Hibiscus chlorotic ringspot virus coat protein
inhibits trans-acting small interfering RNA
biogenesis in Arabidopsis
Chunying Meng,1Jun Chen,2Shou-wei Ding,3Jinrong Peng2
and Sek-Man Wong1,4
1Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, 117545
2Functional Genomics Laboratory, Institute of Molecular and Cell Biology, 138673 Singapore
3Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521,
4Adjunct Investigator, Temasek Life Sciences Laboratory, 1 Research Link, 117604 Singapore
Received 17 March 2008
Accepted 27 May 2008
Many plant and animal viruses have evolved suppressor proteins to block host RNA silencing at
various stages of the RNA silencing pathways. Hibiscus chlorotic ringspot virus (HCRSV) coat
protein (CP) is capable of suppressing the transiently expressed sense-RNA-induced post-
transcriptional gene silencing (PTGS) in Nicotiana benthamiana. Here, constitutively expressed
HCRSV CP from transgenic Arabidopsis was found to be able to rescue expression of the
silenced GUS transgene. The HCRSV CP-transgenic Arabidopsis (line CP6) displayed several
developmental abnormalities: elongated, downwardly curled leaves and a lack of coordination
between stamen and carpel, resulting in reduced seed set. These abnormalities are similar to
those observed in mutations of the genes of Arabidopsis RNA-dependent polymerase 6 (rdr6),
suppressor of gene silencing 3 (sgs3), ZIPPY (zip) and dicer-like 4 (dcl4). The accumulation of
microRNA (miRNA) miR173 remained stable; however, the downstream trans-acting small
interfering RNA (ta-siRNA) siR255 was greatly reduced. Real-time PCR analysis showed that
expression of the ta-siRNA-targeted At4g29770, At5g18040, PPR and ARF3 genes increased
significantly, especially in the inflorescences. Genetic crossing of CP6 with an amplicon-silenced
line (containing a potato virus X–green fluorescent protein transgene under the control of the 35S
cauliflower mosaic virus promoter) suggested that HCRSV CP probably interfered with gene
silencing at a step after RDR6. The reduced accumulation of ta-siRNA might result from the
interference of HCRSV CP with Dicer-like protein(s), responsible for the generation of dsRNA in
Small-RNA-mediated silencing pathways play important
roles in a variety of processes, including defence against
viruses. Viruses in turn evolve mechanisms to interfere
with the host silencing responses for successful infection,
which usually results in differential developmental defects.
The molecular basis of gene-silencing-related abnormal
development is mediated by a group of small RNAs,
including microRNAs (miRNAs) and small interfering
RNAs (siRNAs) (Bartel, 2004; Baulcombe, 2004; Jones-
Rhoades et al., 2006). The miRNAs are a class of small
RNAs similar to siRNAs that are key components of the
complex networks of gene regulatory pathways. As most
plant miRNAs contain near-perfect complementarities
with target sequences, they are thought to function like
siRNAs in guiding target RNA for cleavage (Rhoades et al.,
2002). In Arabidopsis thaliana, there are four Dicer-like
proteins (DCLs) that are responsible for the production of
miRNAs and siRNAs of different sizes. All four DCLs act in
combination to help mediate plant responses to diverse
viral infections (Blevins et al., 2006; Deleris et al., 2006).
The plant miRNAs target a series of genes/gene families
that are important for normal plant development (Kidner
& Martienssen, 2005). Interference in miRNA biogenesis or
miRNA–target interactions lead to developmental abnor-
malities (Bartel & Bartel, 2003; Dugas & Bartel, 2004). One
group of miRNA targets comprises precursors of a subset
A list of the genes tested and the primers used in this study is available
with the online version of this paper.
Journal of General Virology (2008), 89, 2349–2358
2008/002170G2008 SGMPrinted in Great Britain2349
of endogenous small RNAs referred to as trans-acting
siRNAs (ta-siRNAs), which are able to direct the cleavage of
non-identical transcripts. The biogenesis of ta-siRNAs
requires components of both the miRNA and siRNA
pathways in two consecutive phases: after the AGO1–
DCL1–HEN1–HYL1-dependent miRNA-guided cleavage of
the ta-siRNA precursor RNA (TAS), the 59 or 39 cleavage
products are converted into dsRNA in an RDR6–SGS3-
dependent manner;thedsRNAsarethen cleaved byDCL4to
generate ta-siRNAs (Allen et al., 2005; Xie et al., 2005). The
biogenesis pathway of ta-siRNA establishes a link between
the miRNA and siRNA pathways (Peragine et al., 2004;
Vazquez et al., 2004; Allen et al., 2005). ta-siRNAs are
functionally similar to miRNAs in that they regulate the
expression of target genes to which they have limited
sequence similarity, thus maintaining normal plant devel-
opment (Yoshikawa et al., 2005). Six loci have been reported
to generate ta-siRNAs in Arabidopsis: TAS1a, TAS1b, TAS1c
and TAS2 transcripts are targeted by miR173, locus TAS3 is
targeted by miR390 (Allen et al., 2005; Xie et al., 2005) and
locus TAS4 was identified recently by scanning the
Arabidopsis genome for phased clusters of 21 nt and the
TAS4 transcript is targeted by miR828 for its phased
maturation (Rajagopalan et al., 2006).
