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Mol. Cells, Vol. 17, No. 2, pp. 377-380

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A Method of High Frequency Virus-induced Gene SiIencing in
ChiIi Pepper (Capsicum annuum L. cv. Bukang)

Eunsook Chung, Eunsoo Seong, Yeoung-Cheol Kim, Eun 1oo Chung, Sang-Keun Oh, Sanghyeob Lee,
1eong Mee Park, Young Hee 1oung, and Doil Choi*
Plant Genomics Laboratory, Genome Research Center, Korea Research Institute of Bioscience and Biotechnology, Daefeon 305-600,
Korea.

(Received January 26, 2004, Accepted February 11, 2004)

Using a tobacco rattle virus (TRV)-based virus-induced
gene silencing (VIGS) system, expression of phytogene
desaturase (3'6) and ribulose-1,5-bisphosphate car-
boxylase small-subuit (UEF6) genes was suppressed in
1LFRWLDQD EHQWKDPLDQD and pepper plants (&DSVLFXP
DQQXXP L. cv. Bukang). The silenced phenotypes of
pale yellow (UEF6), and photobleached leaves (3'6),
were invariably obvious 2 weeks after inoculation with
the TRV-based vector. In a parallel experiment, the
same set of genes was silenced in 1 EHQWKDPLDQD and
yielded identical phenotypes to pepper 1 week after in-
oculation. Northern blot analyses showed that the en-
dogenous levels of &DUEF6 and &D3'6 transcripts were
dramatically reduced in the silenced leaf tissues. These
observations confirm that the silenced phenotype is
closely correlated with the pattern of tissue expression.
To our knowledge, this is the first high frequency VIGS
method in pepper plants. It should provide a tool for
large-scale gene silencing studies in pepper functional
genomics.

Keywords: C. annuum; N. benthamiana; Pepper; Sup-
pression; TRV; VIGS.


Introduction

Virus-induced gene silencing (VIGS) has been widely
used Ior Iunctional gene characterization in Nicotiana
benthamiana (Lu et al., 2002; RatcliII et al., 2001),
Arabidopsis (Dalmay et al., 2000), tomato (Eckengren et
al., 2003; Liu et al., 2002b) and barley (Holzberg et al.,
2002). In this procedure, a recombinant virus carrying a

* To whom correspondence should be addressed.
Tel: 82-42-860-4340; Eax: 82-42-860-4309
E-mail: doilkribb.re.kr
partial sequence oI the target gene is used to inIect plants.
When the virus spreads systemically, endogenous gene
transcripts are degraded by post-transcriptional gene si-
lencing (PTGS) (Baulcombe, 1999; Hamilton and Baul-
combe, 1999). PTGS occurs via a sequence-speciIic RNA
degradation mechanism caused by double stranded RNA
(dsRNA) (Chuang and Meyerowitz, 2000; Waterhouse et
al., 1998). As in the case oI RNA interIerence in animals,
it is thought that this mechanism involves the processing
oI the double-stranded (ds) RNA to short interIering (si)
RNAs (Zamore, 2001).
Chili pepper (Capsicum annuum L.) is an important
vegetable used as a spice and a source oI red pigment. Ge-
netic transIormation is extremely diIIicult in pepper plants;
either transIormation is not reproducible or the eIIiciency is
very low, even though a number oI pepper transIormation
protocols have been reported (Kim et al., 2001; Li et al.,
2003; Manoharan et al., 1998; Mihalka et al., 2000). In
spite oI these successes, it has been diIIicult to study the
Iunction oI pepper genes because oI the lack oI procedures
Ior stable transIormation. VIGS has been used successIully
to identiIy gene Iunction in plants (Burton et al., 2000;
Kumagai et al., 1995; Liu et al., 2002a; Saitoh et al., 2002).
A large number oI pepper expressed sequence tags (EST)
are available (http://plant.pdrc.re.kr/ks200201/pepper.html)
and an eIIicient reverse genetic tool would permit the iden-
tiIication oI the Iunctions oI many genes. Here we present a
highly eIIicient tobacco rattle virus (TRV)-based VIGS
system as a good alternative to genetic transIormation. This
technique should provide a powerIul means oI identiIying
gene Iunctions that has so Iar been hindered by the low
Irequency oI genetic transIormation.


