Vol. 24, No. 6, 2011 / 671
MPMI Vol. 24, No. 6, 2011, pp. 671–684. doi:10.1094/MPMI-08-10-0183. © 2011 The American Phytopathological Society
Use of a Secretion Trap Screen in Pepper
Following Phytophthora capsici Infection Reveals
Novel Functions of Secreted Plant Proteins
in Modulating Cell Death
Seon-In Yeom,1 Hyang-Ku Baek,1 Sang-Keun Oh,1 Won-Hee Kang,1 Sang Jik Lee,2 Je Min Lee,1
Eunyoung Seo,1 Jocelyn K. C. Rose,2 Byung-Dong Kim,1 and Doil Choi1
1Department of Plant Science, Plant Genomics and Breeding Institute, Seoul National University, Seoul, 151-921, Republic
of Korea; 2Department of Plant Biology, Cornell University, Ithaca, NY 14853, U.S.A.
Submitted 11 August 2010. Accepted 22 January 2011.
In plants, the primary defense against pathogens is mostly
inducible and associated with cell wall modification and
defense-related gene expression, including many secreted
proteins. To study the role of secreted proteins, a yeast-
based signal-sequence trap screening was conducted with
the RNA from Phytophthora capsici-inoculated root of Cap-
sicum annuum ‘Criollo de Morelos 334’ (CM334). In total,
101 Capsicum annuum secretome (CaS) clones were iso-
lated and identified, of which 92 were predicted to have a
secretory signal sequence at their N-terminus. To identify
differences in expressed CaS genes between resistant and
susceptible cultivars of pepper, reverse Northern blots and
real-time reverse-transcription polymerase chain reaction
were performed with RNA samples isolated at different
time points following P. capsici inoculation. In an attempt
to assign biological functions to CaS genes, we performed
in planta knock-down assays using the Tobacco rattle virus-
based gene-silencing method. Silencing of eight CaS genes
in pepper resulted in suppression of the cell death induced
by the non-host bacterial pathogen (Pseudomonas syringae
pv. tomato T1). Three CaS genes induced phenotypic ab-
normalities in silenced plants and one, CaS259 (PR4-l),
caused both cell death suppression and perturbed pheno-
types. These results provide evidence that the CaS genes
may play important roles in pathogen defense as well as
In plants, one of the fiercest battlefields of plant–microbe
interactions is the apoplastic milieu, which includes the cell
wall matrix and associated extracellular environment (Hugot et
al. 2004; Schulze-Lefert 2004). Here, secreted protein popula-
tions, or “secretomes”, play a crucial role in the numerous
complex defense responses that are mounted against pathogens,
(Birch et al. 2006; Kamoun 2006; Lee et al. 2004, 2006a). Dur-
ing early stages of pathogen infection, plants recognize a patho-
gen-secreted elicitor, triggering defense responses through sig-
naling pathways. Secreted proteins are known to be associated
with cell wall thickening, to resist penetration, through cell po-
larization and papilla formation at the site of pathogen attack
(Schmelzer 2002; Schulze-Lefert 2004). Secreted proteins,
such as the classically defined pathogenesis-related (PR) pro-
teins, contribute to the establishment of resistance and sys-
temic acquired resistance in plants (Jones and Takemoto 2004;
Stinzi et al. 1993; Wang et al. 2005).
The oomycete Phytophthora capsici is a soilborne pathogen
of critical food crops, causing root and fruit rot of pepper, to-
mato, and other solanaceous or cucurbitaceous plants (Hausbeck
and Lamour 2004; Walker and Bosland 1999). Recently, the
incidence and infection of P. capsici has increased, leading to
severe economic losses for these crops worldwide (Ristaino
and Johnston 1999). To date, no useful management methods
have been developed, because chemical and biological controls
are limited and ineffective in preventing the spread of P. cap-
sici to pepper crops (Oelke et al. 2003). Although many studies
have reported that the resistance to P. capsici is polygenic and
is controlled by quantitative trait loci (Kim et al. 2008; Lefebvre
and Palloix 1996; Thabuis et al. 2003), little is known about
pepper–oomycete interactions at the molecular and genetic
levels. Furthermore, most genetic and molecular studies have
focused on P. infestans– and P. sojae–host interactions (Birch
et al. 2006; Kamoun 2006; Moy et al. 2004; Tyler 2007).
The defense response in plants is associated with a change
in the repertoire of secreted proteins in response to Phytoph-
thora spp. infection (Hugot et al. 2004; Lee et al. 2006a;
Mithofer et al. 2002). The pepper–P. capsici interaction in
resistant plants also occurs as an intercellular rather than intra-
cellular response. For example, growth of P. capsici in resis-
tant pepper is restricted to the extracellular environment
through cell wall apposition related to secreted proteins; there-
fore, host cells are not severely damaged. In contrast, in sus-
ceptible pepper, P. capsici shows rapid intra- and intercellular
growth, cell walls are degraded, and cells became plasmolyzed
(Hwang et al. 1989; Ilarslan et al. 1996; Lee et al. 2000). A
better understanding of defense mechanisms and, in this case,
extracellular mechanisms may provide new insights into the
interactions between P. capsici and pepper.
Because of the importance of secretory proteins, many re-
searchers have developed high-throughput screening methods
for their detection (Cutler et al. 2000; Jacobs et al. 1997;
Tashiro et al. 1993). Among these, a yeast-based secretion trap
Current address for J. M. Lee: Boyce Thompson Institute for Plant Research,
Ithaca, NY 14853, U.S.A.
Corresponding author: D. Choi; Telephone: +1 82-2-880-4568; Fax: +1 82-
2-873-2056; E-mail: email@example.com
*The e-Xtra logo stands for “electronic extra” and indicates that six sup-
plementary figures and three supplementary tables are published online.
Also, Figures 1, 2, and 6 appear in color online.