Several virus-encoded gene-silencing suppressors have been
reported to cause developmental defects when expressed
constitutively in Arabidopsis as transgenes (Chapman et al.,
2004; Chen et al., 2004; Dunoyer et al., 2004). Transgenic
Arabidopsis containing tobacco etch virus HC-Pro, turnip
yellow mosaic virus (TYMV) p69, beet yellows virus p21
and tomato bushy stunt virus p19 proteins displayed
differential leaf and rosette development, and obvious
flower phenotypes and infertility were also observed
(Chapman et al., 2004; Chen et al., 2004). Some of these
proteins, such as HC-Pro and p69, affect the level of key
gene-silencing components such as DCL1 in the transgenic
plants, which leads to the extensive deviation of a number
of miRNAs (Mallory et al., 2002; Xie et al., 2003; Chen et al.,
2004; Dunoyer et al., 2004). The molecular mechanisms of
silencing suppression have been reported for some
silencing suppressors. p19, p21 and HC-Pro may function
similarly by forming head-to-tail homodimers that seques-
ter siRNA duplexes and prevent them from entering the
RNA-induced silencing complex (RISC) (Vargason et al.,
2003; Ye et al., 2003; Ye & Patel 2005; Lakatos et al., 2006).
Cucumber mosaic virus 2b protein is reported to interact
directly with AGO1 (the core component of RISC) and
block AGO1 cleavage activity to inhibit miRNA pathways
and attenuate RNA silencing (Zhang et al., 2006). Tomato
yellow leaf curl virus V2 protein suppresses gene silencing
through its interaction with the host SGS3 protein (Glick et
Hibiscus chlorotic ringspot virus (HCRSV) is a positive-
sense, single-stranded RNA virus in the genus Carmovirus
(Huang et al., 2000). The coat protein (CP) of HCRSV has
been identified as a strong gene-silencing suppressor,
which suppresses transiently expressed sense-transgene-
induced PTGS, and a complete CP is required for the
effective suppression function (Meng et al., 2006). The
suppression function of CP is also correlated with the host-
induced avirulence of HCRSV during virus evolution
(Meng et al., 2006). Turnip crinkle virus (TCV) is also a
member of the genus Carmovirus. The CPs of TCV and
HCRSV share approximately 30% amino acid sequence
identity. TCV CP induces few or no developmental defects
in Arabidopsis and does not interfere with the accumula-
tion of miRNAs (Chapman et al., 2004; Dunoyer et al.,
2004). TCV CP is reported to interfere with the ta-siRNA
pathway and suppress DCL4 function (Qu et al., 2003;
Deleris et al., 2006; Meng et al., 2006). TCV silencing is
mediated by DCL2 and DCL4, with DCL2 providing
redundant siRNA-processing functions when DCL4 is
suppressed by TCV CP (Deleris et al., 2006). In this study,
we investigated whether HCRSV CP acts on the ta-siRNA
pathway and tried to determine its suppression mech-
anism. We showed that HCRSV CP suppresses gene
silencingwhen it isconstitutively
Arabidopsis. The CP also affected normal plant devel-
opment by interfering with the accumulation of siRNAs,
miRNAs and ta-siRNAs.
Plant materials. Line L1 carried a silenced b-glucuronidase (GUS)
transgene in an A. thaliana ecotype Col-0 background (Elmayan &
Vaucheret, 1998; Mourrain et al., 2000). Lines A and G carried a
silenced potato virus X (PVX)–green fluorescent protein (GFP)
transgene under the control of the 35S cauliflower mosaic virus
(CaMV) promoter (35S–PVX::GFP) and a 35S–GFP transgene in the
background of ecotype C24, respectively. The transformants were
selected in vitro on medium supplemented with 10 mg L-phosphino-
tricin (Melford) l21or 50 mg kanamycin l21, respectively (Dalmay
et al., 2000a). Line G6A was a cross between lines G and A. The
transgenic plants were selected using 50 mg kanamycin l21(Dalmay
et al., 2000a). The L1 line was provided by Dr Herve ´ Vaucheret
(Laboratoire de Biologie Cellulaire, INRA – Centre de Versailles,
France) and lines G and G6A by Professor David Baulcombe
(Department of Plant Sciences, University of Cambridge, UK). The
mutant lines rdr6-11, sgs3-11 and zip-1 were obtained from the
Arabidopsis Biological Resource Center (Ohio State University, OH,
USA). Mutant line dcl4-2 was obtained from Dr James Carrington
(Oregon State University, USA).
Generation of CP- and DCP-transgenic Arabidopsis. The CP
coding sequence of HCRSV (Huang et al., 2000) was PCR amplified
with a 59 leader (59-AAGGAGATATAACA-39) and cloned into
pBI121 by replacing the GUS sequence between the 35S promoter and
the nopaline synthase terminator to yield pBICP (Fig. 1a). The third
amino acid codon (CAG) of CP was mutated to TAG and the start
codon of the overlapping p25 was changed to GTG in pBICP to create
pBIDCP (Fig. 1a). Transgenic Arabidopsis plants were produced by
the flower dip method (Clough & Bent, 1998) using pBICP and
pBIDCP constructs in the wild-type (wt) Col-0 background. After the
seeds had been collected, putative transgenic plants were screened on
MS plates supplemented with 50 mg kanamycin l21.