MateriaIs and Methods

Plasmid construction and $JUR-infiltration A 195-bp EcoRI
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378 Virus-induced Gene Silencing in Pepper
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DNA Iragment corresponding to the 5? region oI C. annuum
phytoene desaturase (CaPDS) cDNA (GenBank accession No.
S29314) was cloned in reverse orientation into the pTRV2 vec-
tor, yielding pTRV2:CaPDS. A 451-bp EcoRI/KpnI DNA Irag-
ment containing part oI the 3? region oI C. annuum ribulose-1,5-
bisphosphate carboxylase small-subunit (CarbcS) cDNA (Gen-
Bank accession No. AE065115) was cloned into the pTRV2
vector in a sense orientation to produce pTRV2:CarbcS.
N. benthamiana and chili pepper (C. annuum L. cv. Bukang)
seeds were germinated and grown in a pot oI soil in a growth
chamber at 25GC with a 16 h light and 8 h dark photoperiod
cycle. Eor Agro-inIiltration, pTRV1, pTRV2 and the recombi-
nant plasmids (pTRV2:CaPDS and pTRV2:CarbcS) were trans-
Iormed into Agrobacterium tumefaciens GV2260 via the Ireeze-
thaw method (An et al., 1988). A 5 ml culture oI each strain was
grown overnight at 30GC in YEP (50 mg/ml oI kanamycin and
50 mg/ml oI riIampicin). The overnight culture was inoculated
into 50 ml oI YEP medium and grown at 30GC shaker overnight,
and the Agro-inIiltration conditions Ior the optimal pepper tran-
sient method (Seong et al., unpublished data) were employed as
described below. The cells were sedimented by centriIugation
(3000 rpm, 15 min, 20GC), resuspended in inIiltration medium
(20 mM citric acid, 2 sucrose, pH 5.2) and adjusted to O.D.
0.50.8. They were then exposed to 200 2m acetosyringone at
2225GC with shaking Ior 35 h. The induced Agrobacterium
mixtures oI pTRV1 and pTRV2, pTRV2:CaPDS or pTRV2:
CarbcS (1:1 ratio) were inIiltrated with a needle-less 1 ml sy-
ringe into cotyledons oI germinating pepper plants and the
5th6th leaves oI 3-week-old N. benthamiana. The Agro-
inIiltrated pepper plants were transIerred to 16GC Ior 1 day and
grown in a growth chamber at 25GC with a 16 h light and 8 h
dark photoperiod cycle.

Extraction of total RNA and RNA blot analysis Total RNAs
were extracted Irom pepper leaves by the LiCl-phenol extraction
method oI Prescott and Martin (1987). The RNA was Iraction-
ated by size on denaturing Iormaldehyde 1.0 (w/v) agarose
gels according to Sambrook et al. (1989), and hybridization and
washes were carried out as described in Church and Gilbert
(1984). To detect CaPDS RNA, we used a 1.6-kbp EcoRI/KpnI
DNA Iragment corresponding to the 3? region oI CaPDS cDNA
(Eig. 1A) and as the CarbcS probe, a 400-bp EcoRI DNA Irag-
ments corresponding to the 5? region oI the CarbcS cDNA (Eig.
1). Probe labeling, blot hybridization and washing conditions
were as previously described (Chung et al., 2003).