672 / Molecular Plant-Microbe Interactions
(YST), using invertase as the reporter gene, has been success-
fully used for screening the plant secretome (Goo et al. 1999;
Klein et al. 1996; Jacobs et al. 1997; Yamane et al. 2005) and
for mining secretory proteins related to host–pathogen interac-
tions (Hugot et al. 2004; Lee et al. 2006a; Oh et al. 2005). This
technique involves ligating a plant-derived cDNA library into a
YST vector fused to the invertase gene (Suc2) lacking the N-
terminal signal peptide. The heterologous cDNA library is trans-
formed into an invertase-deficient yeast mutant. Any yeast trans-
fected with cDNA encoding a secreted protein could secrete an
invertase-fused protein, resulting in growth on medium contain-
ing sucrose as a sole carbon source. Rescued mutant yeast trans-
formants are isolated and the genes encoding the secreted pro-
teins can then be identified.
The purpose of this study was to characterize secreted pro-
teins related to the defense response from a pathogen-resistant
cultivar of pepper following inoculation with P. capsici. We
isolated 101 Capsicum annuum genes encoding secreted pro-
teins using the YST screen and compared the transcript levels
in resistant and susceptible pepper cultivars at different time
points following P. capsici infection. In addition, Tobacco rat-
tle virus (TRV)-based gene silencing assays were performed to
help determine the role of the secreted proteins in plant de-
fense and development. Our study will facilitate a better under-
standing of the molecular functions of those secreted proteins
that modulate the host defense response in plants.
Confirmation of compatible and incompatible interactions
of pepper against P. capsici.
To better understand the spectrum of extracellular defense
responses against pathogen attack, we attempted to isolate
genes encoding secreted proteins modulated during cell death
due to infection by an oomycete pathogen. Specifically, we
inoculated the roots of C. annuum ‘Criollo de Morelos 334’
(CM334) and ‘Chilsungcho’, which are cultivars that are resis-
tant and susceptible, respectively, to P. capsici, with P. capsici
zoospores. Disease symptoms (root rot) were observed in Chil-
sungcho but not in CM334 within 72 h postinoculation (hpi)
(Fig. 1A). To validate these symptoms, we performed a 2,3,5-
triphenyltetrazolium chloride (TTC) reduction assay as a cell
vitality indicator (Chen et al. 2006). Although no differences
were observed above ground during infection until 72 hpi, the
vitality of P. capsici-infected roots changed dramatically be-
tween the two pepper cultivars (Fig. 1B). Differences in TTC
reductase activity in the root of both cultivars were observed at
3 hpi but were significant from 24 to 48 hpi, showing two or
three times more activity in CM334 than in Chilsungcho.
We also determined biomass of P. capsici hyphae in the
infected pepper roots and the expression of PR proteins as
positive markers for P. capsici infection (Silvar et al. 2008).
During infection, transcript levels of the P. capsici elongation
factor 1 (PcEF1) gene were significantly lower in roots of
CM334 than in those of Chilsungcho (Fig. 1C). CaPINII and
CaBPR1 transcripts were more strongly and rapidly induced in
the resistant than the susceptible cultivar (Fig. 1D), confirming
that the two cultivars exhibit compatible and incompatible
interactions, respectively, with P. capsici (Hwang et al. 1989;
Ueeda et al. 2006). Root samples were harvested at various
time points after inoculation of P. capsici and total RNA ex-
tracts were isolated for further study.
Isolation of C. annuum secretome using YST.
To understand the extracellular events during the defense re-
sponse, a cDNA library was constructed in pYST 0-2 vector
(Lee et al. 2006a,b) using pools of mRNA (3, 6, 12, 24, 48, and
72 hpi) from CM334 pepper roots infected with P. capsici. An
invertase-deficient yeast strain (DBY2445) was transformed
with the pepper cDNA library constructed in pYST 0-2 vector
and plasmid DNA was isolated from all colonies that grew on
yeast-peptone-sucrose (YP-Suc) media. To avoid selection of
overlapping clones, the cDNAs selected through the first screen
were then used to identify redundant clones by Southern blot
analysis using amplified inserts as probes, in an iterative step. In
total, 600 yeast transformants were selected from the YST
screen and we identified 101 unique Capsicum annuum secre-
tome (CaS) genes, which we used for further study (Table 1).
The CaS cDNAs were sequenced and the deduced amino
acid sequences were analyzed with the SignalP 3.0 (Bendtsen
et al. 2004), TargetP (Emanuelsson et al. 2007), and PSORT
programs (Nakai and Kanehisa 1992) to confirm the presence
of secretory signal peptide and to predict subcellular localiza-
tion, respectively. Of these, 92 CaS clones were predicted to
encode proteins with signal peptides or signal anchors that
indicate targeting to the secretory pathway or apoplast by at
least one prediction program. The other nine CaS clones were
predicted to encode proteins with no signal sequence or to lo-
calize to microbodies, the cytosol, or nucleus (Table 1).
Functional classification of CaS genes.
All CaS clones were used to search the GenBank nonredun-
dant public sequence database (Table 1) for similarities to
known proteins using the BLASTX program and were classi-
fied into eight functional groups (Fig. 2).
Members of the largest group (27%) were annotated as hypo-
thetical proteins or proteins of unknown function in that they
showed no or low sequence similarities to any protein with char-
acterized functions. Twenty-six CaS genes (26%) shared high
similarities with defense- or stress-related proteins. Several
members of PR protein and antifungal protein families were in-
cluded in this group, which were known to play important roles
in biotic or abiotic stresses (Kim et al. 2002; Oh et al. 1999).
These included a chitinase (Hong and Hwang 2002), a germin-
like protein (Park et al. 2004a), and a ribonuclease (Park et al.
2004b), which were previously characterized as stress-induced
genes in pepper. Thus, these proteins likely have roles in apo-
plastic defense mechanisms.