Genetic crossing of CP-transgenic plants. Crossings between CP
or DCP and L1, A or G6A lines were carried out as described
previously (Chen et al., 2004). After crossing, the introduced
C. Meng and others
2350Journal of General Virology 89
sequences and the effects of CP and DCP on the accumulation of
transgene and amplicon-specific mRNA and siRNAs were detected by
Isolation of total RNA and detection of high- and low-
molecular-mass RNAs (siRNAs, miRNAs and ta-siRNAs).
Isolation of RNAs was carried out as described previously (Li et al.,
2002). To detect the high-molecular-mass RNAs, 5 mg total RNA was
run on a formaldehyde denaturing gel and transferred to a Hybond-N
membrane, followed by detection with a digoxigenin (DIG)-labelled
RNA probe. To detect siRNAs and miRNAs, 50 mg total RNA was run
on a 15% polyacrylamide sequencing gel and detected by32P-end-
labelled DNA oligonucleotides. The probe used for the detection of
miR173 was 59-GTGATTTCTCTCTGCAAGCGAA-39 and for the
detection of ta-siR255 was 59-TACGCTATGTTGGACTTAGAA-39.
Phenotypic analysis of CP-transgenic Arabidopsis. For pheno-
typic analysis, wt Arabidopsis and mutants were grown in 24-well
plant growth trays under conditions of 16 h light/8 h dark at 22 uC.
High humidity was maintained during germination and the early
seedling stage by covering trays with transparent plastic lids. Growth
and developmental parameters were analysed by using at least 30
homologous seeds from each line.
Real-time PCR analysis of CP-transgenic Arabidopsis. Total
RNA for real-time PCR was extracted from leaves and inflorescences
using Trizol reagent (Sigma). Real-time PCR was carried out using a
SYBR Green two-step real-time PCR kit (Applied Biosystems). cDNA
was synthesized using random hexamers. The genes tested and the
primers used are available in Supplementary Table S1 (available with
JGV Online). The real-time PCR primers were designed using Primer
Express v2.0 (Applied Biosystems). The primers for the TAS target
genes were designed to flank the siRNA-directed cleavage sites. The
samples were run on an ABI Prism 7000 Sequence Detection System.
Thermal cycling conditions were one cycle of 50 uC for 2 min and
95 uC for 10 min, followed by 40 cycles of 95 uC for 15 s and 60 uC
for 1 min. Following amplification, a dissociation stage was
performed to check for non-specific PCR product. The 18S rRNA
gene was used as the endogenous control and RNAs from DCP20 were
used as a calibrator sample. The relative abundance of the genes (fold
increase) in CP6 compared with DCP20 was calculated using the
22DDCtmethod (Applied Biosystems, 2008).
HCRSV CP inhibits PTGS induced by a sense-RNA
pBICP but not pBIDCP has been confirmed previously to
function as a PTGS suppressor using Agrobacterium-
mediated transient assays in Nicotiana benthamiana
(Meng et al., 2006). pBICP and pBIDCP were introduced
as transgenes into Arabidopsis (ecotype Col-0). Northern
and Western blot analyses showed that the HCRSV CP
mRNA (Fig. 1b, lanes 2 and 3) and protein (Fig. 1c, lanes 2
and 3) both accumulated at high levels in the CP-
transgenic Arabidopsis, indicating that the HCRSV CP
gene was transcribed and translated in the transgenic
plants. In contrast, DCP-transgenic Arabidopsis did not
express the CP, although the mRNA was transcribed at a
relatively high level (Fig. 1b and c, lane 4). Lines CP6 and
DCP20 were chosen for further studies.
To test whether the constitutively expressed CP was able to
suppress PTGS induced by the sense-RNA transgene, lines
GUS gene was rescued by HCRSV CP in
transgenic Arabidopsis. (a) Constructs used
for transformation of Arabidopsis Col-0. (b)
Detection of CP mRNA from transgenic
Arabidopsis by Northern blotting. Total RNA
(5 mg) from Arabidopsis leaves was detected
using a DIG-labelled CP RNA probe. (c)
Detection of CP expression in transgenic lines
with HCRSV CP polyclonal antibody. (d)
Rescue of silenced GUS expression in line
L1 by 35S–CP. Homozygous line CP6 was
crossed with L1. L1?DCP20 was used as a
control. GUS activity was restored by the CP
transgene. The leaf on the left represents
L1?CP6, whilst the leaf on the right represents
L1?DCP20. (e) Detection of GUS mRNA and
GUS siRNAs in rdr6 (L1 background), L1,
L1?CP6 and L1?DCP20 seedlings. Probes
were DIG-labelled RNA corresponding to the
1.7 kb GUS coding sequence (upper panel)
and the DIG-labelled GUS RNA probes for the
detection of GUS siRNAs (lower panel). Total
RNA (50 mg) was loaded in each lane.
