JOURNAL OF VIROLOGY, July 2002, p. 6815–6824 Vol. 76, No.

13
0022-538X/02/$04.00⫹0 DOI: 10.1128/JVI.76.13.6815–6824.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

P0 of Beet Western Yellows Virus Is a Suppressor of

Posttranscriptional Gene Silencing
S. Pfeffer, P. Dunoyer, F. Heim, K. E. Richards, G. Jonard, and V. Ziegler-Graff*
Institut de Biologie Moléculaire des Plantes du CNRS et de l’Université Louis Pasteur, 67084 Strasbourg, France
Received 19 November 2001/Accepted 8 April 2002

Higher plants employ a homology-dependent RNA-degradation system known as posttranscriptional gene

silencing (PTGS) as a defense against virus infection. Several plant viruses are known to encode proteins that
can suppress PTGS. Here we show that P0 of beet western yellows virus (BWYV) displays strong silencing
suppressor activity in a transient expression assay based upon its ability to inhibit PTGS of green fluorescent
protein (GFP) when expressed in agro-infiltrated leaves of Nicotiana benthamiana containing a GFP transgene.
PTGS suppressor activity was also observed for the P0s of two other poleroviruses, cucurbit aphid-borne
yellows virus and potato leafroll virus. P0 is encoded by the 5ⴕ-proximal gene in BWYV RNA but does not

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accumulate to detectable levels when expressed from the genome-length RNA during infection. The low
accumulation of P0 and the resulting low PTGS suppressor activity are in part a consequence of the suboptimal
translation initiation context of the P0 start codon in viral RNA, although other factors, probably related to the
viral replication process, also play a role. A mutation to optimize the P0 translation initiation efficiency in
BWYV RNA was not stable during virus multiplication in planta. Instead, the P0 initiation codon in the
progeny was frequently replaced by a less efficient initiation codon such as ACG, GTG, or ATA, indicating that
there is selection against overexpression of P0 from the viral genome.

Most if not all plants possess a defense mechanism against
virus infection known as posttranscriptional gene silencing
(PTGS; see reference 44 for a recent review). PTGS is a ho-
mology-dependent RNA degradation system to recognize and
degrade viral RNA (and any homologous transgene mRNA) in
the cytoplasm. Mechanistically similar phenomena exist in an-
imals and fungi (reviewed in references 4, 13, and 35). The
mechanism by which a virus infection triggers PTGS in plants
is not fully understood, but double-stranded RNA is a strong
inducer of PTGS (43) and such a form is produced during
replication of an RNA virus. Production of small RNAs of ⬃21
to 23 nucleotides, called small interfering RNAs (siRNAs),
corresponding to both the plus and minus strands of the target
RNA is associated with PTGS (12, 13, 45). In plants, gene
silencing at one site can trigger PTGS in distant tissues and
across a graft union (29, 39a). The mobile signal, which must
consist at least in part of homologous RNA, is thought to move
from cell to cell through plasmodesmata and systemically via
the vasculature.
Some plant viruses have been shown to encode proteins
which can counteract PTGS (8, 22, 39, 40, 42). These silencing
suppressor proteins may act at different steps in the PTGS
pathway. Thus, (i) the potyvirus helper component-proteinase
(HCPro) interferes with initiation and maintenance of silenc-
ing at a step coincident with or upstream of siRNA production
(1, 7, 16, 23, 24), (ii) the 2b protein of cucumber mosaic virus
(CMV) prevents initiation of PTGS in new growth by inhibit-
ing the long-range PTGS signaling activity (3, 7, 11), and (iii)
p25 from potato virus X (PVX) suppresses production or ac-
* Corresponding author. Mailing address: Institut de Biologie
Moléculaire des Plantes, 12 Rue du Général Zimmer, 67084 Stras-
bourg Cedex, France. Phone: (33) 388 417257. Fax: (33) 388 614442.
E-mail: veronique.ziegler-graff@ibmp-ulp.u-strasbg.fr.
tion of the mobile silencing signal (40). There is evidence that
silencing suppressor proteins are encoded by other plant vi-
ruses as well (39, 42).
The poleroviruses (family Luteoviridae) are obligately trans-
mitted by aphids in a circulative manner and are phloem re-
stricted in their hosts (27). Important members of the genus
include Potato leafroll virus (PLRV; the type member), Beet
western yellows virus (BWYV), and Cucurbit aphid-borne yel-
lows virus (CABYV). The polerovirus genome consists of sin-
gle-stranded plus-sense RNA of ⬃5.7 kb with six recognized
open reading frames (ORFs) (Fig. 1A). The 5⬘-proximal ORFs
0, 1, and 2 are translated from viral RNA and yield P0, P1, and
the translational frameshift protein P1-2. P1 and P1-2 are com-
ponents of the viral RNA-dependent RNA polymerase and are
thought to be translated by ribosomes which scan past the P0
start codon without initiating protein synthesis. The 3⬘-proxi-
mal ORFs 3, 4, and 5 are translated from a subgenomic RNA
and yield the viral capsid proteins and a putative movement
protein (26, 27).