Approximately 13% of the CaS genes were classified as
proteases or protease inhibitors, and included members of the
cysteine protease, subtilisin-like protease, and aspartyl prote-
ase families. Protease/protease inhibitors have been suggested
to play a role in defense and the immune response against
biotic stress (Dunaevskii Ia et al. 2005; van der Hoorn 2008);
however, their specific modes of action and substrates in the
interaction with pathogens are not well understood. A further
14% of the clones were predicted to encode cell wall structural
proteins, including proline-rich proteins, glycine-rich proteins,
extensin, cell adhesion protein, and U-rim protein. Another
group of CaS genes (4%) belonged to a general category of de-
velopment and growth-related proteins, which includes organ-
specific growth protein, ripening-related protein, and expansin-
like protein. The remaining CaS clones (12%) were classified
in the metabolism group, which includes proteins that contrib-
ute to the nutrient reservoir hydrolase, glutamine cyclotrans-
ferase, electron transporters, tonoplast intrinsic protein, and
NADH dehydrogenase. These results suggested that the isolated
CaS genes collectively contribute to both pathogen defense
and the normal range of plant developmental process.
Expression of CaS genes in resistant and susceptible plants
following P. capsici infection.
To address the molecular functions of CaS genes, we per-
formed a reverse Northern blot (dot-blot) assay using RNA
Vol. 24, No. 6, 2011 / 673
Fig. 1. Phenotype in resistant (Capsicum annuum CM334) and susceptible pepper (C. annuum Chilsungcho) roots. A, Symptoms observed at 72 h postinocu-
lation (hpi) in Chilsungcho (susceptible, S) after drenching inoculation with zoospores of Phytophthora capsici (2 × 105 zoospores/ml). B, 2,3,5-Triphenyl-
tetrazolium chloride (TTC) reductase activity was also measured in resistant and susceptible pepper roots as cell vitality indicator. TTC reductase activity
was normalized to that of noninfected root. Values are the means standard deviation. C, Biomass of P. capsici in CM334 and Chilsungcho was assessed by
the expression level of P. capsici elongation factor 1 (PcEF1) gene. CoActin was used as control. D, Expression of pathogenesis-related (PR) genes dur-
ing P. capsici infection in resistant and susceptible pepper cultivars. The CaBPR1 and CaPINII genes were used to confirm the activation of defense-related
674 / Molecular Plant-Microbe Interactions
probes from different time points of infection (0, 3, 6, 12, 24,
48, and 72 hpi). Based on this analysis, altered expression pat-
terns were observed for 68 CaS genes relative to uninfected con-
trols. The differential expression patterns of CaS mRNAs in re-
sistant and susceptible cultivars of pepper following P. capsici
infection are shown in Table 1 and Supplementary Table S1.
Of the 68 CaS genes, 35 were induced in both resistant and
susceptible plant interactions. Although these 35 CaS genes
showed similar expression patterns, 19 CaS genes showed ear-
lier and higher levels of expression in the resistant than in the
susceptible cultivar (Table 1). Conversely, 14 CaS genes showed
earlier and higher levels of expression in the susceptible cultivar
Table 1. Yeast secretion trap clones isolated from Phytophthora capsici-infected pepper roots
Classificationb Patternc Protein name Accessiond E valuee HMM NN
Tobacco mosaic virus (TMV)-induced protein I AAF63515
Class IV chitinase
TMV-induced protein 1-2
Pathogenesis-related protein osmotin
Allergen-like protein BRSn20
Putative defensin AMP1 protein
Wound-induced protein CBP1
Pathogenesis-related protein P2
Pleiotropic drug resistance like protein
Pathogenesis-related protein 4b
Putative gamma-thionin precursor
Pathogenesis-related protein PR-1
Cathepsin B-like cysteine
Aspartyl protease family protein
Matrix metalloprotease 1
Putative subtilisin-like proteinase
Putative proteinase inhibitor II
Putative invertase inhibitor
Ethylene-responsive proteinase inhibitor 1
Trypsin proteinase inhibitor precursor
Putative kunitz-type proteinase inhibitor
Glycine-rich protein Tfm5
Cell wall protein 3
Fasciclin-like AGP 12
Putative membrane protein
(continued on next page)
a Signal peptide was predicted by SignalP3.0. (SA) = signal anchor and no predicted signal peptide was indicated scores as bold. HMM and NN = hidden
Markov model and neural network methods, respectively.
b Classification abbreviations: Defense = defense- or resistance-related proteins, Protease = protease or protease inhibitor, Cell wall = cell wall structure
protein, Growth = development or growth-related protein, and Hypothetical = hypothetical protein or unknown. Capsicum annuum secretome (CaS) names = C.
annuum ‘CM334”–Phytophthora capsici interaction secretome.
c Expression pattern of each gene by dot-blot assay. IB_R+ = induced in both pepper cultivars (resistant and susceptible) but earlier and higher expression in
resistant pepper; IB-S+ = induced in both cultivars but earlier and higher expression in susceptible pepper; IB+ = similar expression in both cultivars; R+ =
induced in resistant pepper but no change or reduced in susceptible pepper; – = repressed in both; nc = transcripts >0.5-fold and <2-fold or no changes in
d Pepper expressed sequence tag database: cacn or caKS accession, Gene Pool; SGN accession, Sol Genomic Network.
e E value from protein blast or blastx; (n) = nucleotide blast.
f Prediction of localization by TargetP program. S = secretory pathway, C = chloroplast, – = any other localization, and * = don’t know. In this analysis,
specificity >0.95 (predefined set of cutoffs that yielded this specificity on the TargetP test sets).
g Sublocalization by PSORT program. Outside = cell wall or extracellular space, N = nucleus, Cyto = cytoplasm, PM = plasma membrane, ER =
endoreticulum, Mt = mitochondrion, Mb = microbody (peroxisome), Too short = amino acid too short for prediction.
Vol. 24, No. 6, 2011 / 675
while 2 CaS genes showed similar expression in both cultivars.
On the other hand, 27 CaS genes were upregulated in the resis-
tant cultivar but showed no significant changes in the susceptible
cultivar. Expression levels of six CaS genes were reduced in
both cultivars, compared with the controls (Table 1). The re-
maining 33 CaS genes showed no significant expression level
changes in this comparison.