1. Expression of transgene-silenced
HCRSV CP inhibits ta-siRNA biogenesis in Arabidopsis
CP6 and DCP20 were crossed with line L1 in which GUS
expression was silenced. GUS staining and Northern blot
analysis showed that high levels of GUS activity (Fig. 1d)
and GUS mRNA (Fig. 1e, upper panel, lane 3) were
detected in the F1 progeny of the L16CP6 cross. In
contrast, 35S–GUS remained silenced in the F1 progeny of
L16DCP20 (Fig. 1d) as well as L16Col-0 (data not
shown). Furthermore, the GUS-specific siRNAs accumu-
lated to high levels in L1 plants and L16DCP20 plants, but
was undetectable in L16CP6 plants (Fig. 1e, third panel,
lanes 2–4). These results indicated that the silencing
suppression activity required the expression of CP, as
shown in CP6. Transcription of CP mRNA alone, as shown
in DCP20, did not suppress PTGS.
Notably, these RNA analyses showed that suppression of
GUS RNA silencing in CP6 plants was as effective as in the
rdr6 mutant, which contains a defective cellular RNA-
dependent RNA polymerase (Fig. 1e, lane 1). In the rdr6
mutant, high levels of GUS mRNA and no GUS siRNA were
detected. This is due to the GUS transgene failing to convert
to dsRNA, resulting in PTGS not being induced. This is in
agreement with the findings of Mourrain et al. (2000).
Rescued GUS expression (Fig. 1d) and the lack of GUS-
specific siRNAs in the progeny of L16CP6 plants (Fig. 1e,
third panel, lanes 3) further indicated that the constitutively
expressed CP6 interferes with PTGS at the early initiation
stage. This result supports the suggestion that HCRSV CP
actively suppresses RNA silencing targeted against transi-
ently expressed 35S-controlled GFP transcripts in GFP-
transgenic N. benthamiana (Meng et al., 2006).
Arabidopsis expressing the HCRSV CP transgene
shows developmental defects resembling those
of mutant lines rdr6-11 and dcl4-2
Many gene-silencing suppressors have been implicated in
abnormal plant development by interfering with the
miRNA/siRNA pathways (Chapman et al., 2004; Chen et
al., 2004; Dunoyer et al., 2004; Zhang et al., 2006). To test
whether the carmovirus HCRSV CP would affect normal
plant development, lines CP6 and DCP20 were used for
phenotypic analysis. The wt line Col-0 was included as a
negative control. The mutant lines rdr6-11, sgs3-11, zip-1
and dcl4-2 were also included as controls. RNA-dependent
polymerase 6 (RDR6) and suppressor of gene silencing 3
(SGS3) are required for PTGS (Dalmay et al., 2000b;
Mourrain et al., 2000; Qu et al., 2005; Schwach et al., 2005)
and the production of ta-siRNAs in Arabidopsis (Peragine
et al., 2004). RDR6 has also been implicated in natural
virus resistance (Quet
ARGONAUTE7 (AGO7) whose primary function is in
the regulation of developmental timing (Hunter et al.,
2003). DCL4 produces 21 nt RDR6-dependent ta-siRNAs
and has been reported to participate in siRNA production
following viral infections (Allen et al., 2005; Gasciolli et al.,
2005; Xie et al., 2005; Yoshikawa et al., 2005; Bouche ´ et al.,
2006; Deleris et al., 2006).
al., 2005). ZIPencodes
In wt Col-0 Arabidopsis, the first two rosette leaves
appeared round and relatively flat (Fig. 2a, row 2). In
contrast, line CP6 showed elongated, downward curled
leaves, which resembled the leaf phenotypes of the rdr6-11,
sgs3-11, zip-1 and dcl4-2 mutants (Fig. 2a, rows 2 and 4).
Transgenic Arabidopsis line DCP20 harbouring DCP
showed the same developmental phenotype as wt Col-0
Arabidopsis. (a) Transgenic Arabidopsis line CP6 showed
incompatible carpel and stamen elongation compared with the
wt Col-0 and DCP20 negative control. The rosette leaves were
elongated and curled down in CP6. The rdr6-11, sgs3-11, zip-1
and dcl4-2 mutant lines were used as controls for phenotypic
analysis. Some of the sepals and petals were removed in rows 1
and 3 to allow observation of the stamens and carpels. All images
were taken under a light microscope. Images of rows 2 and 4 were
taken using a Canon camera model 350D. (b) Pollens and
stigmata developed normally in CP6 and DCP20. CP6 stigmata
failed to be fertilized due to the non-proportional elongation of the
carpel and stamen. The pollens and stigmata of CP6 and DCP20
were observed under a scanning electron microscope. The pollen
grains were coated with gold particles in a sputter coater before
examination under the microscope.
2. Developmentaldefects ofHCRSV CP-transgenic
C. Meng and others
2352 Journal of General Virology 89
(Fig. 2a, row 2). Compared with the wt Col-0 and DCP20,
line CP6 showed early flowering by 2–3 days and reduced
fertilization. Further analysis revealed that the carpels of
CP6 flowers were elongated, which caused a lack of
coordination between stamen and carpel (Fig. 2a, row 1),
resulting in reduced siliques (data not shown). In the
rdr6-11, sgs3-11, zip-1 and dcl4-2 mutants, the carpels
also showed an incompatible elongation with stamens
(Fig. 2a, row 3). The sepals of CP6 flowers appeared
normal under the light microscope compared with wt
(data not shown). The pollens and the stigmata of CP6
were normally developed (no obvious differences com-
pared with those of DCP20) when observed under the
scanning electron microscope (Fig. 2b, columns 1 and 2).