Although P0 has never been detected in polerovirus-infected
plants, there is genetic evidence that it is functionally impor-
tant. Thus, null mutations in the P0 gene of BWYV and PLRV
strongly diminish or abolish viral RNA accumulation in pro-
toplasts and plants (34, 46). In this paper we show that P0 of
BWYV displays strong silencing suppressor activity in a tran-
sient expression assay based upon its ability to inhibit PTGS of
green fluorescent protein (GFP) when expressed in agro-infil-
trated leaves of Nicotiana benthamiana containing a GFP
transgene. Silencing suppressor activity was also observed for
the P0s of CABYV and PLRV. The poor expression of P0
from BWYV viral RNA is in part a consequence of the poor
initiation codon context of the P0 ATG, although other factors
are important as well. Experiments using a BWYV mutant in
which the P0 initiation codon context was optimized indicate

6815
6816 PFEFFER ET AL.
FIG. 1. Schematic representation of the BWYV genome and of
some of the clones used in the study. (A) Genome organization of
BWYV RNA. Numbered rectangles represent ORFs. The ORF for P0
is shaded. The diagonal arrow corresponds to the approximate position
of translation frameshift to synthesize the P1-2 fusion protein men-
tioned in the text. The subgenomic RNA (sgRNA) which is used for
translation of 3⬘-proximal genes is represented by a horizontal arrow
below the map. (B) Structure of PVX-P0 chimera RNA. The white
rectangles (not to scale) represent PVX genes, and the shaded rect-
angle represents the BWYV P0 gene. The black vertical lines corre-
spond to the duplicated PVX subgenomic RNA promoter sequences,
and horizontal arrows below indicate major subgenomic RNAs. The
arrow above the P0 gene indicates the position of the frameshift (fs)
mutation in PVX-P0fs. (C) Structures of the agro-infection vectors
used for expression of different proteins in plants. Only the transcrip-
tion cassette of each vector is shown with the cauliflower mosaic virus
35S transcription promoter and termination sequences indicated.
that overexpression of P0 is deleterious to viral RNA accumu-
lation and that second-site mutations predicted to lower the
efficiency of translation initiation at the P0 start codon were
abundant in the progeny RNA.
MATERIALS AND METHODS
Plasmids and transgenic plants. Full-length BWYV cDNA in pBW0 (20) was
used as template for PCR amplification of the BWYV P0 ORF using appropriate
specific primers. A nonviral EcoRV restriction site was added to the 5⬘ extremity
of each primer. The EcoRV-digested PCR fragment was ligated into EcoRV-
digested pP2C2S (2) to yield pPVX-P0. pPVX-P0fs was produced by filling in of
the XhoI site in the P0 ORF (nucleotide [nt] 255 in BWYV RNA) and recircu-
larization. The aforesaid PCR fragment was also ligated into SmaI-digested
pBin61 (40) to produce pBin-P0. pBin-P0CAB and pBin-P0PL were produced in
a similar fashion by PCR of cloned cDNA from CABYV (31) and the Dutch
isolate of PLRV (a gift of Frank van der Wilk) using appropriate specific
primers. In mutant pBin-P0⫺, a DNA fragment containing a knockout mutation
in the BWYV P0 ORF was obtained by PCR using DNA of the previously
described P0 knockout mutant BW1.6346 (46) as template. pBin-P05⬘wt and
pBin-P05⬘opt were constructed by PCR using an antisense primer complementary
to the 3⬘-terminal sequence of the ORF and a sense primer with the desired
additional 5⬘-terminal sequences built in.
pBin-BW was obtained by inserting a HindIII fragment containing the 35S
terminator sequence from pBin61 into the SalI site of pBinBW0 (5). pBin-
J. VIROL.
BW5⬘opt and pBin-BWP0⫺ were constructed by PCR mutagenesis (14) to gener-
ate a PCR fragment containing the 35S promoter and the mutated BWYV
cDNA sequence extending to the XhoI site at nt 255. This fragment was used to
replace the corresponding sequence in the wild-type clone pBin-BW. The various
plasmids described above were characterized by restriction site analysis, and all
the regions generated by PCR were completely sequenced. Agrobacterium tume-
faciens strains containing pBin-GFP (7) and pBin-HCPro, a pBin61 derivative
harboring the HCPro coding region of potato virus Y, were obtained from D. C.
Baulcombe.
Virus infection and agro-infiltration. N. benthamiana line 16c, which is ho-
mozygous for the GFP transgene, and the Agrobacterium infiltration method
have been described previously (41). For coinfiltrations, each A. tumefaciens
culture was grown to an optical density at 600 nm of 1 and mixed in equal
volumes prior to infiltration. Plants were observed and photographed under UV
light as described previously (41). Capped in vitro bacteriophage T7 RNA poly-
merase runoff transcripts were prepared (7) from SpeI-linearized pP2C2S,
pPVX-P0, and pPVX-P0fs and mechanically inoculated to celite-dusted lower
leaves of N. benthamiana.
RNA and protein analysis. High- and low-molecular-weight RNA fractions
were extracted from plant tissues, separated by agarose or polyacrylamide gel
electrophoresis, and transferred to nitrocellulose (Schleicher-Schuell or Hy-
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Bond-NX [Amersham] membranes) (9, 12). RNA samples for electrophoresis
were adjusted to the same concentration by spectrophotometry, and equal load-
ing was verified by visualization of the bands of ethidium bromide-stained rRNA
(high-molecular-weight RNA) or tRNA (low-molecular-weight RNA) on the
gels. Specific RNAs were detected by hybridization with antisense 32P-labeled
RNA probes corresponding to nt 240 to 664 of the GFP gene, the 3⬘-terminal 150
nt of PVX RNA, and either nt 1 to 376 or the 3⬘-terminal 196 nt of BWYV RNA.
Radioactive signals were detected by autoradiography and quantified using a
FUJIX BAS1000 phosphorimager and MacBAS image analysis software.
Reverse transcriptase PCR (RT-PCR) was performed essentially as described
previously (6). Reverse transcription was primed with an oligonucleotide com-
plementary to the 3⬘-terminal 20 nt of ORF 0 5⬘-terminally tagged with a nonviral
HindIII site. PCR on the reverse transcript was carried out using the above-
mentioned primer and a primer corresponding to the first 20 nt of the BWYV
RNA sequence plus a 5⬘-terminal nonviral HindIII site. The PCR fragment was
digested with HindIII and cloned into HindIII-cleaved pBS⫺ (Stratagene), and
the inserts of randomly selected clones were sequenced.
