by 30 cycles of 94◦ C for 1 min, 60◦ C for 2 min and
72◦ C for 2 min, and a final elongation step of 10 min
at 72◦ C. The full length of the WRKY and ECR genes
were referred to as P. amabilis WRKY1 (PaWRKY1) and
PaECR1, respectively.
Protein subcellular localization
The coding region ofPaWRKY1 cDNA sequence was
amplified using gene-specific primers containing Bgl II
and Spe I restriction sites (Appendix S1). The PaWRKY1
cDNA fragment was cloned into the pCAMBIA1302
vector at the Bgl II and Spe I sites. The PaWRKY1-green
fluorescent protein (GFP ) fusion genes were driven by
the 35S promoter. The fusion construct and empty-vector
control were transformed into onion epidermis cells by
particle bombardment using a Biolistic PDS-1000/He
Physiol. Plant. 2012
device (Bio-Rad, CA). After incubation of transformed
onion epidermis cells for 24 h at 24◦ C, GFP was detected
by a fluorescence microscope equipped with a flu-
orescence module (Leica DMR, Leica Microsystems,
Wetzlar, Germany). Nuclei were stained by adding one
drop of 4 ,6-diamidino-2-phenylindole (DAPI) staining
solution (1 μg ml−1 ) to the onion cells. The onion epi-
dermis cells were incubated in the dark for 15 min,
and DAPI fluorescence images were obtained by UV
illumination of the specimen.
DNA-binding assay in a Y1H system
The interaction of PaWRKY1 protein with the DNA reg-
ulatory element was analyzed by a yeast one-hybrid
(Y1H) assay according to the manufacturer’s protocol
(Clontech Yeast Protocols Handbook, BD Biosciences
Clontech, CA). The open-reading frame (ORF) of the
PaWRKY1 gene was amplified by PCR using the for-
ward (PaWRKY1-Y1H-F ) and reverse (PaWRKY1-Y1H-R)
primers containing the digestion site of the enzymes,
NcoI and BamHI, respectively (Appendix S1). The ORF
of the PaWRKY1 sequence was in-frame fused with
the GAL4 activation domain of the one-hybrid vector,
pGADT7-Rec2, forming pGR-PaWRKY1. The target cis-
acting DNA fragments harboring three tandem repeats of
W box or mutated W box (mW box) elements (Appendix
S1) were fused into the pHIS2.1 vector under the control
of the GAL1 minimal promoter. The promoter regions
(approximately 300 or 700 bps) of the orchid CDPK
(GenBank: EF587760) were also amplified using the
promoter-specific primers (Appendix S1) and included
in the DNA-binding assay. The resulting recombinant
plasmids containing the target promoter were termed
pHIS-W box, pHIS-mW box, pHIS-CDPK300 or pHIS-
CDPK700. Subsequently, the yeast strain Y187 was
co-transformed with the pGR-PaWRKY1 and pHIS-target
promoter. The yeast strains transformed with the above
constructs were grown on synthetic dextrose medium
(SD)/-Trp/-Leu/-His selective medium in the absence
or presence of 30 mM 3-amino-1,2,4-triazole (3-AT) at
30◦ C for 3 days.
Construction of transgenes and
Arabidopsis transformation
The coding region of PaWRKY1 was amplified using Pfu
DNA Polymerase (Promega) with gene-specific primers
containing a HindIII and a Bgl II site (in-frame ATG initi-
ation codon) (Appendix S1). The PaWRKY1 cDNA frag-
ment was cloned into the binary plant expression vector
pCAMBIA1380 under the control of the 35S promoter.
The construct was then introduced into Agrobacterium
tumefaciens strain LBA4404, and transgenic Arabidop-
sis plants were generated by the method of floral-dip
transformation (Clough and Bent 1998). Transformed
plants were selected by growth on Murashige and
Skoog (MS) medium (Duchefa Biochemie, Haarlem, The
Netherlands, Catalog number M0222.0050) and 0.3%
phytagel (Sigma, MO) supplemented with hygromycin
(20 mg l−1 ). Three independent transgenic Arabidopsis
lines were generated and stably expressed the trans-
gene PaWRKY1. The second generation of the transgenic
plant lines homozygous for the transgene was used for
experiments. The control Arabidopsis plants transformed
with the empty vector were also generated in parallel.
Characterization of the transgenic Arabidopsis plants
overexpressing PaWRKY1 was performed by examin-
ing the expression of transgenes using RT-PCR with the
gene-specific primers (Appendix S1).
Microarray analysis and data processing
The Arabidopsis plants were grown on the medium
containing MS (Murashige and Skoog) salt (Duchefa
Biochemie) and 0.3% phytagel (Sigma) in long day
conditions (16-h light and 8-h dark) at 22◦ C. Total RNA
was isolated from 1-week-old seedlings of empty-vector
control and transgenic 35S-PaWRKY1 Arabidopsis
plants with an RNeasy Plant Mini Kit (Qiagen). DNA
contamination was effectively removed by treatment of
the RNA samples with DNase I (Roche, Mannheim,
Germany). Subsequently, the RNA was purified and
concentrated by using the RNeasy MinElute Cleanup Kit
(Qiagen) according to the manual’s instructions. Three
biological replicates were performed in the experiment,
which were grown in the same growth chamber to
minimize experimental and sample-to-sample variation.
Microarray analysis was performed using Affymetrix
microarrays (GeneChip Arabidopsis ATH1 genome
array) containing 22 810 probe sets on a single chip.
Microarray experiments including labeling, hybridiza-
tions and data analysis (one sample per chip) were
carried out according to the manufacturer’s manual. The
data were analyzed with Microarray Suite version 5.0
(MAS 5.0) using Affymetrix default analysis settings and
global scaling as the normalization method. The trimmed
mean target intensity of each array was arbitrarily set to
500. The raw cell intensity data files (GeneChip CEL
files) were imported into Robin software, which is freely
available at http://mapman.gabipd.org/web/guest/home
(Lohse et al. 2010). The data were normalized with the
Robust Multichip Average algorithm and converted to
log2 scale to allow the comparison of the three biolog-
ical replicates performed for each set of experiments.
Significantly different gene expression was detected
based on the rank-product algorithm (Breitling et al.
2004) implemented in the Robin software. The Ben-
jamini and Hochberg algorithm calculates false discov-
ery rates (FDR) that are inherently corrected for multiple
testing (Benjamini and Hochberg 1995). Genes were
considered as being significantly up and downregu-
lated if the FDR value for the corresponding probe
set was smaller than 0.05. To enrich for biologi-
cally relevant changes, only genes with a minimal
fold change of two in all experiments were selected.
Gene ontology analysis was performed with agriGO
software (http://bioinfo.cau.edu.cn/agriGO/) (Du et al.
2010). The microarray data described in this study have
been deposited in the Gene Expression Omnibus and
are accessible through the series accession number,
(GEO:GSE227766) (http://www.ncbi.nlm.nih.gov/geo/
query/acc.cgi?acc=GSE20034).
VIGS of PaECR1 in the orchid plants
Functional analysis of orchid genes by VIGS was
conducted according to the method described by
Lu et al. (2007) with minor modification. The par-
tial coding region of PaECR1 was amplified using Pfu
DNA Polymerase (Promega) with gene-specific primers
PaECR1-VIGS-F and PaECR1-VIGS-R (Appendix S1). A
443-nucleotide fragment corresponding to 409–851
codons of PaECR1 ORF was obtained for subse-
quent cloning. The transcript-derived PaECR1 fragments
were first cloned into pGEM-T easy vector (Promega)
and subcloned into the SmaI-digested pCymMV-pro60
to construct pCymMV-pro60-PaECR1. The pCymMV-
pro60 VIGS vector carrying the PaECR1 was subject to
DNA sequencing to ensure the success of ligation. The
empty plasmid pCymMV-pro60 served as a control. The
pCymMV derivative plasmids were linearized with Spe I.
Infectious RNA transcripts corresponding to the con-
structed vectors of Cymbidium mosaic virus (CymMV)
were synthesized by in vitro transcription with mMES-
SAGEmMACHINE T3 kit (Ambion, TX). The transcribed
RNAs were resuspended in KP buffer (1 M KH2 PO4 and
K2 HPO4 , pH 7.0) and used for inoculation. Infectious
RNA was rubbed onto the second emerging leaves of
24-month-old P. amabilis plants in the presence of a
small amount of carborundum. The orchid plants were
grown in a growth chamber at 22◦ C under 16/8 light/dark
cycle for 2 months before assessment of suppression
of host genes and susceptibility to E. chrysanthemi
infection. Inoculation of the gene-silenced leaf tissues
of the orchid plants with E. chrysanthemi were sim-
ilar to that described in the previous section, except
that the inoculation was performed by needle punctua-
tion (size 0.4 × 13 mm). The primer pairs (PaECR1-full )
Physiol. Plant. 2012
corresponding to the 5 and 3 sequence of complete
PaECR1 ORF were used in RT-PCR to assess the knock-
down efficiency of the host gene by VIGS (Appendix S1).