However, without artificial pollination, only DCP20
stigmata were fertilized (Fig. 2b, column 3). Manual
self-crossing of the pollens to the stigmata of CP6 resulted
in similar fertilization rates compared with those of wt
Col-0. Another CP overexpression line, CP8 (Fig. 1c, lane
3), exhibited the same phenotype as CP6 (data not
shown). However, a closely related gene-silencing sup-
pressor, TCV CP, has been shown to develop only mild
(Chapman et al., 2004) or no (Dunoyer et al., 2004)
developmental defects compared with wt. Comparison of
TCV- and HCRSV-encoded suppressors indicates that
differences in the sequence and structure of suppressors
have substantial impacts on plant development. Changes
in small-RNA accumulation and the level of suppressor
protein expressed in the transgenic plants may account
for the different phenotypes induced by the two CPs of
HCRSV and TCV.
HCRSV CP increases the accumulation of miR171
and miR172 but not miR173
As the miRNA pathway shares some key components with
the siRNA pathway, the suppressors may affect the
accumulation of some miRNAs that are important for
normal plant development. To test whether HCRSV CP
could influence the biogenesis and accumulation of
miRNAs, total RNAs were isolated from rosettes and
flowers, and three miRNAs were detected by Northern blot
hybridization. The mutant line rdr6-11 was included as a
SCARECROW-like (SCL) genes, which encode transcrip-
tion factors that control a wide range of developmental
processes, including radical patterning in roots and
hormone signalling. It downregulates its target by mRNA
cleavage in a similar way to siRNA (Llave et al., 2002;
Reinhart et al., 2002). miR172, however, downregulates
APETALA2 (AP2) and other AP2-like genes to promote
flowering by translational repression (Aukerman et al.,
2003; Chen, 2003). miR173 guides the phased maturation
of ta-siRNAs, including siR255 (Allen et al., 2005; Xie et al.,
2005). All miRNAs showed greater abundance in flowers
than in leaves. Both miR171 and miR172 increased in CP6
isperfectly complementaryto three
flowers (Fig. 3a, lanes 2 and 5), compared with the wt
control (Fig. 3a, lanes 1 and 4) and the rdr6-11 plants (Fig.
3a, lanes 3 and 6), which showed similar phenotypes of leaf
curling and incompatible stamen and carpel elongation to
the CP6 plants. The level of miR173 detected was
comparable in CP6 and Col-0 (Fig. 3a, lanes 1 and 2,
and 4 and 5, respectively).
Fig. 3. Northern blot analyses of miRNA and ta-siRNA accumula-
tion in CP-transgenic Arabidopsis and rdr6-11 in a Col-0
background. (a) Accumulation of miRNA in CP6 and Arabidopsis
mutants. Total RNA (50 mg) from leaves (lanes 1–3) and flowers
(lanes 4–6) of test plants was hybridized with32P-labelled miRNA
sequence-specific antisense DNA probes. RNA samples were
extracted using Trizol reagent from wt (Col-0 background; lanes 1
and 4), CP6 (lanes 2 and 5) and rdr6-11 (lanes 3 and 6),
respectively. Ethidium bromide-stained rRNA is shown as a
loading control. (b) Accumulation of siR255 was reduced in
transgenic Arabidopsis carrying HCRSV CP (CP6). High-molecu-
lar-mass RNA (50 mg) from flowers of CP6 was separated on a
15% sequencing gel and hybridized with a32P-labelled oligonu-
cleotide complementary to miR173 or siR255. RNA from wt Col-0
and mutant line rdr6-11 was used as controls. Ethidium bromide-
stained rRNA is shown as a loading control.
HCRSV CP inhibits ta-siRNA biogenesis in Arabidopsis
HCRSV CP reduces siR255 accumulation
ta-siRNAs resemble miRNAs in plants, acting in trans to
direct cleavage of target mRNAs. As CP6 resembles
mutants in the trans-acting pathway, we tested whether
HCRSV CP interferes with the ta-siRNA pathway. The
accumulation of siR255 was chosen as a marker for ta-
siRNAs. siR255 is one of the cleavage products of TAS1a,
TAS1b and TAS1c, and targets At4g29770 (unclassified)
and At5g18040 (unclassified), respectively. Northern blot
analysis of RNAs extracted from inflorescences showed that
the accumulation of siR255 was significantly reduced in
line CP6 (Fig. 3b, lane 2) compared with wt Col-0 (Fig. 3b,
lane 1). In the rdr6-1 cell line, siR255 was not detected
(Fig. 3b, lane 3). Similar results have been reported for
TCV CP in which the accumulation of siR255 was
downregulated in TCV-inoculated plants or TCV CP-
transgenic Arabidopsis (Bouche ´ et al., 2006; Deleris et al.,
HCRSV CP upregulates ta-siRNA target genes
In the event of reduced ta-siRNA biogenesis, expression of
their target genes should increase correspondingly. To test
this hypothesis, the relative concentrations of various ta-
siRNA target mRNAs from leaves and inflorescences of
CP6 and DCP20 were compared by real-time RT-PCR. The
primers for TAS target genes were designed to flank the
siRNA cutting sites. 18S rRNA was used as an endogenous
control to normalize the RNA input for each sample.
