Journal of General Virology (2009), 90, 519525

Short
Communication

DOI 10.1099/vir.0.006098-0

Inhibition of RNA silencing by the coat protein of

Pelargonium flower break virus: distinctions from
closely related suppressors
Sandra Martnez-Turino and Carmen Hernandez

Correspondence
Carmen Hernandez

Instituto de Biologa Molecular y Celular de Plantas (Consejo Superior de Investigaciones

Cientficas-UPV), Avenida de los Naranjos, 46022 Valencia, Spain

cahernan@ibmcp.upv.es

Received 31 July 2008

Accepted 21 October 2008

Viral-derived double-stranded RNAs (dsRNAs) activate RNA silencing, generating small

interfering RNAs (siRNAs) which are incorporated into an RNA-induced silencing complex (RISC)
that promotes homology-dependent degradation of cognate RNAs. To counteract this, plant
viruses express RNA silencing suppressors. Here, we show that the coat protein (CP) of
Pelargonium flower break virus (PFBV), a member of the genus Carmovirus, is able to efficiently
inhibit RNA silencing. Interestingly, PFBV CP blocked both sense RNA- and dsRNA-triggered
RNA silencing and did not preclude generation of siRNAs, which is in contrast with the abilities
that have been reported for other carmoviral CPs. We have also found that PFBV CP can bind
siRNAs and that this ability correlates with silencing suppression activity and enhancement of
potato virus X pathogenicity. Collectively, the results indicate that PFBV CP inhibits RNA silencing
by sequestering siRNAs and preventing their incorporation into a RISC, thus behaving similarly to
unrelated viral suppressors but dissimilarly to orthologous ones.

RNA silencing is a eukaryotic mechanism for RNA-based

gene regulation that plays essential roles in diverse
biological processes. Though RNA silencing may operate
through different pathways, the triggers in all cases are
double-stranded RNA (dsRNA) or highly structured
single-stranded RNA molecules, the former originated via
an RNA-dependent RNA polymerase (RdRP), which is
processed by RNase III-type Dicer enzymes into small RNA
duplexes of 2125 nt (Bernstein et al., 2001; Hamilton &
Baulcombe, 1999; Zamore et al., 2000). One class of these
small duplexes, termed small interfering RNAs (siRNAs),
mediate an RNA silencing pathway that takes place in the
cytoplasm, which is involved in antiviral defence in
different eukaryotes (Ding & Voinnet, 2007; Waterhouse
et al., 2001). After their generation, these siRNAs are
denatured and one strand is incorporated into a multisubunit endonuclease known as RNA-induced silencing
complex (RISC) (Baumberger & Baulcombe, 2005; Liu
et al., 2004). Within the activated RISC, single-stranded
siRNAs act as guides to bring the complex into contact
with complementary mRNAs and thereby cause their
degradation (Baulcombe, 2005; Hammond et al., 2001).
The associated siRNAs also seem to provide sequence
specificity for a cellular RdRP that generates new dsRNAs
upstream of the initial trigger, leading to secondary siRNA
accumulation and thus amplifying silencing (Lipardi et al.,
2001; Sijen et al., 2001).
A supplementary figure is available with the online version of this paper.

006098 G 2009 SGM

Printed in Great Britain

To counteract RNA silencing-mediated host defences,

many plant and some animal viruses have evolved RNA
silencing suppressor proteins (Qu & Morris, 2005; Voinnet,
2005). These proteins do not share any obvious sequence
or structural similarity across viral groups and might
interfere with the RNA silencing pathway at different stages
(Roth et al., 2004). So far, the best characterized of the viral
suppressors is the p19 protein of tombusviruses. Detailed
studies have demonstrated that this binds siRNAs, thus
making them unavailable for RISC (Lakatos et al., 2004;
Silhavy et al., 2002; Vargason et al., 2003; Ye et al., 2003).
Recent results demonstrate that distinct unrelated viral
suppressors also inhibit silencing by sequestering siRNAs
(Lakatos et al., 2006; Merai et al., 2006), while others might
inactivate silencing by binding dsRNAs without obvious
size selection, which could interfere with Dicer activity
(Chao et al., 2005; Lu et al., 2005; Merai et al., 2005, 2006).
Viral suppressors that apparently exert their action by
different mechanisms have also been described
(Baumberger et al., 2007; Trinks et al., 2005; Wang et al.,
2005; Zhang et al., 2006).
Pelargonium flower break virus (PFBV), genus Carmovirus,
family Tombusviridae, has a single-stranded positive-sense
genomic RNA that encodes two proteins involved in
replication, p27 and its read-through product p86 (the
viral RdRP), two movement proteins, p7 and p12, and the
coat protein (CP) p37 (Rico & Hernandez, 2004). Studies
on turnip crinkle virus (TCV) and hibiscus chlorotic
ringspot virus (HCRSV), both carmoviruses, have shown
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S. Martnez-Turino and C. Hernandez