For Western (immuno)blot analysis, leaf material was ground to a powder in
liquid nitrogen and 200 mg of the powder was further ground with 500 ␮l of
concentrated (2⫻) gel loading buffer (18). Samples were boiled for 5 min and
then centrifuged for 5 min at 6,000 ⫻ g before gel loading. Proteins were
separated by electrophoresis through a denaturing 12% polyacrylamide gel (18)
by inserting an XmnI-EcoRI fragment (nt 104 to 1005) containing most of ORF
0 into pEA305⌬HindIII-3 (28) downstream and in frame with the bacteriophage
␭ cI cistron. The resulting recombinant plasmid, pBW.P9.8, overproduced the
CI-P0 fusion protein in bacteria, and the protein was purified and used to raise
a P0-specific antiserum essentially as previously described (28). Immunoreactive
proteins were visualized by enhanced chemiluminescence (Pierce or Roche).
RESULTS
Enhanced pathogenicity of a PVX chimera expressing
BWYV P0. Previous work has assigned tentative functions to all
of the recognized BWYV proteins except for P0, the protein
encoded by the 5⬘-proximal ORF on the viral RNA (Fig. 1A).
An earlier study showed that the BWYV P0 null mutants were
viable but accumulated 5- to 10-fold lower levels of progeny
RNA than the wild type in inoculated plants and protoplasts
(46). The stimulatory effect of P0 on virus accumulation levels
could indicate that the protein acts as a nonessential enhancer
factor for BWYV RNA replication. Alternatively, P0 could
augment virus accumulation indirectly, for example, by coun-
teracting a host defense mechanism such as PTGS.
If P0 counters a PTGS-related host defense, it would be
expected to augment pathogenicity and/or virus accumulation
levels not only of the cognate virus but of other, unrelated
viruses as well. This has been shown to be the case with HCPro
and CMV 2b, for which expression of each silencing suppressor
VOL. 76, 2002
protein from a PVX or tobacco mosaic virus chimera strongly
exacerbated symptoms compared to infection with the virus
alone (7, 21, 32). To determine whether expression of P0 can
similarly enhance the pathogenicity of another virus, the P0
coding region was inserted into the PVX-based vector pP2C2S
(2) to produce pPVX-P0 (Fig. 1B). Inoculation of N. benthami-
ana with pPVX-P0 transcripts produced necrotic lesions on the
inoculated leaves by 4 days postinfection (Fig. 2A, panel 3).
The appearance of viral RNA in the upper leaves (Fig. 2B, lane
3) was accompanied by necrosis in the petioles and veins and
then in mesophyll tissue (Fig. 2A, panel 3). Death of the upper
leaves and eventually of the entire plant resulted. Northern
blot analysis of RNA extracted from the upper leaves with an
ORF 0-specific probe revealed that the P0 coding sequence
was retained in the progeny viral RNA (Fig. 2B, lane 7).
In contrast, plants inoculated with the transcript of a vector
containing a frameshift-mutated version of the P0 gene
(pPVX-P0fs; Fig. 1B) developed faint chlorotic lesions on the
inoculated leaves and a mild mosaic on the upper leaves (Fig.
2A, panel 4), accompanied by the appearance of chimeric viral
RNA (Fig. 2B, lanes 4 and 8). These symptoms are similar to
those observed following infection with the empty vector
pP2C2S (Fig. 2A, panel 2). We conclude that expression of P0
can enhance the pathogenicity of an unrelated virus and that
this activity is independent of other BWYV genes.
BWYV P0 is a suppressor of PTGS. The foregoing observa-
tions prompted us to further test P0 for its ability to suppress
PTGS. This was done by use of a pBin19-based binary vector to
transiently express P0 (pBin-P0; Fig. 1C) in N. benthamiana
line 16c (7), which contains a transgene encoding GFP. The
leaves and stems of 16c plants normally fluoresce green under
long-wavelength UV light. However, expression of the GFP
transgene can be posttranscriptionally silenced by infiltrating a
lower leaf of a 16c plant with A. tumefaciens harboring a binary
vector (pBin-GFP; Fig. 1C) designed to transiently express a
second copy of the GFP transcript (41). For simplicity, we shall
refer below to each A. tumefaciens culture used in such infil-
trations by the name of the binary vector that it harbors.
The GFP silencing provoked in line 16c by infiltration of
pBin-GFP can be blocked by simultaneous infiltration of a
vector expressing a silencing suppressor protein (40; also see
reference 15). Thus, the infiltrated zone (the patch) on 16c
leaves coinfiltrated with pBin-GFP and pBin-HCPro (pBin-
GFP ⫹ pBin-HCPro) (Fig. 1C), where HCPro is the known
silencing suppressor of potato virus Y (7), became bright flu-
orescent green 5 days postinfiltration (p.i.) (Fig. 3A, panel 2).
Patches which had received pBin-GFP plus the empty vector
pBin61 had undergone silencing of the GFP signal by this time
and appeared deep red (Fig. 3A, panel 1). Similarly, when 16c
leaves where coinfiltrated with pBin-GFP plus pBin-P0, the
infiltrated patch also appeared bright fluorescent green by 5
days p.i. (Fig. 3A, panel 3), indicating that P0, like HCPro, has
silencing suppressor activity. Use of a vector (pBin-P0⫺) con-
taining a nontranslatable version of the P0 cistron in the infil-
tration mix did not provoke suppression of GFP silencing in
the patch (Fig. 3A, panel 4).
Northern blot analysis of GFP transcript levels confirmed
the visual observations of P0-mediated silencing suppression.