Results
Production of ROS and alteration of POX enzyme
activity in orchid leaves infected
with E. chrysanthemi
To understand the early responses of the orchid plants to
E. chrysanthemi, the development of disease symptoms
of directly inoculated leaves was monitored at different
periods of time. At 3- and 6-h post-inoculation, initiation
of tissue maceration was first observed at the site
of inoculation (Fig. 1). At 12 h, the typical tissue
maceration became more obvious and gradually spread
to other parts of the leaf tissues. The progression
of maceration continued throughout 24 h and tissue
necrosis occurred at 48 h. To examine whether plants
inoculated with E. chrysanthemi were producing ROS,
which are an important component during pathogenesis,
the generation of ROS and localization was detected
at 3 h after inoculation. A significant increase in ROS
generation was observed around the primary site of
infection when compared with control leaves mock
inoculated with buffers (Fig. 2A). The results suggested
that ROS production was an early event in orchid leaves
in response to infection with E. chrysanthemi.
To further characterize if the antioxidative enzyme
was involved in the response of the leaf tissues to
E. chrysanthemi infection, POX activity was analyzed.
The enzyme activity of POX was decreased at 3-h
post-inoculation in comparison with control samples
(Fig. 2B). The subsequent return to basal levels of POX
activity was observed at 6 and 12 h. Taken together,
these results suggested that alteration of ROS levels and
antioxidant systems were involved in the early response
of orchid plants to E. chrysanthemi infection.

Isolation of pathogen-induced ESTs in orchid

plants by a SSH strategy
To study the molecular basis of the orchid plants dur-
ing the early stage of E. chrysanthemi infection, the
pathogen-induced ESTs were isolated at 24-h post-
inoculation through the construction of a subtractive
cDNA library. The cDNA populations derived from the
mock-inoculated control and E. chrysanthemi -infected
plants served as the driver and tester, respectively. After
two rounds of subtractive hybridization, the enriched
cDNA fragments were obtained and ranged from about
0.5 to 1.4 kbs with most fragments distributed between
0.8 and 0.9 kbps (Appendix S2). The constructed SSH
Fig. 1. Disease symptoms induced by Erwinia chrysanthemi on
Phalaenopsis amabilis leaves for different time periods. The leaves of
P. amabilis plants at five-leaf stage (24-month-old) were inoculated with
an E. chrysanthemi suspension containing bacteria at the concentration
of 8–9 × 107 CFU. The inoculated plants were kept in a growth room
at high humidity (80–100%) at 28◦ C. Healthy leaves of the same age
(Healthy) and mock-inoculated ones (Mock) from the orchid plants
served as controls. The photographs were taken at different time
points up to 48-h post-inoculation (hpi). The scale bar represents 1 cm.
The experiments were repeated more than three times, and a single
representative photograph is shown.

Physiol. Plant. 2012

 A
Fig. 2. Oxidative burst and antioxidant enzyme activity after infection
with Erwinia chrysanthemi on leaves of Phalaenopsis amabilis plants.
(A) The orchid leaves were inoculated with E. chrysanthemi pathogen
and kept at 28◦ C. Histochemical analysis of ROS in the mock- and
bacterial-inoculated leaves was performed by staining the tissues with
2,7-dichlorofluorescein. The production of ROS was visualized under
a microscope with UV filters at 3-h post-treatment. The same tissues
under bright-field illumination are shown on the bottom panel. The
scale bar represents 0.4 mm. (B) POX activity was analyzed in the
orchid leaves infected with E. chrysanthemi. Six micrograms of protein
were loaded and separated on a 10% native-polyacrylamide gel. The
gels were stained for POX activity with 3,3-diaminobenzidine and
hydrogen peroxide. Similar results were obtained from two independent
experiments.
library containing the pathogen-induced cDNAs was
defined as the Phalaenopsis-Erwinia (PE) EST library
thereafter. In the PE library, a total of 170 SSH clones
were randomly selected for sequencing and assigned
with a PE library number (Appendix S3). The mean size
for the sequenced ESTs from this library was 0.53 kb.
The EST fragments representing the same gene were
counted as a single gene, and a total of 102 unique ESTs
were obtained. The sequence data were deposited in
the EST database in GenBank under accession numbers
(GenBank: HO059275-HO059377) (Appendix S3). By
comparison with the databases using BLASTX, sequences
of the 84 unique ESTs were homologous with the pub-
lished sequences in the databases. A total number of
18 unique ESTs had no significant similarity and were
classified as unknown proteins (Appendix S3).
To obtain a better insight into the identity and possible
functional significance of the pathogen-induced ESTs,
the 84 unique EST sequences were used to annotate the
function of their respective genes within the software
Fig. 3. Functional percentage distribution of the pathogen-induced ESTs
in the orchid leaves at 24-h post-inoculation with Erwinia chrysanthemi.
The ESTs were classified into functional group based on the sequence
alignment using BLASTX and BLAST2GO (Conesa et al. 2005).
program BLAST2GO (Conesa et al. 2005). According to
gene ontology analysis, each clone was distributed into
1 of 16 functional categories related to biological pro-
cesses (Appendix S3). The vast majority of annotations
were involved in oxidation reduction (15%), responses to
pathogens (10%) and metabolic processes (11%) (Fig. 3).
Genes encoding proteins involved in lipid metabolic
processes (6%), generation of precursor metabolites and
energy response to stresses (5%), response to stresses
(4%), lignin biosynthetic processes (4%) and cell wall-
associated enzymes (4%) formed the second largest
groups. The remainder was distributed among sev-
eral processes, including response to metal ions (2%),
signal transduction (2%), transcription factors (2%), vita-
min metabolic processes (2%), cytoskeleton (2%) and
response to JA stimulus (1%). Noticeably, the ESTs in
the category of signal transduction were characterized
as 14-3-3-like protein and calmodulin-binding family
protein (CaMBP) (Appendix S3). Within the category of
transcription factors were found basic leucine zipper,
WRKY and MYB transcriptional regulators. The category
of responses to pathogens consisted of one unique ESTs
with homology to PR proteins. The transcript encoding
ECR (PE7) was the most abundant EST and is related to the
generation of precursor metabolites and energy. Despite
the necrotrophic interaction between the pathogen and
plant, several of the E. chrysanthemi -induced tran-
scripts represented typical pathogen-responsive genes
such as chalcone synthase (CHS ; PE2). Therefore, these
results revealed that the early response of the orchid
to E. chrysanthemi could be largely attributed to the
Physiol. Plant. 2012
 A

Fig. 4. Validation of representative pathogen-induced ESTs by semi-quantitative RT-PCR analysis. (A) Transcript levels of the pathogen-induced ESTs
(WRKY, PE99 and ECR, PE7) were quantified at various time points during infection with Erwinia chrysanthemi. Total RNA was extracted from orchid
leaves harvested at 0, 6, 12, 24 and 48-h post-inoculation (hpi). The transcript level of Paactin (Actin) served as an equal loading control. (B) Expression
patterns of the pathogen-induced ESTs in response to wounding, pathogen infection and defense signaling molecules. Total RNA was isolated from
the leaves of orchids after infection with E. chrysanthemi pathogen (P), treatment with mechanical wounding (W) and exposure to methyl-JA (50 μM)
and CF, respectively. Samples were taken 24 h after each treatment, and the transcript levels were analyzed by semi-quantitative RT-PCR. Transcripts
derived from healthy plants (samples taken prior to treatment) are indicated with the control (C). The signals were quantified with Adobe Photoshop
version 7.0 to develop histograms of signal intensity. The values were normalized with respect to Paactin content. The signal of the first sample on
the panel was defined as 1.0 (arbitrary units), and other abundances are expressed relative to that value. Intensity values in each panel are color
coded to represent the relative fold change in expression. All experiments were performed twice with similar results, and a single representative
autoradiograph is shown.