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ResuIts and Discussion

Development of a TRV based VIGS system A large
number oI chili pepper EST cDNAs are available
(http://plant.pdrc.re.kr/ks200201/pepper.html), and there
have been several attempts to use VIGS Ior studying the
Iunctions oI pepper genes since this method became
widely used (Burton et al., 2000; Liu et al., 2002a; Saitoh















Fig. 1. TRV-based VIGS vectors used in pepper and N. bentha-
miana plants. The TRV-based VIGS vectors are described in Liu
et al. (2002a). TRV cDNA clones were placed between dupli-
cated CaMV 35 S promoters (2 K 35 S) and the nopaline syn-
thase terminator (N) in a T-DNA vector. RdRp, RNA-dependent
RNA polymerase; 16 K, 16 kDa cysteine-rich protein; MP,
movement protein; CP, coat protein; LB and RB, leIt and right
borders oI T-DNA; R, selI-cleaving ribozyme; MCS, multiple
cloning sites. pTRV2:CarbcS (sense orientation) and pTRV2:
CaPDS (antisense orientation) were constructed in order to as-
sess the ability oI TRV vectors to suppress rbcS or PDS in pep-
per and N. benthamiana.


et al., 2002). At the outset, potato virux X (PVX)-
(Hamilton and Baulcombe, 1999) and TRV-based VIGS
(RatcliII et al., 2001) were attempted but we have Iound
that PVX- and TRV-mediated VIGS do not occur at all in
pepper plants.
In order to develop an eIIicient VIGS system, we tested
a modiIied TRV vector (Liu et al., 2002a). TRV is a
bipartite positive sense RNA virus consisting oI RNA1
and RNA2 (MacEarlane, 1999). Eor VIGS, both a pTRV1
vector containing the RNA1 gene driven by a double 35S
cauliIlower mosaic virus (CaMV) promoter in a T-DNA
construct, and a construction vector, pTRV2, are required
(Eig. 1). We constructed pTRV2:CarbcS (sense orienta-
tion) and pTRV2:CaPDS (antisense orientation) (Eig. 1)
to assess the ability oI the TRV vector (a giIt oI Dr.
Dinesh-Kumar oI Yale University) to suppress the expres-
sion oI the endogenous PDS and rbcS genes in pepper and
N. benthamiana plants.

Silencing of 3'6 and UEF6 using a TRV based VIGS
system The newly emerging cotyledons oI germinating
pepper plants were inIiltrated with an Agrobacterium cul-
ture containing pTRV2:CaPDS and pTRV2:CarbcS to-
gether with pTRV1. Silencing was monitored in the upper
leaves oI the plants, and the silencing phenotype appeared
2 weeks aIter Agro-inIiltration (Eig. 2A). As a control, the
Eunsook Chung et al. 379
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A




B







C






Fig. 2. Silencing oI PDS and rbcS in pepper (A) and N. bentha-
miana (B), and Northern blot analysis oI PDS and rbcS expres-
sion in TRV2-inIected and PDS- or rbcS-silenced pepper plants
(C). Non-inoculated, and recombinant TRV-, TRV-CaPDS- or
CarbcS- inIected pepper and N. benthamiana plants are shown
at 21 and 35 days post inoculation (dpi). The photograph was
taken Irom the top. Notice the chlorosis phenocopy as evidence
oI silencing oI rbcS, and the photobleaching phenotype resulting
Irom inhibition oI carotenoid biosynthesis in the PDS-silenced
plants. (C) Eive 2g (rbcS) or IiIteen 2g (PDS) oI total RNA was
analyzed on an RNA gel blot. The blot was hybridized with
32
P-
labeled CarbcS or CaPDS cDNA Iragments. The ethidium bro-
mide-stained gel is shown as an equal loading control.


same Agrobacterium culture was inIiltrated onto lower
leaves oI 3-week-old N. benthamiana plants. The rbcS
and PDS suppression phenotypes were visible 7 days aIter
Agro-inIiltration in 100 oI the N. benthamiana plants
(Eig. 2B). The DNA sequence identity oI rbcS between N.
sylvestris (X01722) and pepper is 87 and that oI PDS
between N. benthamiana (AJ571700) and pepper is 93,
at the nucleotide level. This demonstrates that Agro-
inIiltration oI TRV2:CaPDS and TRV2:CarbcS achieves
perIect gene silencing in N. benthamiana even though the
pepper gene and Nicotiana gene are not 100 identical.
The same silencing phenotype was observed in pepper
and N. benthamiana plants (Eigs. 2A and 2B). RbcS-
silenced leaves displayed chlorotic symptoms such as pale
greening, as previously studied (Jones et al., 1999;
RatcliII et al., 2001), and plants inIected with pTRV2:
CaPDS had symptoms oI photobleaching; this must be-
caused by the absence oI the photoprotective carotenoid
pigments that require phytoene desaturase Ior their syn-
thesis (Kumagai et al., 1995; Ruiz et al., 1998).
Table 1. EIIect oI Agrobacterium cell density on VIGS Ire-
quency in pepper plants.