GFP transcript was abundant in the RNA of patches that had
received pBin-GFP ⫹ pBin-HCPro (Fig. 3B, top, lane 3) and
BWYV P0-MEDIATED PTGS SUPPRESSION 6817
pBin-GFP ⫹ pBin-P0 (Fig. 3B, top, lane 4), but its level was at
least 25-fold lower in patches treated with pBin-GFP ⫹ pBin61
(Fig. 3B, top, lane 2) or pBin-GFP ⫹ pBin-BWP0⫺ (Fig. 3B,
top, lane 5). Conversely, GFP-specific siRNAs (21- to 23-mer)
were readily detected by Northern blotting of RNA from
patches which had received pBin-GFP ⫹ pBin61 and pBin-
GFP ⫹ pBin-P0⫺ (Fig. 3B, bottom, lanes 2 and 5) but were 7
to 10 times less abundant in the patches which had received
pBin-GFP ⫹ pBin-HCPro and pBin-GFP ⫹ pBin-P0 (Fig. 3B,
bottom, lanes 3 and 4).
The high-intensity green fluorescence of the patches which
had received pBin-GFP plus either pBin-P0 or pBin-HCPro is
due to the fact that the GFP transcript in the patch is produced
from pBin-GFP as well as from the transgene and is protected
from PTGS by the presence of the silencing suppressor at 5
days p.i. Observations at later times, however, revealed that
there were significant differences between the behaviors of P0
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and HCPro in the coinfiltration assay. Thus, patches which
received the pBin-GFP ⫹ pBin-P0 mixture remained bright
fluorescent green for up to 25 days (Fig. 3C, panel 3 insert),
whereas the green fluorescence of patches that had received
pBin-GFP ⫹ pBin-HCPro had significantly faded by this time
(Fig. 3C, panel 2 insert). Another difference between P0 and
HCPro concerned the effect on systemic spread of PTGS.
Plants infiltrated with pBin-GFP ⫹ pBin-HCPro did not dis-
play GFP silencing in the upper leaves at 15 days p.i. (Fig. 3C,
panel 2) or later times up to 25 days (data not shown). In
contrast, the upper leaves of 16c plants that had received
pBin-GFP ⫹ pBin-P0 had undergone vein-proximal PTGS of
the GFP signal by 15 days p.i. (Fig. 3C, panel 3) and GFP
silencing spread to the entire leaf thereafter (not shown). This
rate of systemic silencing is similar to that observed in plants
which had received pBin-GFP ⫹ pBin61 (Fig. 3C, panel 1).
Silencing suppressor activity of P0 from other polerovi-
ruses. P0 is the most divergent protein among the polerovi-
ruses (26). Of sequenced poleroviruses, P0 of PLRV displays
the lowest (14%) sequence identity with P0 of BWYV while P0
of CABYV has the highest (23%), with most of the aligned
residues being in the C-terminal region (Fig. 4A). To deter-
mine if P0 from other poleroviruses can suppress GFP silenc-
ing in N. benthamiana line 16c, binary constructs expressing P0
of CABYV and PLRV were created. Both pBin-P0CAB and
pBin-P0PL suppressed GFP silencing in the coinfiltration patch
assay, although the relative intensities of the GFP fluorescence
in the patches (not shown) suggested that P0 of PLRV was a
much less efficient suppressor than the P0s of BWYV and
CABYV. Northern blot analysis confirmed the visual impres-
sion: the GFP transcript in the patch infiltrated with pBin-P0PL
⫹ pBin-GFP (Fig. 4B, lane 5) accumulated 5 to 10 times less
abundantly than those in the patches which had received pBin-
P0CAB ⫹ pBin-GFP (Fig. 4B, lane 4) and pBin-P0 ⫹ pBin-
GFP (Fig. 4B, lane 3). Note that the difference in silencing
suppressor activity of the different P0s does not correlate with
compatibility between the virus and the host used in the patch
coinfiltration assay, since PLRV, like BWYV, infects N.
benthamiana, whereas CABYV does not (19).
Full-length BWYV RNA does not provoke strong silencing
suppression in the coinfiltration assay. P0 is encoded by the
5⬘-proximal gene of BWYV RNA, and the protein is readily
detected when viral RNA is translated in vitro (38). Therefore,
6818 PFEFFER ET AL. J. VIROL.

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FIG. 2. A PVX-P0 chimera is hypervirulent on N. benthamiana. (A) N. benthamiana plants which have been mock inoculated (panel 1) or
inoculated with wild-type PVX transcript (panel 2), PVX-P0 (panel 3), or PVX-P0fs (panel 4). In panel 3, the arrow indicates an inoculated leaf
and the arrowhead indicates an upper leaf with severe systemic necrosis. Photographs were taken 15 days postinfection. (B) Northern (RNA) blot
analysis of viral RNA extracted from upper leaves of the plants shown in panel A and probed with either a PVX RNA-specific probe (lanes 1 to
4) or a probe specific for the BWYV P0 coding region (lanes 5 to 8). The upper band in each lane corresponds to genomic RNA (g). The lower
bands in lanes 2 to 4 are the 3⬘-proximal subgenomic RNAs sg1, sg2, and sg3 (see Fig. 1B). The extra sg1 band in lane 4 compared to lane 3 indicates
that a short deletion occurred in a fraction of the PVX-P0fs progeny RNA in this experiment.