that their CPs are strong suppressors of RNA silencing

(Meng et al., 2006; Qu et al., 2003; Thomas et al., 2003). In
the case of melon necrotic spot virus, the third member of
the genus for which RNA silencing has been investigated,
CP exhibited an apparently weaker suppressor activity and
a weak suppressor function was also reported for the
movement protein p7b (Genoves et al., 2006). Whereas
TCV CP has been proposed to interfere with the processing
of dsRNA mediated by a Dicer enzyme (Deleris et al., 2006;
Merai et al., 2006; Qi et al., 2004; Qu et al., 2003), HCRSV
CP action was determined to occur at or before the dsRNA
generation step (Meng et al., 2006), suggesting that
homologous proteins may block silencing by different
mechanisms. To explore this issue further here, we have
identified and characterized suppressor activity from
PFBV. These results highlight a considerable diversification
in the molecular basis of the suppressor activity of closely
related proteins.
To identify potential RNA silencing suppressors of PFBV,
individual PFBV ORFs were amplified from the infectious
clone pSP18-IC (Rico & Hernandez, 2006) with the
Expand High Fidelity PCR system (Roche) and subsequently inserted under the control of the cauliflower
mosaic virus (CaMV) 35S promoter into the binary vector
pMOG800. Agrobacterium tumefaciens was transformed
with the resulting plasmids or with equivalent binary

constructs (Supplementary Fig. S1, available in JGV

Online) harbouring the green fluorescent protein (GFP)
(Herranz et al., 2005) or the tombusviral p19 (Voinnet
et al., 2003) gene and these were used to infiltrate leaves
from GFP-transgenic Nicotiana benthamiana plants (line
16c) (Ruiz et al., 1998) as described previously (Qu et al.,
2003; Voinnet et al., 2000).
Leaf patches expressing GFP alone or together with any of
the replication or movement proteins showed high levels of
GFP expression at 2 days post-infiltration (p.i.) (data not
shown) which was almost completely silenced at 5 days p.i.
as assessed by observations of GFP fluorescence (Fig. 1a).
However, co-expression of the PFBV CP with GFP resulted
in a sustained, very bright green fluorescence which was
comparable in intensity and persistency to that visualized
in parallel assays with the tombusviral p19, used as a
positive control of suppressor activity (Fig. 1a). Northern
blot hybridization, performed as previously described
(Castano & Hernandez, 2005), confirmed that the
fluorescence patterns reflected changes in the steady-state
levels of GFP mRNA since, at 5 days p.i., GFP mRNA
accumulation was very low in infiltration patches expressing GFP alone or in combination with p27, p86, p7 or
p12, whereas co-expression of GFP and CP gave rise to
GFP mRNA levels equivalent to those observed from
expression of GFP plus p19 (Fig. 1b). As indicated above,

Fig. 1. In planta assay for RNA silencing suppression. N. benthamiana 16c plants were agroinfiltrated with constructs for
expression of GFP either alone or in combination with p27, p86, p7, p12, CP, CPq, CPq.mut or p19. (a) GFP fluorescence at 5
days p.i. in infiltrated leaf patches. (b) Northern blot hybridization for detection of the GFP mRNA or derived siRNAs. Ethidium
bromide staining of RNA is shown as a loading control.
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Journal of General Virology 90

Suppression of RNA silencing by PFBV CP

RNA silencing is always associated with accumulation of

siRNAs. Analysis of siRNAs showed that the levels of GFP
siRNAs were strongly diminished in tissues co-expressing
GFP and PFBV CP, as reported by Martnez de Alba et al.
(2002), which correlated with the high amount of GFP
mRNA detected in these tissues (Fig. 1b). Co-expression of
GFP and p19 also gave rise to an apparent reduction in
siRNA accumulation (Fig. 1b) in agreement with previous
observations (Dunoyer et al., 2004; Voinnet et al., 2003).
Altogether, the results demonstrated that PFBV CP is able
to block sense RNA-induced RNA silencing as efficiently as
p19, a potent viral suppressor.
The CP of PFBV can be divided into an internal RNAbinding domain (R), a shell-forming domain (S) and a
protruding domain (P) (Lommel et al., 2005). Previous
work has shown that adaptation of PFBV to Chenopodium
quinoa results in a 5 aa covariation that affects specific

positions within the R and P domains (Rico et al., 2006).