For more precise monitoring of expression levels of CaS
genes between resistant and susceptible cultivars, we selected
only the differentially expressed CaS genes for quantitative
real-time reverse-transcription polymerase chain reaction
(qRT-PCR) analyses. Based on representative qRT-PCR, gene
expression patterns could be divided into four groups: the first
showed early induction before the onset of the hypersensitive
Table 1. (continued from preceding page)
Classificationb Patternc Protein name Accessiond E valuee HMM NN
Glycine-rich protein 2
Cell wall protein
Glycine-rich protein TomR2
Putative proline-rich protein
Fiber protein Fb34
Putative high-affinity nitrate transporter
NADH dehydrogenase subunit 5
Vacuolar sorting receptor 6
Glutamine cyclotransferase like
Copper ion binding / electron transporter
Receptor like kinase
Receptor protein kinase-like protein
FH protein NFH1
Organ-specific protein P4
Putative auxin-independent growth promoter
Ribosomal protein PETRP
676 / Molecular Plant-Microbe Interactions
response (HR); the second group showed late induction at 12
hpi, and remained at the higher level until 72 hpi; the third
group showed stronger induction in the susceptible than the
resistant cultivar; and the fourth group showed decreased tran-
script levels following P. capsici infection (Fig. 3). Under the
same conditions, the transcripts of CaS102 and CaS113 in-
creased earlier, at 3 hpi, in the resistant cultivar. The CaS290
gene was expressed as early as 3 hpi in both cultivars (Fig.
3A). The time points for appearance of CaS22, CaS148, and
CaS259 transcripts were late stage, from 24 to 72 hpi in both
cultivars (Fig. 3B). Interestingly, some CaS clones (CaS162,
CaS475, and CaS551) were much more upregulated in the sus-
ceptible cultivar, most notably CaS551, whose expression level
was eight times higher (Fig. 3C). Finally, the genes of the fourth
group showed decreased or abolished expression during P.
capsici infection (Fig. 3D). This class, which includes CaS67
(extensin) and CaS203 (putative proline-rich protein [PRP]),
major structural cell wall proteins (Bernhardt and Tierney
2000; Fowler et al. 1999; Niebel et al. 1993), was downregu-
lated but gradually recovered in the resistant cultivar and not in
the susceptible one. Extensin and PRP play a role in the plant
response to pathogen attack (Hematy et al. 2009) and, there-
fore, their downregulation may be involved in effective invasion
of the pathogen (Wei and Shirsat 2006).
The expression patterns of the qRT-PCR analysis largely
agreed with those of the dot-blot assay. Taken together, these
results indicated that the YST technique could be efficiently
used for isolation of pathogen-responsive secreted proteins in
the pepper–P. capsici interaction.
Roles of CaS genes in development and cell death
in pepper plants.
Expression analysis indicated that many CaS genes might
have a role in defense responses (Fig. 3). To test this, a loss-of-
function strategy was taken using the TRV-based virus-induced
gene silencing (VIGS) method for gene knock-down (Chung et
al. 2004; Liu et al. 2002). The VIGS experiments were per-
formed in chili pepper plants with 68 CaS genes.
We observed that silencing of only three CaS genes led to
phenotypic abnormalities at 3 to 4 weeks after VIGS. CaS17-,
CaS221-, and CaS259-silenced pepper plants showed retarded
growth compared with green fluorescent protein (GFP)-
silenced control plants. We also observed delay of cell death
following Pseudomonas syringae pv. tomato T1 inoculation in
8 of 68 CaS gene-silenced cases: CaS75, CaS113, CaS203,
CaS259, CaS270, CaS389, CaS390, and CaS501 (Fig. 4; Sup-
plementary Fig. S3). Among these, CaS259, which showed
both cell death suppression by P. syringae pv. tomato T1 and
phenotypic abnormalities, was selected for validation of the
We measured plant height in CaS259-silenced pepper plants.
The growth of CaS259-silenced pepper showed severe growth
retardation (40% of control) (Fig. 5A). RT-PCR was per-
formed to verify the suppression of CaS259 gene expression in
the silenced plant (Fig. 5A). These results indicate that the
aberrant phenotype correlates with the suppression of tran-
script level of gene CaS259. To validate the effect of CaS259
silencing on pathogen-induced cell death, leaves of the
CaS259-silenced pepper were inoculated with Xanthomonas
axonopodis pv. glycines 8ra (optical density = 0.1), a non-host
pathogen of pepper (Oh et al. 2008; Yi et al. 2009). The cell
death induced by X. axonopodis pv. glycines 8ra was signifi-
cantly delayed in CaS259-silenced pepper plants compared
with that of control plants (Fig. 5C). Ion leakage was also sig-
nificantly reduced compared with that of control plants during
X. axonopodis pv. glycines 8ra inoculation (Fig. 5D).
We also determined the effects of silencing of selected CaS
genes (Fig. 4) in resistant pepper (CM334) following P. capsici
inoculation. Only CaS259-silenced plant significantly showed
reduction in cell death challenged by Phytophthora capsici
Fig. 2. Functional classification of Capsicum annuum secretome (CaS). In all, 101 CaS genes were isolated. Based on the sequence analysis using the BlastX
algorithm with the nr database of the National Center for Biotechnology Information, the genes were classified into eight categories: 27 genes of unknown
function, 26 genes in defense or resistance, 18 genes in development or growth of cell wall structure, 12 genes in metabolism, and 13 genes in protease or
protease inhibitor. The remaining five genes were grouped in signal-related genes and others. Representative clones described in each group are shown in
Vol. 24, No. 6, 2011 / 677
(Supplementary Fig. S4; Fig. 6A). The index of cell death and
P. capsici colonization in CaS259-silenced resistant pepper
were decreased compared with those of control plant (Fig. 6B
and C). The expression levels of CaHin1 and CaCDM1 genes,
cell death markers, were decreased and the CaPR1 gene was
downregulated in the CaS259-silenced resistant pepper follow-
ing pathogen infection (Supplementary Fig. S1). We also per-
formed the VIGS assay of the CaS259 gene in susceptible pep-
per plants following P. capsici inoculation. Disease symptoms
and P. capsici colonization were also decreased in CaS259-si-
lenced susceptible pepper compared with those of control (Fig.