molecular networks involved in redox homeostasis,
signal transduction, transcriptional regulation, energy,
metabolism and responses to pathogens.
Expression validation of the pathogen-induced
ESTs by semi-quantitative RT-PCR
A subset of genes were selected for semi-quantitative
RT-PCR based on their putative functions and the results
of the SSH differential screening. The expression analysis
was to confirm the validity of the pathogen-induced ESTs
and also to determine more precise timing of expression
for selected genes of interest. The steady-state transcript
level of ECR (PE7) and WRKY transcription factor (PE99)
increased at 6-h post-inoculation and remained elevated
for 48 h (Fig. 4A). To gain more insight into the roles
of the pathogen-induced ESTs, the expression of the
ECR and WRKY genes in response to various defense
signaling molecules was investigated. The leaves of
the orchid plants were treated with wounding, defense
signaling molecules such as JA and CF or inoculated
with E. chrysanthemi. As expected, a significant increase
in the expression levels of the ECR and WRKY genes
was observed at 24 h after inoculation (Fig. 4B). The
results indicated that the expression of the ECR and
WRKY genes was mainly influenced by the infection
with E. chrysanthemi instead of wounding. Treatment
of orchid leaves with CF or JA had subtle effects on
the expression of the two genes (Fig. 4B). The results
suggested that the induced expression of the pathogen-
induced ECR and WRKY genes was specific to infection
with E. chrysanthemi.
A total of 10 additional pathogen-induced ESTs cor-
responding to a range of functional categories were
also selected for characterization (Appendix S4). The
most prominent classes were those involved in oxidation
reduction, including short chain alcohol dehydrogenase
protein (ADH ; PE5), methylenetetrahydrofolate reduc-
tase (MTHFR; PE9) and GST (PE26). The genes encoding
protein with putative functions in responses to pathogens
such as PR protein 10c (PR10c ; PE1), CHS (PE2) and
PDR-like ABC transporter (ABC ; PE19) were selected.
The expression of acetyl-coenzyme A carboxylase (ACC ;
PE97) and caffeic acid O-methyltransferase (COMT ;
PE12) belonging to the categories of energy and lignin
biosynthetic processes were examined. The ESTs related
to signal transduction (CaMBP ; PE114) and basic leucine
zipper transcription factors (bZIP ; PE61) were also
subjected to expression analysis. The gene expression

Physiol. Plant. 2012

profiles examined by semi-quantitative RT-PCR analysis the ECRs from other plant species. The PaECR1 contains
were closely in accordance with the SSH screening. 82 amino acids at the C-terminal region with homology
to steroid-5-α-reductase, which catalyzes the reduction
of testosterone to dihydrotestosterone (Kohlwein et al.
Isolation of the full-length cDNAs encoding WRKY
2001). The functional residues on the enzyme active
transcription factor and ECR from P. amabilis
sites such as Glutamine 89, Lysine 144 and Arginine 145
The cDNA fragments encoding the C-terminus of a (Paul et al. 2007) are conserved in the orchid PaECR1.
WRKY transcription factor (PE99) and ECR (PE7) were A putative NADPH-binding domain (Song et al. 2009) is
identified initially as pathogen-induced ESTs from the also present in the PaECR1.
SSH cDNA library (Appendix S3). It suggested a role
for the orchid WRKY and ECR in the host responses to Subcellular localization and DNA-binding activity
E. chrysanthemi. To better characterize the pathogen- of the PaWRKY1 transcription factor
induced WRKY transcription factor and ECR, the cor-
responding full-length cDNAs of the two genes were To determine the subcellular localization of the
isolated using RACE. A full-length cDNA clone with 748 PaWRKY1 protein, the GFP gene was fused to PaWRKY1
nucleotides in length was obtained and subsequently under the control of the Cauliflower mosaic virus 35S
designated as PaWRKY1. The nucleotide sequence of promoter (Fig. 6A). In the transient expression assay,
the PaWRKY1 cDNA contained an ORF that would 35S-PaWRKY1-GFP and an empty-vector control were
introduced into onion epidermal cells by particle bom-
encode a protein of 170 amino acids with a predicted
bardment. The cells were stained with DAPI to reveal the
molecular weight of 20 kDa and an isoelectric point of
nuclei and then examined using a fluorescence micro-
9.72. The full-length PaECR1 transcript encoded a pro-
scope. GFP signals of 35S-PaWRKY1 were detected in
tein of 310 residues with an approximate molecular mass
the nuclei of onion cells (Fig. 6A), whereas the empty-
of 36 kDa and a calculated isoelectric point of 9.46. The
vector controls were uniformly distributed throughout
gene sequences encoding PaWRKY1 and PaECR1 were
the epidermal cells (data not shown). These data thus
deposited in GenBank under the accession numbers
indicate that PaWRKY1 is a nuclear protein that may
(GenBank: HM596074 and HM596076, respectively).
act as a transcription factor to regulate the expression of
Sequence analysis and comparison with data banks
downstream genes.
confirmed the high similarity and identity of this orchid
WRKY transcription factors are thought to function by
PaWRKY1 protein to members of group IIc WRKY pro-
binding their cognate TTGACC/T W-box cis-elements
teins of higher plants (Eulgem et al. 2000). The PaWRKY1
in the promoter regions of target genes and activating
protein sequence showed significant homology to
or repressing their expression (Rushton et al. 2010). To
Triticum aestivum TaWRKY (Mangelsen et al. 2008),
examine the DNA-binding capacity of PaWRKY1 for
Solanum tuberosum StWRKY1 (Dellagi et al. 2000), Fra-
binding to the W-box elements, a series of Y1H assays
garia ananassa FaWRKY1 (Encinas-Villarejo et al. 2009),
were performed. Two DNA fragments containing W-box
Arabidopsis thaliana AtWRKY75 (Devaiah et al. 2007)
sequences were generated; one had a triple tandem
and Brassica napus BnWRKY75 (Yang et al. 2009) with
repeat of the wild-type W box (W box; TTGAC) and
percentages of identity ranging from 77 to 86%. The
the other had the repeat of an mW box (TTGAA). In
multiple sequence alignment of PaWRKY1 with mem-
addition, two promoter-containing fragments (300 and
bers of the group IIc WRKY proteins in plants was
700 bps in length) derived from an orchid gene encoding
generated (Fig. 5A). There was little sequence conserva-
a CDPK (PaCDPK) (Tsai et al. 2007) were included in the
tion at the N-terminal region among the WRKY proteins.
experiment. Detection of protein–DNA-binding activity
The C-terminal part of the coding regions was highly
by growth performance of the yeast cells showed that
conserved and contained signature motifs characteristic
PaWRKY1 possessed specific DNA-binding ability to the
of WRKY transcription factors. The PaWRKY1 protein
promoter of wild-type W box, but not to those of mutated
consisted of one putative WRKY domain, together with W box and PaCDPK (Fig. 6B). The result indicated that
a potential nuclear localization signal and a zinc finger- the PaWRKY1 was able to recognize and interact with
like motif in the C-terminal region (C -X4 -C -X23 -H -X1 -H ) the W-box elements.
(Fig. 5A). In addition, amino acid sequence alignment
of PaECR1 with orthologs from Gossypium hirsutum
Potential regulatory roles of the
(Song et al. 2009), A. thaliana, Oryza sativa, Nicotiana
pathogen-induced PaWRKY1
benthamiana (Park et al. 2005) and Vitis vinifera was
generated (Fig. 5B). The PaECR1 protein shared a high To better characterize PaWRKY1-mediated gene reg-
degree of sequence identity (approximately 70%) with ulatory networks, the transgenic Arabidopsis plants

Physiol. Plant. 2012

 A

Fig. 5. Amino acid sequence alignment and conserved domains of PaWRKY1 and PaECR1. (A) The deduced PaWRKY1 (HM596075) polypeptide
was aligned with five closely related group IIc WRKY proteins in plants, including Triticum aestivum TaWRKY (ABN43184), Solanum tuberosum
StWRKY 1 (CAB97004), Fragaria ananassa FaWRKY1 (ACH88751), Arabidopsis thaliana AtWRKY75 (NP_196812) and Brassica napus BnWRKY75
(ACI14409). The black boxes indicate identical residues and gray boxes indicate conservative substitutions. Hyphens indicate gaps introduced to
optimize alignments. The numbers on the left indicate the amino acid residues in the PaWRKY1 protein sequence. The WRKYGQK peptide stretch
is highlighted in red. Putative nuclear localization signals are indicated with black stars ( ) under the sequences. The two conserved cysteine and
histidine residues making up the potential zinc finger motif are marked with green. The black triangles ( ) and circles ( ) indicate amino acids
conserved within the motif A ([K/R]EPRVAV[Q/K]T[K/V]SEVD[I/V]L) and motif 3 (KAKKxxQK) described for subgroup IIc of the WRKY family, respectively
(Eulgem et al. 2000). The alignments were generated by the BOXSHADE website (http://www.ch.embnet.org/software/BOX_form.html). (B) Amino
acid sequence alignment of PaECR1 (GenBank accession number HM596076) with orthologs from Gossypium hirsutum (ABV60089), A. thaliana
(NP_191096), Oryza sativa (NP_001042030), Nicotiana benthamiana (AAY17262) and Vitis vinifera (XP_002283594). The ECR protein sequences with
homology to steroid-5-α-reductase domain are underlined. Blue shades mark the residues identified as critical for function of fatty acid elongation
(Paul et al. 2007). A putative NADPH-binding domain at the C-terminal region (Song et al. 2009) is denoted with squares ( ).