This C. quinoa-selected CP variant (CPq) was also cloned
into pMOG800 and co-expressed with GFP in agroinfiltration assays, to assess whether serial transfer through the
local host causes a reduction in the ability of the protein to
block RNA silencing, as reported for HCRSV CP (Meng
et al., 2006). The GFP fluorescence at 5 days p.i. was
indistinguishable from that observed in leaf patches
expressing GFP plus CP. As expected, Northern blot
analysis showed a high increase in the levels of GFP mRNA
which correlated with a low accumulation of GFP-specific
siRNAs (Fig. 1). These results indicate that the antisilencing activity of PFBV CP is not affected by the amino
acid substitutions fixed in C. quinoa, and suggested that the
local host does not necessarily impose restrictions on
viruses that diminish their suppressor function as a strategy
to limit the spread of infection. Interestingly, during
amplification of the CPq gene, a nucleotide substitution,

Fig. 2. Effects of the expression of PFBV CP variants from a PVX vector on symptom severity and viral accumulation. (a)
Symptoms in systemic N. benthamiana and N. clevelandii leaves 15 days after inoculation with a PVX vector containing no
insert (pPVX202) or containing the PFBV CP gene (pPVX-CP), the gene of the C. quinoa-selected CP (pPVX-CPq) or the
latter one with a mutation leading to the change P323L (pPVX-CPq.mut). (b) Northern blot analysis of total RNA from
N. benthamiana or N. clevelandii plants infected after inoculation with pPVX202 or with pPVX-CP, pPVX-CPq or pPVXCPq.mut. The probes that were employed are indicated on the right and the positions of the PVX genomic (gRNA) and
subgenomic (sgRNAs) RNAs are marked on the left.
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521

S. Martnez-Turino and C. Hernandez

which resulted in an amino acid change (P323L) in the P

domain, was accidentally introduced into one of the clones.
Co-expression of this protein variant, named CPq.mut
with GFP showed that the single amino acid substitution
was sufficient to abolish the silencing suppressor function
of the PFBV protein (Fig. 1).
Silencing suppressors are commonly involved in enhancement of viral pathogenicity and accumulation of viruses
(Voinnet et al., 1999), thus many of them have been shown
to accentuate symptoms when expressed by a heterologous
virus (Pruss et al., 1997; Valli et al., 2008; Zhou et al.,
2006). To investigate this, the PFBV CP gene was cloned in
a PVX vector (pPVX202; Sablowski et al., 1995) and the
resulting construct (pPVX-CP) was used to mechanically
inoculate N. benthamiana and Nicotiana clevelandii plants.
Constructs with the CPq (pPVX-CPq) and CPq.mut
(pPVX-CPq.mut) genes inserted were also tested. In both
hosts, heterologous CP or CPq expression induced stunting
and necrosis at 810 days post-inoculation, in contrast
with the vein clearing and mild chlorotic mosaic elicited by

wild-type PVX202 (Fig. 2a). The necrosis of systemic leaves

and stems was followed by death of the plants, showing
that expression of CP (or CPq) markedly accentuates
pathogenicity of the unrelated PVX. Remarkably, symptoms induced by pPVX-CPq.mut were indistinguishable
from those observed with pPVX202 (Fig. 2a). Northern
blot analysis revealed that CP and CPq, but not CPq.mut,
greatly enhanced PVX accumulation, even though the
corresponding inserts were retained to similar extents in
the recombinant viruses (Fig. 2b). The results showed that
increasing pathogenicity and RNA silencing suppression
activities of PFBV CP are intimately connected.
To obtain further insight into the mechanism of PFBV CP
suppression of RNA silencing, we examined whether PFBV
CP was blocking conversion of sense GFP to dsRNA or
inhibiting silencing downstream of dsRNA generation. To
address this, PFBV CP was co-expressed in N. benthamiana
leaves with GFP and double-stranded GFP (dsGFP), the
latter was transcribed from a binary construct that
harbours both sense and antisense sequences of GFP

Fig. 3. Exploration of the molecular basis of silencing suppression by PFBV CP. (a) Inhibition of dsRNA-triggered RNA
silencing. Leaves of N. benthamiana 16c were agroinfiltrated with construct combinations for expression of GFP plus dsGFP
alone () or together with CP or p19. GFP fluorescence at 5 days p.i. in infiltrated patches is shown on the left and Northern
blot detection of GFP mRNA or siRNAs extracted from the same patches is shown on the right. (b) Gel mobility shift assay.
32
P-Labelled double-stranded siRNAs were incubated alone () or with crude extracts from mock-agroinfiltrated (Mock) leaves
or agroinfiltrated leaves expressing PFBV CP, CPq, CPq.mut or p19. All binding reactions were performed using 22 ml of
extract, except p19 for which 5 ml was used. The positions of free siRNA and of proteinsiRNA complexes are indicated on the
right.
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Suppression of RNA silencing by PFBV CP