VOL. 76, 2002 BWYV P0-MEDIATED PTGS SUPPRESSION 6819

we asked whether full-length BWYV RNA can elicit silencing

suppression in the patch coinfiltration assay. We have shown
previously that agro-inoculation of plants with pBinBW0, a
pBin19-based binary construct containing full-length BWYV
cDNA behind a 35S promoter, leads to production of viral
RNA transcripts which can then auto-replicate to give rise to a
typical virus infection (5, 20). For use in the patch assays,
pBinBW0 was slightly modified by addition of a 35S transcrip-
tion termination sequence downstream of the BWYV cDNA
to produce pBin-BW (Fig. 1C). Preliminary experiments dem-
onstrated that infiltration of N. benthamiana leaves with A.
tumefaciens harboring pBin-BW also triggered a virus infec-
tion. The infiltrated plants developed typical symptoms of
BWYV infection, viral RNA and capsid proteins were detected
in the patches and upper leaves, and the plants could serve as
an inoculum source for aphid-mediated transmission of
BWYV to other plants (data not shown).

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Coinfiltration of line 16c leaves with pBin-GFP plus
pBin-BW produced suppression of GFP silencing (Fig. 5A,
panel 3). However, the intensity of GFP fluorescence in the
patches was much lower than in patches treated in parallel with
the pBin-GFP ⫹ pBin-P0 combination (Fig. 5A, panel 1). This
difference was borne out by Northern hybridization analysis of
GFP transcript levels in the patches. The level of GFP tran-
script in the RNA from the pBin-GFP ⫹ pBin-BW patches
(Fig. 5B, lane 3) was reproducibly 2 to 3 times higher than in
patches infiltrated with pBin-GFP ⫹ pBin-P0⫺ (Fig. 5B, lane
2) or with pBin-GFP ⫹ pBin-BWP0⫺, a construct in which the
wild-type P0 sequence in the viral cDNA was replaced with the
P0⫺ sequence (Fig. 5B, lane 4). However, the level of GFP
transcript accumulation in the pBin-GFP ⫹ pBin-BW patches FIG. 4. The P0s of the poleroviruses CABYV and PLRV have
silencing suppressor activity. (A) Sequence similarity between the dif-
was 15- to 20-fold lower than in patches coinfiltrated with ferent P0s. The P0 sequences of CABYV and PLRV were aligned
pBin-GFP ⫹ pBin-P0 (Fig. 5B, lane 1). No suppression of GFP pairwise to the BWYV P0 sequence by using the MultAlign interface.
silencing was visible in the upper leaves of 16c plants infiltrated (B) Northern (RNA) blot analysis of high-molecular-weight RNA ex-
with pBin-GFP ⫹ pBin-BW at 2 to 3 weeks p.i. (data not tracted from the infiltrated patch of 16c leaves which had received a
shown), although it should be noted that very weak silencing mixture of bacteria harboring pBin-GFP plus one of the following:
empty vector pBin61 (lane 2), pBin-P0 (lane 3), pBin-P0CAB (lane 4),
suppressor activity confined to cells of the phloem compart- or pBin-P0PL (lane 5). The RNA loaded in lane 1 came from a non-
ment would have been difficult to detect. We conclude that the infiltrated 16c leaf. The blot was hybridized with a probe specific for
silencing suppressor activity displayed by P0 is much lower GFP mRNA.
when the P0 gene is introduced into the patch in its “normal”
environment in the viral genome than when it is translated
from a monocistronic construct.
P0 is poorly expressed from full-length BWYV RNA. In view antiserum. An ⬃29-kDa band that comigrated with a P0 in
of the failure of pBin-BW to support strong GFP silencing in vitro translation product (data not shown) was readily immu-
the agro-infiltrated zone, we next attempted to measure the nodetected in protein extracted from patches which had re-
level of P0 accumulation in leaves that were agro-infiltrated ceived pBin-GFP ⫹ pBin-P0 (Fig. 5C, lane 1) but could not be
with pBin-BW. To do this, total protein was extracted from detected in protein of patches which had received pBin-GFP ⫹
patch tissue 5 days p.i. and analyzed by Western (immuno)blot pBin-BW (Fig. 5C, lane 3). No P0 was detected in similar
analysis for P0 accumulation using a P0-specific polyclonal experiments when tissue that was agro-infiltrated with pBin-

FIG. 3. Expression of BWYV P0 in N. benthamiana line 16c suppresses PTGS of the GFP transgene. (A) Leaves of line 16c plants
agro-infiltrated with bacteria mixtures harboring pBin-GFP plus one of the following: empty vector pBin61 (panel 1), pBin-HCPro (panel 2),
pBin-P0 (panel 3), or pBin-P0⫺ (panel 4). Photographs were taken with long-wavelength UV light 5 days p.i. (B) Northern (RNA) blot analysis
of high-molecular-weight RNA (upper panel) and low-molecular-weight RNA (lower panel) extracted from the agro-infiltrated zone (patch) of 16c
leaves such as those shown in panel A. The patches had received bacterial mixtures harboring pBin-GFP plus one of the following: empty vector
pBin61 (lane 2), pBin-HCPro (lane 3), pBin-P0 (lane 4), or pBin-P0⫺ (lane 5). The RNA loaded in lane 1 came from a noninfiltrated 16c leaf.
The blots were hybridized with a probe specific for GFP mRNA. In the lower panel, the mobilities of 32P-labeled oligodeoxynucleotides of the
indicated length are shown to the left. (C) Line 16c plants 15 days p.i. of bacterial mixtures harboring pBin-GFP plus one of the following: pBin61
(panel 1), pBin-HCPro (panel 2), or pBin-P0 (panel 3). Upper leaves in panels 1 and 3 show vein-proximal silencing of the GFP signal. The inserts
show an agro-infiltrated leaf from each plant harvested at the same time.