7). These data suggested that one of the pathogen-responsive
secreted proteins identified, CaS259, has an important role in
both normal growth and the modulation of cell death induced
by pathogen in pepper plants.
The goal of this study was to isolate the secreted proteins of
pepper resistant to P. capsici infection for comparison of tran-
script profiles between susceptible and resistant cultivars. We
isolated 101 secreted proteins using the YST system from P.
capsici-infected pepper roots. Of the 101 unique sequences, 92
(92%) contained a predicted secretory signal peptide. The other
Fig. 3. Representative quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of several Capsicum annuum secretome (CaS) clones. Total
RNAs were isolated at different time points following inoculation of Phytophthora capsici into roots of resistant and susceptible pepper plants. qRT-PCRs
were performed on cDNA using gene-specific primers for each CaS clone. Each bar represents the value of relative gene expression at different time points
following inoculation of P. capsici for indicated CaS gene, between resistant pepper (black bar, CM334) and susceptible pepper (white bar, Chilsungcho)
plants. The expression of CaS genes was normalized to the expression of CoActin. Values were calculated for CaS genes following three replications and
standard deviations are shown. Similar results were obtained from at least two independent experiments. One representative experiment is shown.
678 / Molecular Plant-Microbe Interactions
nine CaS genes might be “false positives”, because some trun-
cated proteins could exhibit unnaturally exposed N-terminal
hydrophobic or highly basic regions and, thus, be artificially se-
creted, or they might be secreted through noncanonical path-
ways (Rose and Lee 2010). Nevertheless, the rate of signal
sequence recovery is high compared with previous studies,
showing 76 to 83% using other signal-sequence trap technolo-
gies with tobacco or Arabidopsis (Goo et al. 1999; Hugot et al.
2004). Several CaS genes (CaS1, CaS2, CaS67, CaS203,
CaS388, and CaS507) encode well-known secreted cell wall or
extracellular proteins, which confirmed that our experimental
system worked for the pepper–Phytophthora interaction (Table
1). However, the large proportion of unknown secreted proteins
(27 genes) suggests that many functions of secreted proteins still
remained to be elucidated.
We did not find any pathogen-derived secreted proteins by
computational analysis of these clones after searching numerous
DNA databases. In this regard, our results differed from those of
Lee and associates (2006a), who reported that the same YST
system was valuable for the identification of both host- and
pathogen-derived secreted proteins involved in the interaction
between tomato (Solanum lycopersicum) and the oomycete P.
infestans. This difference might reflect the susceptible or resis-
tant interaction between pathogen and plant host. In our study,
we constructed the YST cDNA library from resistant pepper fol-
lowing P. capsici infection, which may have resulted in unde-
tectable levels of pathogen biomass compared with that of the
susceptible interaction used by Lee and associates (2006a). It is
also possible that the greater abundance of plant cDNA encoding
secreted proteins prevented detection of pathogen-derived
cDNAs, which are relatively rare.
The transcript levels of a number of CaS genes were signifi-
cantly changed in resistant or susceptible cultivars following P.
capsici infection. The differences in CaS transcripts could be
grouped by temporal changes and by transcript levels between
the susceptible and resistant cultivars (Fig. 3). These results
are consistent with the observations of Richins and associates
(2010), who showed global gene expression profiles using mi-
croarrays in resistant and susceptible pepper cultivars infected
by P. capsici. The gene expression profiling allowed us to de-
termine differentiation of expression at the molecular level be-
tween resistance and susceptibility to pathogen.
To select CaS genes related to the defense response to
pathogen, 68 CaS genes affected by pathogen infection were
selected for gene silencing. We observed only three CaS genes
that induced significant morphological change in silenced pep-
per plants, which were assumed to be defense-related proteins
and not cell wall structure-related proteins (Table 1; Figs. 3
and 4). According to in silico analyses, 18 CaS clones were
predicted to be cell wall structural protein or growth- or devel-
opment-related protein. However, when CaS genes were si-
lenced in Nicotiana benthamiana, 31 of 68 CaS genes (approxi-
mately 50%) showed morphological abnormalities, such as
curly leaves, stunted growth, and severe developmental defects
(data not shown). It could be that the efficiency of gene silenc-
ing in pepper is less uniform than in N. benthamiana (Dong et
al. 2007; Liu et al. 2004).
Gene silencing experiments also revealed that eight CaS
genes compromised cell death following Pseudomonas syrin-
gae pv. tomato T1 infection, to less than 20% relative to the
control (Fig. 4). Of these, three (CaS113, CaS270, and
CaS501) are classified in the hypothetical protein/unknown
function group. Two CaS genes (CaS259 and CaS390) belong
to PR gene families and defensin, which are well known to act
on plant defense showing anti-bacterial or antifungal activity,
respectively (Pelegrini and Franco 2005; van Loon et al.
2006). The remaining CaS genes can be classified within the
groups of cell wall structure (CaS203, putative PRP), metabo-
lism (CaS389, hydrolase), and development- or growth-related
proteins (CaS75, putative ROX1) (Cecchetti et al. 2007).
Fig. 4. Hypersensitive response (HR) of representative Capsicum annuum secretome (CaS) gene-silenced pepper following bacterial pathogen (Pseudomo-
nas syringae pv. tomato T1). CaS gene-silenced pepper plants were infiltrated with non-host bacterial pathogen P. syringae pv. tomato T1 (optical density at
600 nm = 0.05). The HR symptoms were taken at 2 days postinoculation (upper panel). Delay of HR by P. syringae pv. tomato T1 was scored by the mean
percentages of sites showing cell death (lower panel). Standard deviations were scored from 15 infiltration site per line, comprising three leaves from five
independent plants. Similar results were obtained from at least two independent experiments. One representative experiment is shown.