Physiol. Plant. 2012

 A

Fig. 6. Subcellular localization and DNA-binding activity of PaWRKY1. (A) The PaWRKY1-GFP fusion protein was transiently expressed by introducing
plasmid DNA using particle bombardment into onion epidermal cells. The photographs were taken in the dark field for green fluorescence and under
bright light for the morphology of the cell. Staining with DAPI indicated the localization of cell nuclei. The empty vector (pCAMBIA1302) harboring
the GFP construct served as control. (B) DNA-binding activity to the W-box elements was analyzed by a Y1H assay using PaWRKY1 as the target
protein (pGR-PaWRKY1) and three W boxes as bait (pHIS-W box). The bait construct bearing a mutated W box (pHIS-mW box) was included in the
experiment. The bait sequences from the promoter region of the orchid CDPK with approximately 300 or 700 bases in length (pHIS-CDPK300 or
700) were also subject to analysis. The constructed pGR- and pHIS-derived vectors were transformed into the Y187 strain of yeast, and were grown
on selective synthetic dextrose medium, SD/-Trp/-Leu/-His or SD/-Trp/-Leu/-His plus 30 mM 3-AT, respectively. The empty pGR-vector served as the
negative control. All experiments were performed twice with similar results.

were generated by constitutive expression of the of PaWRKY1 accumulated to a significant level in

PaWRKY1 gene under the control of the 35S promoter.
Subsequently, the transgenic Arabidopsis plants harbor-
ing the 35S-PaWRKY1 construct were characterized
by analyzing overexpression of the PaWRKY1 trans-
gene using semi-quantitative RT-PCR. The transcripts
the transgenic Arabidopsis plants when compared with
the control plants transformed with the empty vector
(Appendix S5). The PaWRKY1-overexpressing trans-
genic lines did not exhibit any apparent phenotypic
abnormalities compared with vector control plants (data

Physiol. Plant. 2012

not shown). The PaWRKY1-overexpressing Arabidop-
sis plants were subjected to microarray analysis using
the Arabidopsis GeneChip ATH1. The gene expres-
sion profiles of transgenic Arabidopsis plants expressing
PaWRKY1 were compared with those of vector control
plants.
As a result, the expression levels of 50 genes
were upregulated more than twofold in the transgenic
PaWRKY1 Arabidopsis when compared with those in
vector control plants. In contrast, the gene expression
levels of seven genes were downregulated less than
0.5-fold (Table 1). According to the gene ontology anal-
ysis by agriGO (Du et al. 2010), the largest functional
categories involved in the biological processes were
those in response to stimuli (Appendix S6). A gene
encoding cell wall bound POX was significantly down-
regulated in the transgenic Arabidopsis plants when
compared with the vector control plants. The microarray
analysis also revealed a substantial number of stress-
responsive genes with altered expression levels in the
transgenic plants. These genes were regulated by diverse
stimuli such as heat, drought, salt and other stresses. The
findings suggested that the PaWRKY1 transcription factor
was potentially involved in regulating the expression of
genes encoding stress-related proteins in the transgenic
Arabidopsis plants.
Functional analysis of PaECR1 gene by VIGS in the
leaves of P. amabilis plants
The Erwinia-induced PaECR1 (PE7) was the most abun-
dant transcripts in the EST library (Appendix S3). The
expression analysis of PaECR1 suggested that the gene
was predominately induced by E. chrysanthemi infection
in the leaf tissues. To better characterize the function of
PaECR1 in the response to E. chrysanthemi infection,
the orchid leaf tissues with suppression of endoge-
nous PaECR1 transcripts were generated by VIGS. The
PaECR1 cDNA fragment containing the partial sequence
of ORF was cloned into the CymMV-based VIGS vec-
tors (Fig. 7A). The leaf tissues of the orchid plants
were inoculated with the resultant CymMV-PaECR1
construct to trigger VIGS. The effect of VIGS on
endogenous PaECR1 mRNA levels was examined by
semi-quantitative RT-PCR using leaf RNA samples
(Fig. 7B). At 14-h post-inoculation with E. chrysan-
themi, the pathogen-induced PaECR1 gene expression
was compromised in the PaECR1-silenced orchid lines
in comparison with the empty-vector control plants.
Interestingly, the leaf tissues from the PaECR1-silenced
orchid lines were more susceptible to E. chrysanthemi
infection than that of vector control plants according
to the CFU (Fig. 7B). The progression of maceration
Physiol. Plant. 2012
symptoms caused by E. chrysanthemi was more rapidly
in the PaECR1-silenced lines than that of control plants
(data not shown). The data indicated that E. chrysanthemi
was able to accumulate to higher level in the leaf tissues
of PaECR1-silenced orchid in comparison with that of
empty-vector control. The relatively high susceptibility
to E. chrysanthemi in PaECR1-silenced leaf tissues were
consistent and observed in other VIGS-PaECR1 orchid
plant lines (Fig. 7B). The results demonstrated the func-
tional significance of PaECR1 in the orchid plants when
infected with E. chrysanthemi.
Discussion
Understanding the complex transcriptional changes
occurring in orchids in response to soft rot Erwinia is
important for efficient management of this pathogen.
Transcriptomics is a powerful approach for the global
analysis of plant–pathogen interactions (Fekete et al.
2009, Wang et al. 2009, Avrova et al. 2003, Sarowar
et al. 2011). To gain further information on the molecular
basis in orchids upon E. chrysanthemi infection, this
study describes the first large-scale investigation into
the pathogen-induced genes during the host–pathogen
interaction. SSH strategy was conducted to establish an
EST library enriched for genes expressed in the orchid
plants at the early stage of infection. A total of 102 unique
pathogen-induced ESTs were successfully generated and
annotated to different functional categories, especially
those associated with redox regulation, responses to
pathogens, metabolic process and lipid synthesis.
Expression profiles of the selected ESTs were consistent
with the SSH-based analysis, further strengthening the
reliability of the results. As anticipated, several of
the pathogen-induced ESTs were related to general
responses of plants to pathogen attack such as PR10c,
CHS and cell wall-associated enzymes (Appendix S3).
On the basis of the functional analysis of PaECR1 gene,
it is tempting to speculate that derivatives of the very
long chain fatty acid (VLCFA) biosynthesis pathway may
participate in the regulation of the pathogenesis. Taken
together, these data may reflect an intricate signaling
system, involving a number of signal compounds and
interacting signal pathways, regulating the expression of
pathogen-responsive genes in the orchid plants.
Necrotrophic pathogens appear to stimulate ROS
accumulation so as to trigger host cell death and allow
the pathogens to access nutrients, contributing to survival
and disease development. In pear leaves, inoculation
with E. amylovora resulted in superoxide accumulation,
lipid peroxidation and electrolyte leakage (Torres 2010).
In accordance with the report, collapse and macera-
tion of the orchid cells induced by E. chrysanthemi
Table 1. List of selected genes changed transcriptionally in transgenic PaWRKY1 Arabidopsis plants. The gene expression profiles of transgenic
35S-PaWRKY1 Arabidopsis were compared with those of control plants transformed with the empty vector. On the basis of microarray analysis,
genes with expression level that is more than twofold higher or less at an FDR below 0.05 are shown. Rank products analysis reveals that most
significantly differential expressed genes display the lowest RP value. AGI, Arabidopsis genome initiative; FC, fold change; RP/Rsum, rank product;
FDR, false discovery rate.