(At4g29770), TAS1b (At5g18040), TAS2 PPR and TAS3
ARF3, were all upregulated, especially in the inflorescences
of CP6 (Fig. 4a), supporting the proposal that upstream ta-
siRNA biogenesis was inhibited. Among the four genes
tested, TAS1b and TAS3 ARF3 accumulated to a relatively
higher level than the other two genes in CP6 leaves (Fig. 4a).
As normal regulation of TAS3 target genes by TAS3 ta-
siRNAs is required for proper leaf development (Gasciolli
et al., 2005; Adenot et al., 2006), the enhanced accumula-
tion of ARF genes may explain the abnormal leaf
phenotypes in CP6.
Other genes that are involved in gene-silencing-related
pathways were also tested, including DCL1, DCL2, DCL3,
DCL4, DRB4, RDR6 and AGO1. Most of the genes tested
increased in the CP6 line, especially in the inflorescences
(Fig. 4b). Among these genes, DCL1, which is important
for miRNA and ta-siRNA biogenesis, increased by 1.8-fold,
DCL2 and DCL3 both increased by around 1.5-fold in
inflorescences, and DCL4, which is crucial for ta-siRNA
biogenesis, increased by 1.3-fold in leaves. DRB4, which
interacts with DCL4 in vivo and functions in the ta-siRNA
pathway, remained unchanged in both leaves and inflor-
escences. RDR6 remained unchanged in leaves but
increased by more than 1.5-fold in the inflorescences.
AGO1 selectively recruits miRNAs and siRNAs into RISC
(Baumberger & Baulcombe, 2005) and was enhanced 2.5-
fold in the inflorescences.
HCRSV CP may interfere with RNA silencing at a
step after RDR6
RDR6 plays a crucial role in gene-silencing-related path-
ways by converting ssRNA into dsRNA. To determine
whether HCRSV CP interferes with RDR6, CP6 and DCP20
were each crossed with the A. thaliana lines A and G6A
(Dalmay et al., 2000a). In line A (Amp243, which carries
the 35S–PVX::GFP amplicon), initiation of PTGS is
(Dalmay et al., 2000a, b), corresponding to the genetically
determined PTGS initiation pathway. In line G6A
Fig. 4. Comparison of gene expression in
leaves and inflorescences. Total RNA was
extracted, using Trizol reagent, from the leaves
and inflorescences of CP6 and DCP20 plants.
Reverse transcription was carried out using
random hexamer primers. The resulting cDNAs
were amplified using gene-specific primers
(see Supplementary Table S1, available in JGV
Online) and fold changes were measured
using real-time PCR. CP6 was the target
sample and DCP20 was used as the cal-
ibrator; 18S rRNA was chosen as an endo-
genous control. The relative abundance of the
genes (fold increase) in CP6 compared with
DCP20 was calculated using the 2”DDCt
method. (a) Accumulation of ta-siRNA target
gene transcripts in CP6 and DCP20. (b)
Accumulation of key gene transcripts involved
in the RNA interference-related pathways.
C. Meng and others
2354 Journal of General Virology 89
(homologous line resulting from crossing of line A and the
GFP-overexpression line GFP142), PTGS is initiated by the
replicating PVX::GFP and maintained by the 35S–GFP
transgene (Dalmay et al., 2000b); host RDR6 is required for
the strong PTGS in G6A (Dalmay et al., 2000b).
The F1 plants of A6CP6 and (G6A)6CP6 were both of
similar size and the GFP fluorescence was weak; no
apparent differences were observed compared with the
wild-type and DCP20-crossed controls (data not shown)
under a UV (312 nm) lamp (VL-6LM; Vilber Lourmat). By
RNA blot analysis, we detected a significant increase in the
accumulation of PVX::GFP genomic and subgenomic
RNAs in both A and G6A plants (Fig. 5, upper panel,
lanes 2 and 5) after they were crossed with line CP6, but
not with DCP20 or wt (Fig. 5, upper panel, lanes 1, 3, 4 and
6). Higher levels of GFP siRNAs were also detected in the
progeny of both A6CP6 and (G6A)6CP6 crosses (Fig. 5,
third panel, lanes 2 and 5) compared with those in the
control plants (Fig. 5, third panel, lanes 1, 3, 4, and 6),
suggesting that HCRSV CP is able to rescue the genetically
silenced 35S–PVX::GFP in line A and the epigenetically
silenced 35S–GFP and 35S–PVX::GFP in line G6A.
Similar results have been reported for the TYMV silencing
suppressor p69 and CaMV p6 suppressor (Chen et al.,
2004; Love et al., 2007). The increase in both PVX::GFP
viral RNA and GFP siRNA can be explained, as HCRSV CP
is unable to inhibit degradation of pre-existing dsRNAs,
whilst viral replication produces a continuous supply of
dsRNAs. These results are different from the inverse
correlation of GUS mRNA and siRNA in the L16CP6
cross (Fig. 1e, lane 3).
In the CP66(G6A) line, the GFP mRNA level (Fig. 5,
upper panel, lane 5) was much weaker than the PVX RNAs.
In contrast, the RDR6 mutant line rdr6(G6A) accumu-
lates much more GFP mRNAs than PVX RNAs because the
RDR6-dependent PTGS is disabled in rdr6(G6A) (Dalmay
et al., 2000b). Comparing the GFP mRNA and siRNA
accumulation in CP66(G6A) and rdr6(G6A) suggests
that HCRSV CP probably interferes with gene silencing at a
step after RDR6; HCRSV CP and RDR6 might act at
different steps of the same pathway.