Vol. 24, No. 6, 2011 / 679
To validate these findings, the CaS259 gene was chosen and
the gene-silenced pepper was infected with a non-host patho-
gen, X. axonopodis pv. glycines 8ra. The cell-death delay was
determined by scoring for ion leakage (Fig. 5D). In addition,
the cell death symptoms and Phytophthora capsici infection
were reduced in both the resistant and susceptible pepper
plants following silencing of CaS259 gene (Figs. 6 and 7). In
contrast, biomass of P. capsici was increased in CaS259 tran-
sient overexpressed N. benthamiana compared with that of the
control (Supplementary Fig. S6). These data suggested that the
function of CaS259 might be related to susceptibility factor for
P. capsici, or else the pleiotropic effects of CaS259-silencing
in pepper have rendered the plant more resistant, showing cell-
The CaS259 protein showed a high degree of similarity to
PR4 (Supplementary Fig. S2). The PR4 family has been known
to have potent antifungal and antimicrobial activity in vitro
against a wide range of pathogens (Fiocchetti et al. 2008; Li et
al. 2010; Zhu et al. 2006). This family of proteins is also modu-
lated by pathogen infection, as well as by defense-signaling
molecules (Bertini et al. 2003; Park et al. 2001). Expression of
PR4 has also been known to be developmentally controlled in an
organ-specific manner in healthy pepper (Park et al. 2001). Re-
cently, PR4 proteins from various plants have been described as
having RNase or DNase activity (Caporale et al. 2004; Guevara-
Morato et al. 2010; Li et al. 2010), much of which is secreted to
the apoplast, resulting in the breakdown of DNA and RNA and
consequent HR cell death (Mittler and Lam 1997). In addition,
Fig. 5. Effects of Capsicum annuum secretome (CaS)259 silencing in pepper. A, Phenotype of CaS259-silenced pepper plants (upper panel). The picture was
taken at 6 weeks after silencing. Semi-quantitative reverse-transcription polymerase chain reaction analysis for expression analysis of CaS259 gene in
Tobacco rattle virus (TRV)-green fluorescent protein (GFP) and TRV-CaS259 (N-terminal) infiltrated pepper. The level of actin was used as control (lower
panel). B, Plant height of CaS259-silenced plants represented as comparison of plant height. The plant height was measured at 3 weeks after the onset of
virus-induced gene silencing. In total, 15 plants were measured and data are indicated as means standard deviation. Similar results were obtained from
three independent experiments. C, Cell death of CaS259-silenced pepper plants following non-host pathogen (Xanthomonas axonopodis pv. glycines 8ra)
inoculation. Xanthomonas axonopodis pv. glycines 8ra was infiltrated as 1 × 108 CFU/ml (optical density at 600 nm = 0.1). The picture was taken at 2 dpi
under normal and UV light. D, Cell death by X. axonopodis pv. glycines 8ra (as in C) was quantified by measuring the ion leakage of inoculated regions.
Data represent means of six leaf discs (1 cm in diameter) and error bars represent standard deviations. The experiments were repeated three times with simi-
lar results. Asterisks indicate difference of significant level as determined by Student’s t test (P < 0.05).
680 / Molecular Plant-Microbe Interactions
Guevara-Morato and associates (2010) suggested that the nucle-
ase activity of C. chinense PR4 might contribute to the deple-
tion of RNA and DNA fragments during cell death, as in pro-
grammed cell death and necrosis.
However, the roles of plant PR4 proteins in relation to cell
death and development have not yet been elucidated. The nucle-
ase function of PR4 can reasonably explain cell death delay in
CaS259-silenced pepper following inoculation with Pseudomo-
nas syringae pv. tomato T1 and X. axonopodis pv. glycines 8ra
(Figs. 4 and 5C) or with Phytophthora capsici (Figs. 6A and
7A). To our knowledge, this is the first report showing that
CaS259, a CaPR4-like protein, plays an essential role in the
regulation of development and the cell death that can result from
pathogen infection (Figs. 5, 6, and 7). These observations pro-
vide evidence that CaS genes play an important role in pathogen
defense as well as plant development.
Our data strongly suggest that studies using these secreted
proteins might contribute to understanding the fundamental
basis of plant innate immunity that occurs at the interface of
pathogen and host cell. Further investigations into the role of
each gene could provide insights into some of the unknown
functions of the plant secretome in pathogen defense and nor-
mal growth and development.
MATERIALS AND METHODS
C. annuum CM334 (resistant to P. capsici) and C. annuum
Chilsungcho (susceptible to P. capsici) were used for P. capsici
infection experiments. C. annuum ‘Bukang’ was used for
VIGS experiments and followed by bacterial pathogen infec-
tion. All plants were grown in a growth chamber at 25C under
a cycle of 16 and 8 h of light and darkness, respectively, and
transported in a Magenta box (7.2 by 7.2 by 10 cm3; SPL Life
Science, Gyeonggi-do, Korea) for P. capsici infection (Kim et
Pathogen preparation and inoculation.
Preparation of P. capsici inocula was described previously
(Kim et al. 2008). P. capsici Leon, ‘Pa23’, was grown in 3.9%
Fig. 6. Effects of Capsicum annuum secretome (CaS)259 gene silencing following Phytophthora capsici infection in resistant pepper. A, CaS259-silenced
‘CM334’ pepper was infiltrated on leaves by P. capsici as 2 × 105 zoospores/ml. The picture was taken at 5 days postinoculation (dpi). B, Percentages of sites
showing cell death following P. capsici infection of leaves of CaS259-silenced CM334. In total, 10 leaves per line, comprising two leaves from five
independent plants, were scored at 4 dpi. Data points represent the means standard deviation. Similar results were obtained from three independent
experiments. One representative experiment is shown. C, Quantitative reverse-transcription polymerase chain reaction of P. capsici colonization levels in
CaS259-silenced CM334 and control pepper cultivars. Total RNA was extracted from P. capsici-infected regions at 4 dpi. Expression of the P. capsici Ef1
(PcEf1) gene was normalized to the expression of CaActin. Values were calculated with biological three replications as standard deviations.