AGI number Product encoded FC RP/Rsum FDR

Responses to pathogens
AT5G48570 Peptidyl-prolyl cis–trans isomerase 6.46 11.69 0.0000
AT5G24160 Squalene monoxygenase 6 4.37 31.55 0.0000
AT5G40380 Cysteine-rich receptor-like protein kinase 42 2.58 102.76 0.0059
AT1G05680 UDP-glucoronosyl/UDP-glucosyl transferase family protein 2.43 130.90 0.0078
AT3G04110 Putative glutamate receptor 2.24 151.98 0.0110
AT1G29500 Auxin-responsive protein 2.21 160.10 0.0113
AT1G31580 Cell wall protein ECS1 2.10 190.24 0.0138
AT4G33550 Lipid transfer protein family 2.25 192.17 0.0139
AT3G51240 Flavanone 3-hydroxylase 2.24 210.84 0.0158
AT4G23570 Phosphoprotein phosphatase (SGT1A) 2.13 221.32 0.0178
AT5G05270 Chalcone-flavanone isomerase family protein 2.06 226.54 0.0191
AT1G29440 Auxin-induced protein 2.06 229.03 0.0188
AT5G24150 Squalene monooxygenase 2.01 253.11 0.0219
AT5G53120 Spermine synthase 2.00 289.11 0.0282
AT3G16670 MGL6 0.13 3.62 0.0000
AT5G64120 Cell wall bound peroxidase 0.38 54.18 0.0075
AT4G12480 Putative lipid transfer protein 0.41 89.06 0.0143
Response to heat
AT5G12030 Heat shock protein 17.6 A 7.04 13.34 0.0000
AT1G07400 Heat shock protein 17.8 kDa class I 6.06 20.86 0.0000
AT5G12020 Heat shock protein 17.6 kDa class II 4.77 35.42 0.0025
AT3G12580 Heat shock protein 70 4.16 38.88 0.0022
AT3G46230 Class I small heat shock protein 4.07 44.97 0.0020
AT5G52640 Cytosolic heat shock protein AtHSP90.1 4.11 45.51 0.0018
AT5G51440 Mitochondrial small heat shock protein 3.41 63.61 0.0046
AT4G12400 Stress-inducible protein 3.41 65.07 0.0040
AT2G29500 Heat shock protein 17.6 kDa class I 3.46 87.65 0.0035
AT1G74310 Heat shock protein 101 3.25 111.87 0.0054
AT2G32120 Heat shock protein 70 2.63 124.02 0.0065
AT2G22240 Myo-inositol-1-phosphate synthase 2.53 124.56 0.0061
AT2G20560 DNAJ heat shock family protein 2.93 152.70 0.0105
AT3G09350 Arabidopsis orthologs of the human Hsp70-binding protein 1 2.75 160.06 0.0116
AT2G26150 Heat shock factor A2 2.78 178.84 0.0128
AT2G47180 Galactinol synthase 2 2.29 194.35 0.0139
AT3G09440 Heat shock cognate 70 kDa protein 3 2.11 220.17 0.0180
AT2G25140 Heat shock protein 100 2.10 227.94 0.0188
AT1G54050 Heat shock protein 17.4 kDa class III 2.42 244.52 0.0218
AT1G30070 SGS domain-containing protein 2.11 262.90 0.0248
Response to cold, drought, salt and other stresses
AT2G29450 Glutathione transferase TAU 5 3.36 63.84 0.0043
AT2G42540 Cold-regulated protein cor15a precursor 3.33 69.05 0.0035
AT3G05640 Protein phosphatase 2C 2.96 97.27 0.0048
AT5G52310 Low temperature-induced 78 3.01 103.33 0.0057
AT1G72930 Disease resistance protein (TIR-NBS class) 3.10 119.55 0.0065
AT4G14400 Accelerated cell death 6 2.51 121.81 0.0070
AT1G01120 3-Ketoacyl-coasynthase 1 2.53 122.05 0.0068
AT1G02820 Late embryogenesis abundant 3 family protein 2.83 124.28 0.0062
AT1G22770 Gigantea protein 2.47 126.97 0.0066
AT5G66400 Drought-induced 8 2.69 139.04 0.0087
AT1G10370 Early responsive to dehydration 9 2.42 149.88 0.0107
AT4G08950 Phosphate-induced 1 2.16 176.87 0.0129
AT1G02400 Gibberellin 2-oxidase 2.16 183.78 0.0135