HCRSV CP has been shown to be a strong gene-silencing
suppressor, and the reduced suppression function of the
CP after serial passages may be correlated with the
avirulence of HCRSV in Hibiscus (Meng et al., 2006).
Most of the suppressors identified are pathogenicity
determinants, and many of them have been reported to
show developmental defects when expressed as transgenes
in Arabidopsis. These defects are linked with the interfer-
ence of endogenous small-RNA pathway(s). In this paper,
we investigated the effects of HCRSV CP on the
accumulation of selected miRNAs, ta-siRNAs and genes
related to gene-silencing pathways and plant development.
Different silencing-suppression assays may result in
different interpretations of suppression mechanisms, and
stable expression assays are mostly free of complications
(Roth et al., 2004). HCRSV CP-transgenic Arabidopsis was
created to test the constitutively expressed CP on
suppression of gene silencing. Genetic crosses of HCRSV
CP-transgenic Arabidopsis with the GUS-silenced L1 line
restored GUS expression, confirming the function of
HCRSV CP as a silencing suppressor in the Arabidopsis
The Arabidopsis CP-transgenic line CP6 showed several
developmental abnormalities (Fig. 2a), similar to those of
RDR6, SGS3, ZIP and DCL4 mutants (Peragine et al.,
2004). CP8 showed the same developmental defects as CP6.
In contrast, another CP-transgenic line with marginal levels
of CP expression showed no difference in phenotype when
compared with wt (data not shown), suggesting that the
severity of the abnormalities was correlated with the level
of CP expression in the transgenic plants. This is in
agreement with the observation in turnip mosaic virus HC-
Pro- and TYMV p69-transgenic Arabidopsis (Chen et al.,
2004; Dunoyer et al., 2004). Developmental phenotypes in
Arabidopsis have been correlated with strong gene-silencing
suppressor activity (Chapman et al., 2004). However, the
molecular basis may be more complex. By comparing the
Fig. 5. Suppression of amplicon-induced PTGS by the CP
transgene. Transgenic line CP6 was crossed with lines A and
G?A. Col-0 and line DCP20 were crossed with lines A and G?A
as negative controls. All plants analysed were hemizygous F1
plants. Five micrograms (upper panel) or 50 mg (lower panel) total
RNA was separated and transferred to Hybond-N membrane,
followed by hybridization with a DIG-labelled GFP RNA probe.
Ethidium bromide-stained rRNA and 5S rRNA are shown as
loading controls. gRNA, Genomic RNA; sgRNA, subgenomic
HCRSV CP inhibits ta-siRNA biogenesis in Arabidopsis
results of this study with those of TCV CP, we have clearly
demonstrated that such a correlation is not universally
applicable to all virus suppressors. TCV belongs to the
same genus as HCRSV. TCV CP is a strong suppressor that
completely abolishes siRNA accumulation in N. benthami-
ana (Qu et al., 2003). Transgenic Arabidopsis expressing
TCV CP shows no significant developmental defects and
miRNA accumulation remains unchanged (Chapman et
al., 2004; Dunoyer et al., 2004). However, the accumulation
of miR171 and miR172 increased in the HCRSV CP6
inflorescences (Fig. 3a). Arabidopsis expressing CaMV 2b
protein from a severe strain (FNY) was reported to cause
significant accumulation of miRNAs and to display
obvious developmental defects (Zhang et al., 2006).
However, CaMV 2b from mild strains (LS and Q) was
reported to have a mild effect on miRNA-guided functions
and plant development (Chapman et al. 2004; Lewsey et al.,
Compared with wt Col-0, the accumulation of miR171 and
miR172 was enhanced in the CP6 line, especially in the
flowers (Fig. 3a). The downregulation of SCL genes by
miR171 may contribute to the phenotype of CP6, as SCL
genes control a wide range of developmental processes,
such as hormone signalling (Llave et al., 2002; Reinhart et
al., 2002). The enhanced accumulation of miR172 may
change the expression pattern of AP2, which leads to
morphological changes in flowers. The reduction in AP2
gene expression in the carpel region may reduce inhibition
of C gene expression by the A gene, which may contribute
to the abnormal growth of carpels and stamens. The AP2
mutant showed transformation of sepals into carpels and
petals into stamen (Drews et al., 1991). Therefore, the
phenotype observed in CP6 may involve other genes (e.g.
flower homeotic genes) that act in concert with miR172 for
its abnormal development.