Vol. 24, No. 6, 2011 / 681
potato dextrose agar medium for 7 days (27C, in the dark)and
mycelial plugs (8 mm in diameter) were cut from the pe-
riphery and cultured on V8 juice agar media (20% V8 juice,
0.4% CaCO3, and 1.8% agar) for 5 days. The mycelia were
scraped and incubated under light for 2 days to promote spo-
rangium formation. The plate was flooded with sterile water
and incubated at 4C for 1 to 2 h; then, plates were placed at
28C for 30 min. The released zoospores were counted by
Fig. 7. Effects of Capsicum annuum secretome (CaS)259 gene silencing following Phytophthora capsici infection in susceptible pepper A, Disease symptom
development on the leaves after P. capsici inoculation (2 × 105 zoospore/ml). The picture was taken at 7 days postinoculation (dpi) under normal (upper
panel) and UV light (lower panel). Red circles indicate the site of P. capsici inoculation. Colored dotted lines (blue or yellow) indicate disease symptoms. B,
P. capsici colonization in CaS259-silenced susceptible pepper was assessed by quantitative reverse-transcription polymerase chain reaction of the P. capsici
Ef1 (PcEf1) gene. Total RNA was extracted from P. capsici-infected leaves at 4 dpi. Expression of PcEf1 gene was normalized to the expression of
CaActin. Values were calculated with biological three replications as standard deviations. C, Leaf area covered with Phytophthora blight lesion (%) in
CaS259-silenced pepper and control (Tobacco rattle virus [TRV]-green fluorescent protein [GFP]-infiltrated pepper) after inoculation with P. capsici. Data
points represent the means standard deviation from disease symptoms of 20 leaves (TRV-GFP) and 32 leaves (TRV-CaS259). Similar results were
obtained from two independent experiments. One representative result is shown.
682 / Molecular Plant-Microbe Interactions
hemacytometer and the concentration adjusted to 2 × 106 zoo-
spores/ml with sterile water.
Five-week-old pepper plants were inoculated with P. capsici
zoospores. For root inoculation, 10 ml of 106 zoospores/ml
(final concentration 2 × 105) was introduced into the Magenta
box. Growth conditions were maintained at 27C with cycles
of 16 and 8 h of light and darkness, respectively. P. capsici-
infected pepper roots were collected at 0, 3, 6, 12, 24, 48, and
72 hpi. Pseudomonas syringae pv. tomato T1 and X. axonopo-
dis pv. glycines 8ra were grown overnight in liquid Luria-Ber-
tani (LB) medium. The bacterial cultures resuspended in 10
mM MgCl2 were introduced into pepper leaves by pressure in-
filtration using a needless syringe (Oh et al. 2008; Yi et al.
TTC reduction assay.
The TTC reduction assay was modified from the methods of
Chen and associates (2006). P. capsici-infected roots from
each time point, including noninfected roots, were washed
with sterile water for 10 min before TTC tests. Fresh roots
(each 300 mg) were incubated with 5 ml of 0.6% TTC (Sigma-
Aldrich, St. Louis) in 50 mM phosphate buffer (pH 7.4) for 22
h at 30C in the dark. Roots were then washed twice with ster-
ile water. Formazan (reduced TTC) was extracted twice from
the roots with 95% EtOH at 80C for 30 min. Combined ex-
tracts were adjusted to a final volume of 15 ml and absorbance
was read at 490 nm (model DU 730; Beckman Coulter, Fuller-
ton, CA, U.S.A.). Assays were performed three times in each
Construction of YST cDNA library.
Isolation of total RNAs from resistant and susceptible pep-
per roots following Phytophthora capsici infection were per-
formed by the method of Choi and associates (1996) and
mRNAs from resistant pepper roots were purified by the
oligotex mRNA minikit (Qiagen, Chatsworth, CA, U.S.A.).
The pYST vector system used for library construction was as
previously described (Lee et al. 2006b). The HybriZAP cDNA
synthesis kit (Stratagene, La Jolla, CA, U.S.A.) was used for
random-primed cDNA synthesis and a random primer (5-GA
including a NotI restriction enzyme site (underlined), was used
for first-strand synthesis. After second-strand synthesis, liga-
tion with an EcoRI adaptor, 5-end phosphorylation with T4
polynucleotide kinase, digestion with NotI, and cDNA size-
fractionation was performed using the approximately 300- to
1,000-bp gel elution fraction. The cDNAs were ligated to an
equimolecular mixture of the EcoRI- and NotI-digested pYST
0, 1, and 2 vectors. TOP10 electrocompetent cells (Invitrogen,
Carlsbad, CA, U.S.A.) were used for transformation of the
ligation mixture. After transformation, the cells were plated on
LB agar plates including ampicillin at 50 mg/ml. Plasmid
DNA was isolated from a pooled sample of the transformants
(YST library) using the Perfect prep plasmid midi kit (Eppen-
dorf, Hamburg, Germany).
Yeast transformation, selection, and sequencing.
YST library plasmids were transformed into an invertase-de-
ficient yeast mutant strain, DBY2445 (Saccharomyces cere-
visiae, MAT, suc2-9, lys2-801, ura3-52, ade2-101) by the
YEASTMAKER Yeast Transformation System2 (BD Biosci-
ence, San Jose, CA, U.S.A.). Transformants were selected on
YP-Suc medium (1% Bacto yeast extract, 2% Bacto peptone,
and 2% agar), and incubated at 30C for 8 to 9 days. Colonies
were restreaked on an YP-Suc medium. After incubation at
30C for 2 days, plasmids were isolated from the yeast colo-
nies. The cDNAs selected from the first screen were used for
identifying redundant clones using Southern blot assay, in an
iterative step. The plasmids were then transformed into DH10b
Escherichia coli electrocompetent cells, and the isolated plas-
mids were sequenced using the ADH1 primer (5-TCCTCGTC
ATTGTTCTCGTTCC-3) (Lee et al. 2006b). All rescued plas-
mids from E. coli were retransformed into yeast to reconfirm
the ability to grow on sucrose selection medium at least twice.