Physiol. Plant. 2012

Table 1. Continued
AGI number Product encoded
AT5G15960 KIN1 cold and ABA inducible protein
AT4G24960 Eukaryote-specific ABA- and stress-inducible gene
AT2G34810 FAD-binding domain-containing protein
AT5G57560 Xyloglucosyl transferase
AT1G04220 3-Ketoacyl-coasynthase 2
AT1G20450 Early responsive to dehydration 10
AT2G33380 Responsive to dessication 20
AT5G37260 MYB family transcription factor Circadian 1 (CIR1)
AT4G02380 Senescence-associated gene 21
AT2G40670 Putative two-component response regulator protein
AT1G73330 Drought-repressed 4
was preceded by accumulation of ROS (Figs 1 and 2).
Alteration in POX activity has been reported in various
plant–pathogen interactions. POX activity is expected
to reduce the level of ROS by metabolizing hydrogen
peroxides, but POX is also capable of various ’oxidase’
reactions leading to ROS generation (Nanda et al. 2010).
In this study, the necrotrophic bacterium, E. chrysan-
themi, induced in orchids a rapid production of ROS
and a concomitant alteration in antioxidative systems
(Fig. 2). Accumulation of ROS was detected mainly at
the site of inoculation during the early stage of patho-
genesis. Especially, the build-up of ROS levels was in
correlation with the decrease in POX activity during
the early stages post-inoculation (Fig. 2). The results sug-
gested that the initial effect of infection was the reduction
of POX activity resulting in the increase in ROS produc-
tion. Furthermore, a subset of pathogen-induced ESTs
associated with the oxidation regulation was also rep-
resented in the library. Among them there are genes
that encode antioxidant enzymes such as CAT, GST
and GR (PE40, PE26 and PE89; See Appendix S3). The
abundance of antioxidant enzymes in the library indi-
cated that the redox status in the attacked orchid plants
had been disturbed. One pathogen-induced EST encod-
ing an enzyme with homology to lipoxygenase (LOX,
PE163; Appendix S3) was also isolated, suggesting a
role of lipid peroxidation in the orchid– Erwinia interac-
tion (Porta and Rocha-Sosa 2002). Taken together, the
ROS production, POX activity and antioxidant-related
ESTs provide information on an alteration in redox status
that might coordinate regulation of molecular responses
during the orchid– Erwinia interaction.
Upon infection of host plants by pathogens, a complex
network of signaling components and transcription
factors relays the information in the plant cell to
the nucleus, where specific transcriptional responses
is triggered. Members of several transcription factor
families (bZIP, WRKY, MYB, ERF and Whirly) have been
shown to participate in the regulation of plant responses
Physiol. Plant. 2012
FC RP/Rsum FDR
2.52 184.91 0.0132
2.29 185.20 0.0130
2.14 233.45 0.0201
2.63 243.99 0.0221
2.16 267.94 0.0251
2.33 275.56 0.0254
2.56 300.86 0.0297
0.34 45.04 0.0150
0.45 81.34 0.0133
0.45 92.18 0.0125
0.49 137.74 0.0270
to pathogens (Eulgem 2005). Several lines of evidence
have indicated the potential involvement of WRKY
family transcription factors in plant– Erwinia interactions.
For example, Erwinia- or CF-induced expression of
WRKY genes was observed in various plant species such
as potato, apple and Arabidopsis (Dellagi et al. 2000,
Li et al. 2004, Baldo et al. 2010, Sarowar et al. 2011).
Dual functionality has also been suggested for WRKY
transcription factors. Arabidopsis plants overexpressing
AtWRKY41 exhibited enhanced resistance toward
virulent Pseudomonas but decreased resistance toward
E. carotovora (Higashi et al. 2008). This study described
a full-length orchid gene (PaWRKY1) encoding a protein
with sequence homology to members of the plant
WRKY family (Fig. 5). Functional analysis of PaWRKY1
in the orchid plants was performed to address its role
in the pathogenesis. However, the pCymMV-PaWRKY1
recombinant viruses were unable to trigger VIGS in the
orchid plants (data not shown). This newly characterized
gene is the first WRKY factor described in orchids and
belongs to group IIc of the WRKY family. Nuclear
localization and a DNA-binding activity assay indicated
that PaWRKY1 is a functional protein characteristic of
WRKY transcription factors (Fig. 6). The closest sequence
homologs of PaWRKY1, the potato StWRKY, is also
involved in the host response to Erwinia (Dellagi et al.
2000). These findings collectively suggest a regulatory
role for group IIc proteins of the WRKY family in
plant– Erwinia interactions.
VLCFAs (fatty acids with chain lengths ranging from
20 to 36 carbons) and their derivatives are essential
biological components found in lipid, suberin and cutic-
ular waxes in plants (Raffaele et al. 2009). The role
of VLCFAs in plant responses to Erwinia infection has
not been reported previously. In this study, analysis of
gene expression demonstrated that a number of genes
encoding proteins involved in the VLCFA biosynthesis
pathway were induced in orchid plants during infection
by E. chrysanthemi. Two genes coding for key enzymes
 A responses against E. chrysanthemi (Fig. 7). Indeed, recent
B sphingolipids. The plant cuticle is believed to provide
Fig. 7. Functional analysis of PaECR1 in Phalaenopsis amabilis plants
by VIGS. (A) Schematic of the PaECR1 cDNA structure and the VIGS
construct containing the cDNA fragments. The box indicates the ORF
region of PaECR1. The primer sets (PaECR1-full) corresponding to the 5
and 3 end of the PaECR1 coding region were used in RT-PCR to assess
the knockdown efficiency of the host gene by VIGS. (B)Characterization
of the PaECR1-silenced leaf tissues in the orchid plants by VIGS.
Knockdown effects of PaECR1 gene expression were determined at
14 h after inoculation with Erwinia chrysanthemi. The empty-vector
control plants served as a control as compared with the PaECR1-silenced
plants. The PaECR1 and Paactin cDNA fragments were amplified by
RT-PCR using the gene-specific primers (Appendix S1). Assessment of
susceptibility status was performed in the PaECR1-silenced orchid plants
when infected with E. chrysanthemi. The infected leaf tissues derived
from the PaECR1-silenced and vector control plants were collected at
14-h post-inoculation. The susceptibility was determined in accordance
with CFU. The number of lines indicates the samples from individual
plants carrying empty vector or PaECR1 VIGS constructs. These results
are representative of experiments conducted in triplicates with similar
expression patterns.
that catalyze the intermediate steps of VLCFA biosynthe-
sis, ACC and ECR (Roudier et al. 2010), were represented
in the pathogen-induced EST library (PE7 and PE97;
Appendix S3). In particular, the orchid ECR was found
to be the most abundant transcripts in the library sug-
gesting a role for VLCFAs in the host response to Erwinia
infection. Gene expression validation of the orchid ACC
and ECR confirmed the induction of these genes in
response to E. chrysanthemi infection (Fig. 4; Appendix
S4). Noticeably, functional analysis of PaECR1 gene in
the orchid plants clearly demonstrated its roles in defense
findings indicate that the VLCFA biosynthesis pathway
has been associated with plant defense through differ-
ent aspects (Raffaele et al. 2009). VLCFAs contribute to
biosynthesis of the plant cuticle and the generation of
an efficient barrier against the majority of pathogens that
colonize the plant surface and it represents a passive
defense (Reina-Pinto and Yephremov 2009). Sphin-
golipids are key components of the membrane and may
also serve as signaling molecules during plant–pathogen
interactions. Accumulating evidence implies that sphin-
golipids play a crucial role in plant innate immunity
including triggers of programmed cell death and elicitor-
induced defense mechanisms (Takahashi et al. 2009).
Therefore, this study implies that the orchid ACC and
ECR may lead to alteration in cuticle structure or sphin-
golipid synthesis, which in turn contributes to a defense
response against E. chrysanthemi. ECR is required for
the synthesis of the VLCFA-containing lipids that is in
association with deception for pollinator attraction in
the orchid family (Schlüter et al. 2011). The finding
in this study also revealed the unique orchid– Erwinia
interaction mediated by the pathogen-inducible PaECR1.
It has been demonstrated in Nicotiana benthamiana
and Arabidopsis that disruption of the ECR genes
affected the VLCFA-mediated cell expansion during
plant morphogenesis (Park et al. 2005, Zheng et al.
2005). Analyses of the Arabidopsis ECR mutants also
provide in planta evidence that the ECR is involved
in VLCFA biosynthesis for the production of wax, seed
storage triacylglycerols and sphingolipids. Accordingly,
it was speculated that silencing of PaECR1 gene
in the orchid leaf tissues would lead to alteration
of VLCFA-derived metabolites such as wax and
sphingolipids. It is reasonable to assume that epicuticular
wax composition was responsible for the altered