6820 PFEFFER ET AL.
FIG. 5. Down-regulation of P0 levels and silencing suppressor ac-
tivity from genome-length BWYV RNA. (A) Leaves of 16c plants
agro-infiltrated with bacteria mixtures harboring pBin-GFP plus one of
the following: empty vector pBin-P0 (panel 1), pBin-P0⫺ (panel 2),
pBin-BW (panel 3), or Pbin-BWP0⫺ (panel 4). Photographs were taken
with long-wavelength UV light 5 days p.i. (B) Northern (RNA) blot
analysis of high-molecular-weight RNA extracted from the agro-infil-
trated patches of the leaves shown in panel A. The blot was hybridized
with a probe specific for GFP mRNA. (C) Western (immunoblot)
analysis of total protein from the infiltrated patches of the leaves
shown in panel A. The blot was probed with a polyclonal antiserum
specific for BWYV P0. The position of P0 is indicated to the right.
GFP ⫹ pBin-BW was sampled at other times (1 to 7 days p.i.;
data not shown).
To obtain a measure of the sensitivity of our immunodetec-
tion assay for P0, an experiment was carried out in which
different amounts of a protein extracted from a pBin-GFP ⫹
pBin-P0 patch extract were diluted into a healthy leaf extract
and then carried through the P0 Western blot procedure. The
results (not shown) indicated that the limit of detection for P0
in our assay is one-tenth the amount of P0 present in the
undiluted extract. Thus, the level of P0 accumulation in
patches that have been infiltrated with pBin-BW is at least 10
times lower than the level in leaves infiltrated with pBin-P0.
It may be argued that the low accumulation of P0 in the
J. VIROL.
pBin-BW patches relative to that in the pBin-P0 patches could
be due to differences in the amounts of transcript produced
from the two vectors. To test this hypothesis, Northern blots of
total RNA from patches which had received pBin-GFP ⫹
pBin-BW or pBin-GFP ⫹ pBin-P0 were hybridized with an
antisense RNA probe specific for the P0 ORF. In three inde-
pendent experiments there was 240 to 275 times more radio-
activity associated with the genome-length (⬃5.7-kb) RNA
species in the patches which had received pBin-BW (Fig. 6C,
lane 6) than with the ⬃0.8-kb transcript in the pBin-P0-infil-
trated tissue (Fig. 6C, lane 3). We do not know what fraction
of the 5.7-kb species corresponds to primary transcript (pro-
duced by transcription of pBin-BW DNA) and what fraction
corresponds to BWYV RNA produced by replication of the
primary transcript. Nor do we know if the 5.7-kb primary
transcript and BWYV RNA are translated with similar effi-
ciency (see Discussion). Nevertheless, the aforesaid observa-
Downloaded from jvi.asm.org at SCD UNIV LOUIS PASTEUR on June 4, 2010
tions rule out the possibility that the poor accumulation of P0
in the patches infiltrated with pBin-BW relative to the P0 level
in the pBin-P0-infiltrated patches is related in a straightfor-
ward manner to poor accumulation of the longer messenger
species.
The 5ⴕ-noncoding region of BWYV RNA regulates P0 ex-
pression levels. The foregoing observations illustrate that P0
accumulation levels are low when the protein is expressed from
full-length viral RNA compared to the levels attained from the
monocistronic construct pBin-P0. A significant difference be-
tween pBin-BW and pBin-P0 is that the transcribed sequence
immediately upstream of the P0 initiation codon in the former
corresponds to the BWYV 5⬘-noncoding region. In pBin-P0,
on the other hand, the corresponding sequence is an artificial
sequence of nonviral origin (see Fig. 6A for 5⬘-noncoding se-
quences of the different constructs). To determine the effect of
the upstream sequence on P0 expression, the 31-residue viral
5⬘-noncoding region was inserted into pBin-P0 immediately
upstream of the P0 initiation codon to produce pBin-P05⬘wt
(Fig. 6A). Leaf patches that were agro-infiltrated with pBin-
GFP ⫹ pBin-P05⬘wt accumulated GFP transcript to levels sim-
ilar to those found in patches infiltrated with pBin-GFP ⫹
pBin-P0 (Fig. 6B, lanes 3 and 4). However, P0 was not detect-
able in the pBin-GFP ⫹ pBin-P05⬘wt patches by Western blot
analysis (Fig. 6D, lane 4), although it was readily detected in
the pBin-GFP ⫹ pBin-P0 patches (Fig. 6D, lane 3). Thus,
insertion of the viral 5⬘-noncoding sequence upstream of the
P0 cistron reduced accumulation of P0 to below the level of
detection by Western blotting, although the amount of the
protein produced was still sufficient to efficiently suppress GFP
silencing (Fig. 6B, lane 4).
The sequence immediately surrounding an initiation codon
has a strong influence on the efficiency of translation initiation
in eukaryotes. A purine (usually A) in position ⫺3 relative to
the first nucleotide of the AUG is of primary importance for
efficient translation initiation, while a G in position ⫹4 has a
lesser but still significant stimulatory effect (17). Examination
of the different 5⬘-noncoding sequences (Fig. 6A) reveals that
the initiation codon in pBin-P0 is in a fairly good context with
an A at position ⫺3 but that the initiation codon context in
pBin-P05⬘wt (and in viral RNA) is poor (TTGATGC; P0 initi-
ation codon underlined). To determine if the poor expression
of P0 from the latter is a consequence of the suboptimal ini-
VOL. 76, 2002
FIG. 6. Effect of initiation codon context on expression of P0. (A) Se-
quence of the region immediately surrounding the P0 initiation codon in
the constructs. The first 33 residues of each transcript (designated
n30. . .ATC) is derived from the vector. The first residue of the 31-nt viral
5⬘-noncoding region in pBin-P05⬘wt and pBin-P05⬘opt is indicated by a dot
above the sequence. Underlined residues correspond to positions ⫺3 and
⫹4 relative to the A residue in the P0 initiation codon. Residues modified
in pBin-P05⬘opt are shown in bold. (B) Northern (RNA) blot analysis of
high-molecular-weight RNA extracted from agro-infiltrated patches of
16c leaves which had received bacterial mixtures harboring pBin-GFP
plus one of the following: empty vector pBin61 (lane 2), pBin-P0 (lane 3),
pBin-P05⬘wt (lane 4), pBin-P05⬘opt (lane 5), pBin-BW (lane 6), or pBin-
BW5⬘opt (lane 7). The RNA loaded in lane 1 came from a noninfiltrated
16c leaf. The blot was hybridized with a probe specific for GFP mRNA.