YST clones were sequenced (NICEM, Korea) and sequence
similarity was determined using the National Center for Bio-
technology Information and Phytophthora Functional Genom-
ics Database. To identify the gene annotation, the sequences of
the CaS clones were compared with pepper expressed sequence
tag databases (Gene Pool and Sol Genomic Network). DNA
sequences were translated into amino acid sequence using the
ExPasy translation tool (Appel et al. 1994). Signal peptides
were predicted by the SignalP3.0 program (Bendtsen et al.
2004) and subcellular localization was predicted using the Tar-
getP (Emanuelsson et al. 2000) and PSORT programs (Nakai
and Horton 1999).
Northern blot analysis.
Total RNA (10 µg) was electrophoresed on 1.2% formalde-
hyde agarose gels and blotted onto Hybond-N
(Amersham Biosciences, Piscataway, NJ, U.S.A.). The mem-
branes were hybridized with [-32P]-labeled partial cDNA
fragments of YST clones at 65C overnight. After hybridiza-
tion, the membranes were washed with 2× SSC (1× SSC is
0.15 M NaCl plus 0.015 M sodium citrate) (pH 7.2) and 0.1%
sodium dodecyl sulfate (SDS) at 65C for 15 min, with 1×
SSC and 0.1% SDS at 65C for 15 min, and with 0.5× SSC
and 0.1% SDS at 65C for 15 min. Membranes were exposed
to X-ray film at –80C for 3 to 24 h.
Approximately 2 µg of pYST plasmid DNA containing
cDNA-encoding signal peptide (containing approximately 100
ng of each cDNA) was used for reverse Northern blotting.
Dot-blot hybridization was performed using the Bio-Dot SF
microfiltration apparatus (Bio-Rad, Hercules, CA, U.S.A.) fol-
lowing the manufacturer’s protocol, with slight modification.
Fourteen RNA probes were generated: time course RNA (0, 3,
6, 12, 24, 48, and 72 h) from resistant and susceptible pepper
roots after P. capsici infection. Total RNA (5 µg each sample)
was used for template cDNA synthesis using the SUPER-
SCRIPT II RNase–Reverse-Transcriptase system (Invitrogen)
in a mixture with anchor primer (oligo-dT), dNTP mixture mi-
nus dCTP, and [-32P]-dCTP (3,000 Ci/mmol) (Amersham
Biosciences). The labeled probes were separated from unincor-
porated nucleotides using mini-Quick Spin DNA columns
(Roche Applied Science, Mannheim, Germany). Membranes
were prehybridized at 65C for 3 h in hybridization buffer (0.5
M sodium phosphate [pH 7.2], 7% SDS, 1 mM EDTA). La-
beled cDNA probe was mixed with fresh buffer and hybridized
at 65C for at least 16 h. After hybridization, blots were
washed twice in 2× SSC and 0.1% SDS at 65C for 5 min,
once in 1× SSC and 0.1% SDS at 65C for 15 min, and twice
0.1× SSC and 0.1% SDS at 65C for 10 min. The washed
membranes were exposed to a BAS imaging plate (Fujifilm,
Tokyo) and quantified by scanning the plate with a Fujix
For qRT-PCR, total RNA (5 µg) was reverse-transcribed using
the SUPERSCRIPT II RNase–Reverse-Transcriptase system
(Invitrogen). Triplicate samples were analyzed using a Rotor-
Gene 6000 apparatus (Qiagen) with SYBR Green (Invitrogen),
according to the manufacturer’s instructions. The relative quan-
Vol. 24, No. 6, 2011 / 683
titation of gene expression was calculated by the relative stan-
dard curve method (Larionov et al. 2005). The CaActin gene
was used to normalize expression levels, and noninoculated
pepper plants were used as controls for expression of the target
genes (Supplementary Table S2). Expression levels were re-
ported as mean values with standard errors.
Construction of the TRV-CaS vector and VIGS in pepper.
CaS genes in the pYST vector were digested with EcoRI
and KpnI and cloned into a TRV-based gene silencing vector
(pTRV2) via the same enzyme sites. Average insert size was
approximately 370 nucleotides. The pTRV2 vectors containing
CaS genes were transformed into Agrobacterium sp. strain
GV2260 by the freeze-thaw method (An 1987) and the TRV-
based VIGS on pepper was performed as described by Chung
and associates (2004).
Measurement of ion leakage.
The measurement of ion leakage was performed as described
by Lee and associates (2010). Two days after inoculation with
bacterial pathogen, three leaf discs (1 cm in diameter) were
floated on 5 ml of distilled water for 2 h at room temperature.
Electrical conductivity was measured using a conductivity me-
ter (model 455C; Istek, Seoul). To release whole electrolytes
from leaf discs, samples were autoclaved, cooled to room tem-
perature, and measured with a conductivity meter. Ion leakage
was expressed as percent leakage to conductivity of control
This work was supported by a grant from the Crop Functional Genomic
Center (CG1132) and the National Research Foundation (project number
2010-0015105) of MEST of Korean Government. S.-I. Yeom is a scholar-
ship grantee (S2_2009_000_0210_1) from the Korea Student Aid Founda-
tion (KOSAF). J. Rose was supported by a grant from the National Sci-
ence Foundation (NSF) Plant Genome program (DBI-0606595). B.-D.
Kim and D. Choi contributed equally as corresponding authors.
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AUTHOR-RECOMMENDED INTERNET RESOURCES
Center for Biological Sequence Analysis TargetP prediction server:
Gene Pool server: genepool.kribb.re.kr
National Center for Biotechnology Information: www.ncbi.nlm.nih.gov
Oomycete Genomic databse: www.oomycete.org/ogd
PSORT WWW server: psort.nibb.ac.jp
SignalP3.0 server: www.cbs.dtu.dk/services/SignalP
Sol Genomics Network website: sgn.cornell.edu