interaction with the pathogens, but it is also possible
that lipids derived from the VLCFA biosynthesis pathway
may have played an important role in defense signaling.
Interference with PaECR1 in the VLCFA pathways
may increase the susceptibility of the orchid plants to
E. chrysanthemi infection.
In conclusion, the host transcriptional responses of
the orchid plants were initially triggered either by
E. chrysanthemi attack or by its corresponding elicitor,
which contained cell wall-degrading enzymes. During
the early stage of pathogenesis, the increase in ROS
production occurred at the site of infection. Alteration
in cellular redox homeostasis through the antioxidant
enzyme systems (i.e. CAT, GR, GST and POX)
might contribute to activation of signal transduction.
The pathogen-induced signals may affect activity of
Physiol. Plant. 2012
transcription factors, including bZIP, MYB and WRKY.
The ECR-mediated VLCFA biosynthesis pathway is
potentially involved in the defense responses and unique
in the orchid– Erwinia interaction. In addition, several
genes whose homologs were detected during this study
have not been characterized for their possible roles in the
pathogenesis. Such genes could account for differences
in gene expression specific to the orchid plants and
need to be examined further. The determination of the
changes in VLCFA-derived metabolites is required to
get more insight into the pathogenesis. The findings
in this study provide valuable information not only
in understanding overall gene expression in orchids
during E. chrysanthemi infection, but also in identifying
potential targets for development of disease control
strategies.
Acknowledgements – This study was funded by grants from
the Taiwan National Science Council (project number NSC
96-2317-B-006-005) and the Council of Agriculture (project
number 91-3.1.3-Z3). This work was also supported by a
grant from the Ministry of Education, Taiwan (’Landmark
Project Grant’ for National Cheng Kung University’s
‘Top-University Project’, B024). The Affymetrix GeneChip
assays were performed by the Affymetrix Gene Expression
Service Lab (http://ipmb.sinica.edu.tw/affy/), supported by
Academia Sinica, Taiwan. Expression profiles and data
mining were carried out with a system provided by
the Bioinformatics Core for Genomic Medicine and
Biotechnology Development at the National Cheng Kung
University, supported by a National Science Council grant
(NSC 97-3112-B-006-011).
References
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z,
Miller W, Lipman DJ (1997) Gapped BLAST and
PSI-BLAST: a new generation of protein database search
programs. Nucleic Acids Res 25: 3389–3402
Asai S, Yoshioka H (2008) The role of radical burst via
MAPK signaling in plant immunity. Plant Signal Behav
3: 920–922
Austin S, Lojkowska E, Ehlenfeldt MK, Kelman A,
Helgeson JP (1988) Fertile interspecific somatic hybrids
of Solanum –a novel source of resistance to Erwinia soft
rot. Phytopathology 78: 1216–1220
Averyanov A (2009) Oxidative burst and plant disease
resistance. Front Biosci (Elite Ed) 1: 142–152
Avrova AO, Venter E, Birch PR, Whisson SC (2003)
Profiling and quantifying differential gene transcription
in Phytophthora infestans prior to and during the early
stages of potato infection. Fungal Genet Biol 40: 4–14
Bains PS, Bisht VS, Lynch DR, Kawchuk LM, Helgeson JP
(1999) Identification of stem soft rot (Erwinia carotovora
Physiol. Plant. 2012
subspecies atroseptica) resistance in potato. Am J Potato
Res 76: 137–141
Baker B, Zambryski P, Staskawicz B, Dinesh-Kumar SP
(1997) Signaling in plant–microbe interactions. Science
276: 726–733
Baldo A, Norelli JL, Farrell RE Jr, Bassett CL,
Aldwinckle HS, Malnoy M (2010) Identification of genes
differentially expressed during interaction of resistant
and susceptible apple cultivars (Malus×domestica) with
Erwinia amylovora. BMC Plant Biol 10: 1
Barras F, Vangijsegem F, Chatterjee AK (1994)
Extracellular enzymes and pathogenesis of soft-rot
Erwinia. Annu Rev Phytopathol 32: 201–234
Bell KS, Sebaihia M, Pritchard L, Holden MT, Hyman LJ,
Holeva MC, Thomson NR, Bentley SD, Churcher LJ,
Mungall K, Atkin R, Bason N, Brooks K, Chillingworth T,
Clark K, Doggett J, Fraser A, Hance Z, Hauser H,
Jagels K, Moule S, Norbertczak H, Ormond D, Price C,
Quail MA, Sanders M, Walker D, Whitehead S,
Salmond GP, Birch PR, Parkhill J, Toth IK (2004)
Genome sequence of the enterobacterial phytopathogen
Erwinia carotovora subsp. atroseptica and
characterization of virulence factors. Proc Natl Acad Sci
USA 101: 11105–11110
Benjamini Y, Hochberg Y (1995) Controlling the false
discovery rate – a practical and powerful approach to
multiple testing. J R Stat Soc B Met 57: 289–300
Beyer WF Jr, Fridovich I (1987) Assaying for superoxide
dismutase activity: some large consequences of minor
changes in conditions. Anal Biochem 161: 559–566
Brader G, Tas E, Palva ET (2001) Jasmonate-dependent
induction of indoleglucosinolates in Arabidopsis by
culture filtrates of the nonspecific pathogen Erwinia
carotovora. Plant Physiol 126: 849–860
Bradford MM (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal Biochem 72:
248–254
Breitling R, Armengaud P, Amtmann A, Herzyk P (2004)
Rank products: a simple, yet powerful, new method to
detect differentially regulated genes in replicated
microarray experiments. FEBS Lett 573: 83–92
Clough SJ, Bent AF (1998) Floral dip: a simplified method
for Agrobacterium-mediated transformation of
Arabidopsis thaliana. Plant J 16: 735–743
Collier SM, Moffett P (2009) NB-LRRs work a ‘‘bait and
switch’’ on pathogens. Trends Plant Sci 14: 521–529
Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M,
Robles M (2005) Blast2GO: a universal tool for
annotation, visualization and analysis in functional
genomics research. Bioinformatics 21: 3674–3676
Dellagi A, Helibronn J, Avrova AO, Montesano M,
Palva ET, Stewart HE, Toth IK, Cooke DE, Lyon GD,
Birch PR (2000) A potato gene encoding a WRKY-like
transcription factor is induced in interactions with
 Erwinia carotovora subsp. atroseptica and
Phytophthorainfestans and is coregulated with class I
endochitinase expression. Mol Plant Microbe Interact
13: 1092–1101
Dellagi A, Segond D, Rigault M, Fagard M, Simon C,
Saindrenan P, Expert D (2009) Microbial siderophores
exert a subtle role in Arabidopsis during infection by
manipulating the immune response and the iron status.
Plant Physiol 150: 1687–1696
Devaiah BN, Karthikeyan AS, Raghothama KG (2007)
WRKY75 transcription factor is a modulator of
phosphate acquisition and root development in
Arabidopsis. Plant Physiol 143: 1789–1801
Du Z, Zhou X, Ling Y, Zhang Z, Su Z (2010) agriGO: a GO
analysis toolkit for the agricultural community. Nucleic
Acids Res 38(suppl): W64–W70
Encinas-Villarejo S, Maldonado AM, Amil-Ruiz F, de los
Santos B, Romero F, Pliego-Alfaro F, Munoz-Blanco J,
Caballero JL (2009) Evidence for a positive regulatory
role of strawberry (Fragaria×ananassa) Fa WRKY1 and
Arabidopsis At WRKY75 proteins in resistance. J Exp Bot
60: 3043–3065
Eulgem T (2005) Regulation of the Arabidopsis defense
transcriptome. Trends Plant Sci 10: 71–78
Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The
WRKY superfamily of plant transcription factors. Trends
Plant Sci 5: 199–206
Fagard M, Dellagi A, Roux C, Perino C, Rigault M,
Boucher V, Shevchik VE, Expert D (2007) Arabidopsis
thaliana expresses multiple lines of defense to
counterattack Erwinia chrysanthemi. Mol Plant Microbe
Interact 20: 794–805
Fekete C, Fung RW, Szabó Z, Qiu W, Chang
L, Schachtman DP, Kovács LG (2009) Up-regulated
transcripts in a compatible powdery mildew-grapevine
interaction. Plant Physiol Biochem 47: 732–738
Gozzo F (2003) Systemic acquired resistance in crop
protection: from nature to a chemical approach. J Agric
Food Chem 51: 4487–4503
Hammerschmidt R (2004) Turning a pathogenicity factor
against the pathogen. Physiol Mol Plant Pathol 65:
57–58
Hammond-Kosack KE, Parker JE (2003) Deciphering
plant-pathogen communication: fresh perspectives for
molecular resistance breeding. Curr Opin Biotechnol
14: 177–193
Hancock JT, Desikan R, Clarke A, Hurst RD, Neill SJ
(2002) Cell signalling following plant/pathogen
interactions involves the generation of reactive oxygen
and reactive nitrogen species. Plant Physiol Biochem
40: 611–617
Higashi K, Ishiga Y, Inagaki Y, Toyoda K, Shiraishi T,
Ichinose Y (2008) Modulation of defense signal
transduction by flagellin-induced WRKY41 transcription
factor in Arabidopsis thaliana. Mol Genet Genomics
279: 303–312
Hou S, Yang Y, Zhou JM (2009) The multilevel and
dynamic interplay between plant and pathogen. Plant
Signal Behav 4: 283–293
Jones JD, Dangl JL (2006) The plant immune system.
Nature 444: 323–329
Kazemi-Pour N, Condemine G,
Hugouvieux-Cotte-Pattat N (2004) Thesecretome of the
plant pathogenic bacterium Erwinia chrysanthemi.
Proteomics 4: 3177–3186
Kohlwein SD, Eder S, Oh CS, Martin CE, Gable K,