(C) Northern (RNA) blot analysis of the same samples shown in panel B,
using a probe specific for the P0 coding region. The bands corresponding
to 5.7-kb BWYV RNA (genomic RNA) and 0.8-kb P0 mRNA are labeled
to the right. (D) Western (immunoblot) analysis of total protein from the
infiltrated patches. The blot was probed with a polyclonal antiserum
specific for BWYV P0. The position of P0 is indicated to the right. Lane
5 was loaded with one-third the amount of protein extract loaded in the
other lanes.
BWYV P0-MEDIATED PTGS SUPPRESSION 6821
tiation codon context, a P0-expressing construct in which the
poor context was replaced with the optimal sequence AC-
CATGG was created (pBin-P05⬘opt; Fig. 6A). Leaf patches
infiltrated with pBin-GFP ⫹ pBin-P05⬘opt accumulated GFP
transcript to high levels (Fig. 6B, lane 5), and P0 was readily
detected by Western blot analysis (Fig. 6D, lane 5).
A mutation to optimize P0 translation initiation efficiency
was not stable during virus multiplication. The optimal P0
initiation codon context described above was also introduced
into full-length BWYV cDNA to produce pBin-BW5⬘opt. Leaf
patches infiltrated with this construct plus pBin-GFP accumu-
lated GFP transcript to high levels (Fig. 6B, lane 7), indicating
that more P0 was produced than when the wild-type construct
pBin-BW was used in the infiltration mix (compare lanes 6 and
7 in Fig. 6B). Nevertheless, P0 still did not accumulate in
amounts permitting its detection by Western blot analysis (Fig.
6D, lane 7).
Downloaded from jvi.asm.org at SCD UNIV LOUIS PASTEUR on June 4, 2010
Northern blot analysis of the RNA from the patches (at 5
days p.i.) and from upper leaves (21 days p.i.) of plants infil-
trated with pBin-BW5⬘opt revealed the presence of viral RNA
in both locations (Fig. 7A). The level of accumulation of viral
RNA in the patches infiltrated with pBin-BW and pBin-
BW5⬘opt (Fig. 7A, lanes 2 and 3) was ⬃100 times higher than in
the upper leaves (Fig. 7A, lanes 4 and 5; note that a longer
autoradiographic exposure time was used for lanes 4 and 5).
The difference in viral RNA accumulation levels in the patches
and upper leaves is presumably due to the fact that agro-
infiltration transfers DNA to almost all cells in the infiltrated
zone (23; O. Voinnet, personal communication), whereas virus
infection in the upper leaves is confined to cells of the phloem
compartment. The pBin-BW patches contained 5 times more
viral RNA than the pBin-BW5⬘opt patches (Fig. 7A, lanes 2 and
3; see also Fig. 6C, lanes 6 and 7). In the upper leaves the
relative accumulation levels were 2:1 (Fig. 7A, lanes 4 and 5).
Thus, optimization of the P0 initiation codon context does not
enhance viral RNA accumulation but, on the contrary, has the
opposite effect.
To assess the stability in planta of the codon context muta-
tions, total RNA was isolated from the upper leaves of three
plants infected with pBin-BW5⬘opt and the region around the
P0 initiation codon was cloned after amplification by specific
primer-directed RT-PCR. Sequence analysis of a number of
randomly selected clones revealed that the ACC triplet up-
stream of the ATG codon was conserved in all cases but that
the ATG codon itself had frequently undergone mutation (Fig.
7B). Similar analysis on progeny virus RNA from plants that
had been infiltrated with the wild-type construct pBin-BW
revealed no such modifications (data not shown). For plant 1,
the ATG codon had been mutated to ACG in all 11 clones
analyzed. For plant 2, the P0 ATG had been replaced with
either ACG, ATA, or GTG in 11 of the 14 clones analyzed and
3 clones retained the BW5⬘opt sequence (Fig. 7B). ACG and
GTG have been reported to initiate translation in plants but
with only about 15% the efficiency of translation initiation at
an ATG codon, while the translation initiation efficiency of
ATA was 5% that of ATG (10).
Analysis of the progeny viral RNA in plant 3 produced a
somewhat different result. Twelve of fourteen clones analyzed
contained a GTG or TTG substitution in the P0 initiation
codon, but, unexpectedly, the G residue in position ⫹4 had
6822 PFEFFER ET AL.
FIG. 7. BWYV RNA in which the P0 translation initiation codon
context has been optimized undergoes second-site mutations in the
initiation codon. (A) Northern (RNA) blot analysis of viral genome-
length RNA in N. benthamiana 16c plants agro-infiltrated with
pBin-BW (lanes 2 and 4) or pBin-BW5⬘opt (lanes 3 and 5). The RNA
shown in lane 1 was extracted from a healthy leaf. High-molecular-
weight RNA was isolated from patches 5 days after infiltration (lanes
2 and 3) or from upper leaves 21 days after infiltration (lanes 4 and 5).