Based on the phenotypic similarities of CP6 with those of
ta-siRNA pathway mutants, interference in the ta-siRNA
pathway was suspected. We observed that siR255 accu-
mulation was greatly reduced in the CP6 line or not
detectable in the rdr6-11 line (Fig. 3b). The reduced siR255
in CP6 may result from changes at three stages: (i) the level
of miR173, which is responsible for the in-phase matura-
tion of ta-siRNA siR255; (ii) the downstream RDR6–SGS3-
dependent conversion of the miR173 cleavage product into
dsRNAs; and (iii) the DCL4 cleavage step. It has been
reported that DCL4 interferes with the biogenesis of ta-
siRNAs including siR255 but not the level of miR173
(Yoshikawa et al., 2005; Bouche ´ et al., 2006), or that it
marginally increased the level of miR173 (Xie et al., 2005).
miR173 was detected in the same sample used for siR255 in
Northern blotting. The level of miR173 remained constant
in line CP6 inflorescences compared with wt Col-0 (Fig. 3b,
lanes 1 and 2). The constant miR173 level indicates that the
reduced siR255 level in CP6 might be the direct
consequence of reduced dsRNAs or inhibition of DCL4
by HCRSV CP.
siR255 is the cleavage product of TAS1a, TAS1b and TAS1c
(Allen et al., 2005; Xie et al., 2005). Target gene analysis
(Fig. 4a) in CP6 showed that the expression levels of the
genes for TAS1a (At4g29770), TAS1b (At5g18040), TAS2
PPR and TAS3 ARF3 were all significantly increased. These
data further support our hypothesis that HCRSV CP
interferes with the ta-siRNA pathway. TAS3 specifies leaf
polarity by targeting ARF3 and ARF4, which in turn
downregulate the FILAMENTOUS FLOWER (FIL) gene
(Garcia et al., 2006). Based on the developmental roles of
TAS target genes, it is possible that these varied ta-siRNA
targets may be crucial for the abnormal development in
In addition to the contribution of endogenous small RNAs,
the expression of some other genes that are closely involved
in silencing pathways may also be changed by the
expression of HCRSV CP. Through real-time PCR analysis,
the genes for the four DCLs and RDR6 and AGO1 were
shown to be expressed at higher levels in CP6, especially in
the inflorescences (Fig. 4b). The increased accumulation of
these gene transcripts indicates that the overall antiviral
silencing in CP6 might have been enhanced. The fact that
DCL4 is expressed at about the same level or higher in the
CP6 plants suggests that the CP might inhibit DCL4
directly, given that siR255 is reduced in CP6-transgenic
RDR6 is not only indispensable for transgene-induced gene
silencing, it also plays a vital role in the biogenesis of ta-
siRNAs and in the natural host antiviral response. HCRSV
CP enhanced the level of viral RNA in lines A and G6A
(Fig. 5). The replication of PVX overcomes viral dsRNA-
induced degradation and/or 35S–GFP (in the case of
G6A)-induced silencing by the host plant. These results
indicate that silencing in the genetically silenced GFP in
line A and epigenetically silenced GFP in line G6A was
reversed. Comparing the siRNA accumulation patterns in
L16CP6 with A6CP6 and (G6A)6CP6, an inconsistent
inverse correlation of PVX::GFP mRNAs and GFP siRNAs
was observed (Fig. 5, lanes 2 and 5). The increase in GFP
siRNAs in the A6CP6 and (G6A)6CP6 crosses suggests
that large amounts of GFP-containing RNA were pro-
duced, some of which were cleaved into GFP siRNAs,
indicating that some Dicer and AGO1 proteins are still
functioning, despite the presence of HCRSV CP sup-
pressor. The presence of PVX and its replication provides a
continuous source of dsRNAs, which accounts for the
increased accumulation of GFP and PVX siRNAs in the
A6CP6 and (G6A)6CP6 crosses. However, HCRSV CP
(G6A)6CP6. The GFP mRNA accumulation pattern
compared with the PVX genomic RNA and subgenomic
RNA levels in (G6A)6CP6 was different from that of
rdr6(G6A) (Dalmay et al., 2000b), suggesting a block in
gene silencing at a step after RDR6.
the RDR6 functionin
DCLs in Arabidopsis act in combination to counteract RNA
virus and DNA virus invasion, with RNA viruses mainly
C. Meng and others
2356Journal of General Virology 89
affected by DCL4 and DNA viruses targeted by all four
DCLs (Blevins et al., 2006; Deleris et al., 2006). CaMV is
reported to silence host gene expression through the
coordinated action of four DCLs in Arabidopsis (Moissiard
& Voinnet, 2006). The accumulation of viral RNAs is
significantly increased in DCL mutants, especially double
or triple mutants (Deleris et al., 2006). With DCL4
suppressed by TCV CP, TCV was targeted by DCL2 and
produced 22 nt siRNAs in Col-0 (Deleris et al., 2006).
Taking our fingdings together, we postulate that HCRSV
CP interferes with PTGS and ta-siRNA biogenesis at the
RNA recognition step downstream of RDR6, which is
common to the ta-siRNA pathway and amplicon-induced
silencing pathway. The phenotypes observed in CP6 may
be a sum effect of the interplay by miRNAs, ta-siRNAs or
unknown endogenous siRNA pathways and other tran-
scriptional or translational regulation mechanisms.
We thank Dr Peter Palukaitis of the Scottish Crop Research Institute,
UK, and Dr Hao Yu of the National University of Singapore for
helpful discussions; the Arabidopsis Biological Resource Center for
providing seeds of the rdr6-11, sgs3-11 and zip-1 mutants, Dr Herve ´
Vaucheret for the L1 line, Dr David Baulcombe for the amplicon
lines, Dr James Carrington (Oregon State University, USA) for
mutant line dcl4-2 and Dr Milton Zaitlin from Cornell University,
USA, for editing the manuscript. The work was supported by
National University of Singapore research grants R-154-000-252-112
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