Bacikova D, Dunn T (2001) Tsc13p is required for fatty
acid elongation and localizes to a novel structure at the
nuclear-vacuolar interface in Saccharomyces cerevisiae.
Mol Cell Biol 21: 109–125
Li J, Brader G, Palva ET (2004) The WRKY70 transcription
factor: a node of convergence for jasmonate-mediated
and salicylate-mediated signals in plant defense. Plant
Cell 16: 319–331
Liau CH, Lu JC, Prasad V, Hsiao HH, You SJ, Lee JT,
Yang NS, Huang HE, Feng TY, Chen WH, Chan MT
(2003) The sweet pepper ferredoxin-like protein (pflp)
conferred resistance against soft rot disease in Oncidium
orchid. Transgenic Res 12: 329–336
Lohse M, Nunes-Nesi A, Kruger P, Nagel A, Hannemann J,
Giorgi FM, Childs L, Osorio S, Walther D, Selbig J,
Sreenivasulu N, Stitt M, Fernie AR, Usadel B (2010)
Robin: an intuitive wizard application for R-based
expression microarray quality assessment and analysis.
Plant Physiol 153: 642–651
Lu HC, Chen HH, Tsai WC, Chen WH, Su HJ, Chang DC,
Yeh HH (2007) Strategies for functional validation of
genes involved in reproductive stages of orchids. Plant
Physiol 143: 558–569
Mangelsen E, Kilian J, Berendzen KW, Kolukisaoglu UH,
Harter K, Jansson C, Wanke D (2008) Phylogenetic and
comparative gene expression analysis of barley
(Hordeum vulgare ) WRKY transcription factor family
reveals putatively retained functions between monocots
and dicots. BMC Genomics 9: 194
McDowell JM, Woffenden BJ (2003) Plant disease
resistance genes: recent insights and potential
applications. Trends Biotechnol 21: 178–183
Montillet JL, Chamnongpol S, Rusterucci C, Dat J, van de
Cotte B, Agnel JP, Battesti C, Inze D, Van Breusegem F,
Triantaphylides C (2005) Fatty acid hydroperoxides and
H2 O2 in the execution of hypersensitive cell death in
tobacco leaves. Plant Physiol 138: 1516–1526
Nanda AK, Andrio E, Marino D, Pauly N, Dunand C
(2010) Reactive oxygen species during
plant-microorganism early interactions. J Integr Plant
Biol 52: 195–204
Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N,
Estelle M, Voinnet O, Jones JD (2006) A plant miRNA
Physiol. Plant. 2012
 contributes to antibacterial resistance by repressing
auxin signaling. Science 312: 436–439
Nicaise V, Roux M, Zipfel C (2009) Recent advances in
PAMP-triggered immunity against bacteria: pattern
recognition receptors watch over and raise the alarm.
Plant Physiol 150: 1638–1647
Nomura K, Melotto M, He SY (2005) Suppression of host
defense in compatible plant– Pseudomonas syringae
interactions. Curr Opin Plant Biol 8: 361–368
Norman C, Vidal S, Palva ET (1999)
Oligogalacturonide-mediated induction of a gene
involved in jasmonic acid synthesis in response to the
cell-wall-degrading enzymes of the plant pathogen
Erwinia carotovora. Mol Plant Microbe Interact 12:
640–644
Norman-Setterblad C, Vidal S, Palva ET (2000) Interacting
signal pathways control defense gene expression in
Arabidopsis in response to cell wall-degrading enzymes
from Erwinia carotovora. Mol Plant Microbe Interact 13:
430–438
Palva TK, Holmstrom KO, Heino P, Palva ET (1993)
Induction of plant defense response by exoenzymes of
Erwinia carotovorasubsp. carotovora. Mol Plant Microbe
Interact 6: 190–196
Park JA, Kim TW, Kim SK, Kim WT, Pai HS (2005)
Silencing of NbECR encoding a putative enoyl-CoA
reductase results in disorganized membrane structures
and epidermal cell ablation in Nicotiana benthamiana.
FEBS Lett 579: 4459–4464
Paul S, Gable K, Dunn TM (2007) A six-membrane-
spanning topology for yeast and Arabidopsis Tsc13p, the
enoylreductases of the microsomal fatty acid elongating
system. J Biol Chem 282: 19237–19246
Porta H, Rocha-Sosa M (2002) Plant lipoxygenases.
Physiological and molecular features. Plant Physiol 130:
15–21
Raffaele S, Leger A, Roby D (2009) Very long chain fatty
acid and lipid signaling in the response of plants to
pathogens. Plant Signal Behav 4: 94–99
Reina-Pinto JJ, Yephremov A (2009) Surface lipids and
plant defenses. Plant Physiol Biochem 47: 540–549
Roudier F, Gissot L, Beaudoin F, Haslam R, Michaelson L,
Marion J, Molino D, Lima A, Bach L, Morin H, Tellier F,
Palauqui JC, Bellec Y, Renne C, Miquel M, Dacosta M,
Vignard J, Rochat C, Markham JE, Moreau P, Napier J,
Faure JD (2010) Very-long-chain fatty acids are involved
in polar auxin transport and developmental patterning in
Arabidopsis. Plant Cell 22: 364–375
Rushton PJ, Somssich IE, Ringler P, Shen QJ (2010) WRKY
transcription factors. Trends Plant Sci 15: 247–258
Sarowar S, Zhao Y, Soria-Guerra RE, Ali S, Zheng
D, Wang D, Korban SS (2011) Expression profiles of
differentially regulated genes during the early stages of
apple flower infection with Erwinia amylovora. J Exp Bot
62: 4851–4861
Physiol. Plant. 2012
Schlüter PM, Xu S, Gagliardini V, Whittle E, Shanklin
J, Grossniklaus U, Schiestl FP (2011) Stearoyl-acyl
carrier protein desaturases are associated with floral
isolation in sexually deceptive orchids. Proc Natl Acad
Sci USA 108: 5696–5701
Sjahril R, Chin DP, Khan RS, Yamamura S, Nakamura I,
Amemiya Y, Mii M (2006) Transgenic Phalaenopsis
plants with resistance to Erwinia carotovora produced
by introducing wasabi defensin gene using
Agrobacterium method. Plant Biotechnol 23: 191–194
Song WQ, Qin YM, Saito M, Shirai T, Pujol FM,
Kastaniotis AJ, Hiltunen JK, Zhu YX (2009)
Characterization of two cotton cDNAs encoding
trans-2-enoyl-CoA reductase reveals a putative novel
NADPH-binding motif. J Exp Bot 60: 1839–1848
Takahashi Y, Berberich T, Kanzaki H, Matsumura H,
Saitoh H, Kusano T, Terauchi R (2009) Unraveling the
roles of sphingolipids in plant innate immunity. Plant
Signal Behav 4: 536–538
Tek AL, Stevenson WR, Helgeson JP, Jiang J (2004)
Transfer of tuber soft rot and early blight resistances
from Solanum brevidens into cultivated potato.
TheorAppl Genet 109: 249–254
Torres MA (2010) ROS in biotic interactions. Physiol Plant
138: 414–429
Torres MA, Jones JD, Dangl JL (2006) Reactive oxygen
species signaling in response to pathogens. Plant Physiol
141: 373–378
Toth IK, Bell KS, Holeva MC, Birch PRJ (2003) Soft rot
Erwiniae: from genes to genomes. Mol Plant Pathol 4:
17–30
Tsai TM, Chen YR, Kao TW, Tsay WS, Wu CP, Huang DD,
Chen WH, Chang CC, Huang HJ (2007) PaCDPK1, a
gene encoding calcium-dependent protein kinase from
orchid, Phalaenopsis amabilis, is induced by cold,
wounding, and pathogen challenge. Plant Cell Rep 26:
1899–1908
Tyler BM (2009) Entering and breaking: virulence effector
proteins of oomycete plant pathogens. Cell Microbiol
11: 13–20
Vidal S, deLeon IP, Denecke J, Palva ET (1997) Salicylic
acid and the plant pathogen Erwinia carotovora induce
defense genes via antagonistic pathways. Plant J 11:
115–123
Vidal S, Eriksson ARB, Montesano M, Denecke J, Palva ET
(1998) Cell wall-degrading enzymes from Erwinia
carotovoracooperate in the salicylic acid-independent
induction of a plant defense response. Mol Plant
Microbe Interact 11: 23–32
Wang X, Tang C, Zhang G, Li Y, Wang C, Liu B, Qu Z,
Zhao J, Han Q, Huang L, Chen X, Kang Z (2009)
cDNA-AFLP analysis reveals differential gene expression
in compatible interaction of wheat challenged with
Pucciniastriiformis f. sp. tritici. BMC Genomics 10: 289
Wegener B, Olsen O (2004) Heterologous
pectatelyaseisoenzymes are not different in their effects
on soft rot resistance in transgenic potatoes. Physiol Mol
Plant Pathol 65: 59–66
Yang B, Jiang Y, Rahman MH, Deyholos MK, Kav NN
(2009) Identification and expression analysis of WRKY
transcription factor genes in canola (Brassica napus L.)
in response to fungal pathogens and hormone
treatments. BMC Plant Biol 9: 68
Zhao YF, Blumer SE, Sundin GW (2005) Identification of
Erwinia amylovora genes induced during infection of
immature pear tissue. J Bacteriol 187: 8088–8103
Zheng H, Rowland O, Kunst L (2005) Disruptions of the
Arabidopsis enoyl-CoA reductase gene reveal an
essential role for very-long-chain fatty acid synthesis in
cell expansion during plant morphogenesis. Plant Cell
17: 1467–1481
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Appendix S1. List of primers and expected lengths of
PCR products described in this study.
Edited by R. Terauchi
Appendix S2. Electrophoresis analysis of the subtracted
cDNA fragments from the orchid leaves after infection
with Erwinia chrysanthemi.
Appendix S3. List of early pathogen-induced transcripts
in Phalaenopsis amabilis plants infected by Erwinia
chrysanthemi.
Appendix S4. Validation of representative pathogen-
induced ESTs by semi-quantitative RT-PCR analysis.
Appendix S5. Characterization of transgenic Arabidop-
sis plants overexpressing the orchid PaWRKY1 gene.
Appendix S6. Gene ontology categorization of the
differentially expressed genes in transgenic Arabidopsis
plants overexpressing the orchid PaWRKY1 gene.
Please note: Wiley-Blackwell are not responsible for
the content or functionality of any supporting materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
Physiol. Plant. 2012