separated by an intron and thus generating GFP dsRNA

(Takeda et al., 2002). Combinations of GFP plus dsGFP,
either alone or with p19, were also included as controls.
The fluorescence in patches expressing GFP plus dsGFP
dropped to undetectable levels at 5 days p.i., but patches
co-expressing PFBV CP showed considerable fluorescence,
which increased in subsequent days, and a strong
fluorescence was detected in p19-expressing patches
(Fig. 3a). Consistent with the fluorescence patterns,
accumulation of GFP mRNA in the PFBV CP-expressing
patches was notably higher than in those expressing just
GFP and dsGFP, indicating that PFBV CP was able to
interfere with dsRNA-triggered RNA silencing (Fig. 3a).
GFP mRNA accumulation was greatly enhanced by p19
expression, in agreement with previous reports (Csorba
et al., 2007). A large amount of siRNA accumulated in
tissues co-expressing GFP and dsGFP alone or together
with p19 (Fig. 3a). This was expected, since the tombusviral
suppressor specifically binds siRNAs and, therefore, does
not inhibit Dicer activity, in contrast with other suppressors that bind to long dsRNAs and compromise their
Dicer-mediated processing, as seen with TCV CP (Chao et
al., 2005; Lingel et al., 2005; Lu et al., 2005; Merai et al.,
2005, 2006). Remarkably, cleavage of dsGFP to siRNAs was
inhibited neither by PFBV CP, which allowed siRNAs to
accumulate at levels similar to those observed in the
presence of p19, nor in the absence of suppressor proteins
(Fig. 3a). These results strongly suggested that PFBV CP
does not interfere with Dicer activity and targets the
silencing machinery downstream of siRNA generation,
thus differing from the CPs of TCV and HCRSV.
The results above indicated that the mechanism for silencing
inhibition of PFBV CP could involve sequestering of
siRNAs, as reported for different suppressors including
p19. To explore this possibility, N. benthamiana leaves were
separately agroinfiltrated with constructs for expression of
CP, CPq and CPq.mut. In addition, mock-infiltrated leaves
and infiltrated leaves expressing p19 were used as negative
and positive controls, respectively. Crude extracts were
prepared 3 days p.i. and incubated with 32P-labelled
synthetic siRNAs according the protocol described by
Merai et al. (2006), and the resulting products were resolved
on SDS-PAGE gels (Fig. 3b). As expected, p19 extracts
caused a shift in siRNA mobility, whereas no shifts were
observed with mock extracts. Interestingly, extracts containing PFBV CP or CPq, both functional in silencing
suppression, also showed siRNA binding activity, giving
rise to a complex that migrated more slowly than that
generated by p19 extracts. In contrast, those containing
CPq.mut, which had no detectable suppressor function,
failed to form complexes with siRNAs, even when the
amount of extract was increased in the binding mixtures
(Fig. 3b and data not shown). Collectively, the results
presented here strongly indicate that the silencing inhibition
activity of PFBV CP relies on the ability of the protein to
sequester siRNAs, as has been shown for phylogenetically
unrelated suppressors but not for orthologous ones. In
http://vir.sgmjournals.org

addition, our results might imply that TCV CP and PFBV

CP have diverged in their dsRNA-binding properties, with
PFBV CP acquiring size-selection separately from the sizeindependent dsRNA-binding of TCV CP. It has been
suggested that selection of siRNAs for binding may reduce
host damage, as the corresponding suppressor will not
interfere with endogenous long dsRNAs or long structured
RNAs (Baulcombe & Molnar, 2004; Vargason et al., 2003). It
has also been suggested that the expression of sizeindependent silencing suppressors might be down-regulated
by the virus (Merai et al., 2006). In the case of TCV CP, this
down-regulation is probably accomplished by capsid
formation which reduces available suppressor levels
(Zhang & Simon, 2003). In this scenario, it is tempting to
speculate that capsid formation plus selective binding to
siRNAs by PFBV CP might account for the low viral
accumulation and the frequent absence of symptoms in
PFBV-infected plants.

Acknowledgements
We are indebted to Vicente Pallas and Kazuyuki Mise for generously
providing us with binary plasmids for GFP and dsGFP expression,
respectively, and to David Baulcombe for N. benthamiana 16c and
binary plasmid for p19 expression. We gratefully thank Jose Antonio
Navarro for his valuable advice on agroinfiltration assays and to
Dolores Arocas for excellent technical assistance. This research was
supported by grants AGL2003-04249 (MCyT) and BFU2006-11230
(MEC) and by grant ACOM06/210 (GV). S. M.-T. was the recipient of
a predoctoral fellowship from the Generalitat Valenciana.

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