rbcS-silencing Irequency
chlorotic plants / total
plants
PDS-silencing Irequency
photobleaching / total
plants
0.5 O.D.
0.8 O.D.
12 / 12
14 / 14
12 / 12
13 / 14
* Agrobacterium cell density was adjusted to O.D. 0.5 or 0.8 in
inIiltration medium (20 mM citric acid, 2 sucrose, pH 5.2) be-
Iore VIGS. The number oI plants with the silenced phenotype
was determined 21 dpi.


To see iI Agrobacterium cell density aIIects the eIIi-
ciency oI VIGS in pepper, we compared the eIIect oI
Agrobacterium cells adjusted to optical density (O.D.) 0.5
and 0.8 (Table 1). As shown in Table 1 silencing was
highly eIIicient at both densities. As previously described
by Ruiz et al. (1998), either the antisense orientation or
sense orientation oI genes in the TRV2 vector gave the
same silencing eIIiciency.
The modiIied TRV-based VIGS (Liu et al., 2002a)
caused more rapid and uniIorm suppression in N. bentha-
miana than the original TRV system (RatcliII et al., 2001).
TRV also induces VIGS in tomato plants (Liu et al.,
2002b). Here we have demonstrated that TRV induces
high-Irequency VIGS in pepper plants, so the Iactors re-
sponsible Ior the eIIiciency oI VIGS need to be consid-
ered. The low temperature treatment aIter Agro-inIiltration
may well be one oI the reasons Ior the high eIIiciency.
Even though silencing eIIiciency was 50 to 90 in tomato
plants (Liu et al., 2002), the eIIiciency oI VIGS was greater
at lower temperature (between 1620GC) (Ekengren et al.,
2003). Another Iactor may be the Agro-inIiltration medium
(20 mM citric acid, 2 sucrose, pH 5.2). This has been
recommended Ior eIIicient genetic transIormation oI tree
plants, which are usually diIIicult to transIorm (Seong et
al., 2003). Plant stage is also thought to be another impor-
tant Iactor; germination-stage pepper plants being advan-
tageous Ior eIIicient VIGS. Transient expression experi-
ments in pepper plants have indicated that GUS integra-
tion eIIiciency was better in young plants than older
plants (Seong et al., unpublished data). The contribution
oI these and other Iactors to the eIIiciency oI VIGS in
pepper plants is currently under investigation.
To conIirm that the silencing phenotype was the result
oI reduced PDS and rbcS mRNA levels, we perIormed
Northern blot analyses (Eig. 2C). As expected, the PDS
transcript level was dramatically reduced in the photo-
bleached PDS-silenced samples. Similarly, rbcS-silenced
plants showing chlorosis accumulated much Iewer rbcS
mRNA transcripts.
In summary, we present a rapid and eIIicient TRV-based
VIGS system in pepper plants. Silencing oI PDS and rbcS
380 Virus-induced Gene Silencing in Pepper
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expression in pepper plants was close to 100 eIIicient 2
weeks aIter Agro-inoculation and lasted through their liIe
cycle. At the mRNA level, a clearcut reduction in PDS and
rbcS expression was observed in the silenced plants. This
method will be useIul Ior Iunctional analysis oI genes oI
interest and Ior advances in the Iunctional genomics oI
pepper.


Acknowledgments We thank Dr. S. P. Dinesh-Kumar (Yale
University) Ior the pTRV1 and pTRV2 vectors. This work was
supported by grants Irom the Crop Eunctional Genomics Center
oI the 21st Century Erontier Research Program oI the Ministry
oI Science and Technology.

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