The blot was hybridized with a radioactive probe complementary to the
3⬘-terminal 196 nt of BWYV RNA. The positions of the genomic RNA
and a subgenomic RNA (sgRNA) are shown to the left. An autora-
diographic exposure time of 1 h was used to visualize lanes 1 to 3, and
an exposure time of 16 h was used for lanes 4 and 5. The subgenomic
RNA was difficult to detect in lanes 4 and 5 due to rRNA interference
(30). (B) Sequence in the vicinity of the P0 initiation codon for cloned
RT-PCR products obtained from viral progeny RNA in the upper
leaves of three plants agro-infiltrated with pBin-BW5⬘opt. The number
of clones containing the indicated sequence relative to the number of
clones analyzed is shown to the right. Positions modified to optimize
codon context are underlined, and second-site mutations in the prog-
eny RNA are in bold type.
been replaced by T in all 14 clones (Fig. 7B). This creates a
TAA termination codon immediately following the P0 start
site, which will presumably terminate any translation that is
initiated there. Taken together, our findings thus indicate that
there is selection during propagation of virus derived from
pBin-BW5⬘opt for appearance of second-site mutations at the
P0 initiation codon (or immediately downstream) which will
strongly down-regulate accumulation levels of P0.
J. VIROL.
DISCUSSION
In this paper we have demonstrated that P0 of BWYV has
PTGS suppressor activity, a property shared by the P0s of at
least two other poleroviruses. The silencing suppressor activity
of P0 appears to be of the same basic type as that of the
paradigm silencing suppressor HCPro, although several possi-
bly significant differences were noted. Thus, the suppression
induced by pBin-P0 in the patch assay is long lasting, with the
infiltrated patch remaining bright fluorescent green for as long
as 25 days. In contrast, the silencing suppression induced by
HCPro faded over a period of 8 to 15 days p.i. This observation
suggests that P0 either is a more efficient suppressor of silenc-
ing or is more stable than HCPro in planta. Another distinction
between P0 and HCPro concerns their effects on systemic
silencing in the coinfiltration patch assay. In our hands, HCPro
blocked the appearance of PTGS in upper leaves of infiltrated
Downloaded from jvi.asm.org at SCD UNIV LOUIS PASTEUR on June 4, 2010
16c plants, whereas BWYV P0 had no effect on the rate of
GFP silencing in upper leaves (Fig. 3C). Our findings thus
suggest that P0 interacts with a component of the silencing
machinery responsible for cell-autonomous silencing but (un-
like HCPro) does not interfere with production and delivery of
the mobile silencing signal. It should be pointed out, however,
that the behavior of a silencing suppressor in the coinfiltration
patch assay does not necessarily reflect its behavior in other
systems. Thus, it has been observed that ␤-glucuronidase-
transgenic tobacco in which PTGS of ␤-glucuronidase had
been lifted by crossing with tobacco expressing an HCPro
transgene from tobacco etch potyvirus still produced a mobile
silencing signal which could operate across a graft junction
(24). (However, the reported ability of HCPro to traffic from
cell to cell [33] could complicate the interpretive comparison of
data from the two types of experiment.) Nevertheless, it is
evident that characterization of P0-transgenic plants will be of
interest in the future, although production of such plants may
be hindered by the reported cytopathic effects of P0 when
expressed in this manner (37).
When the monocistronic construct pBin-P0 was used in
agro-infiltration, P0 accumulated in amounts that were readily
detectable by immunoblot analysis of patch proteins and strong
PTGS suppressor activity was observed. Patches infiltrated
with the full-length viral cDNA construct pBin-BW, on the
other hand, displayed low but detectable PTGS suppressor
activity and did not accumulate amounts of P0 which we could
detect by immunoblot analysis. Our findings indicate that the
inefficient accumulation of P0 in the latter case is due in part
to the suboptimal context of the P0 initiation codon in the viral
RNA sequence, since replacement of the near-optimal P0 ini-
tiation context in pBin-P0 with the viral sequence in pBin-
P05⬘wt reduced P0 accumulation levels to below the level of
detection, i.e., at least 10-fold (Fig. 6D, lane 4). Nevertheless,
enough P0 was still produced from pBin-P05⬘wt to strongly
suppress PTGS of GFP (Fig. 6B, lane 4), which was not the
case for infiltration with pBin-BW (Fig. 6B, lane 6). Since the
P0 initiation codon context is identical in the two constructs, an
additional factor or factors must contribute to further diminish
P0 accumulation levels in the pBin-BW patches.
There are several (not necessarily exclusive) possibilities
which could account for the above observations. First, since the
pBin-BW transcript is infectious, cells in the infiltrated patches
VOL. 76, 2002
will contain other viral gene products in addition to P0. Pos-
sibly, one of these gene products down-regulates the transla-
tion or stability of P0. Another possibility is that the viral
replication process itself is responsible for the inefficient accu-
mulation of P0. For example, most of the viral RNA in patches
infiltrated with pBin-BW could be sequestered in replication
complexes and be inaccessible to the translation machinery.
Finally, we have shown that abundant amounts of viral ge-
nome-length RNA accumulate in the patches infiltrated with
pBin-BW. The bulk of this RNA is probably authentic viral
RNA (formed by replication of the primary transcript) and
hence will possess a 5⬘-terminal genome-linked protein (VPg
[25, 36]). The presence of VPg could impede ribosome access
to the P0 initiation codon on the viral RNA relative to the
nonreplicating transcripts produced from constructs such as
pBin-P0 and pBin-P05⬘wt, which are presumably capped. To
summarize, our observations indicate that the unfavorable P0
initiation codon context is partially responsible for the poor
expression of P0 from full-length viral RNA but that an addi-
tional factor (or factors), probably linked to the viral replica-
tion cycle, is important as well.
As noted in the introduction, null mutations in the P0 gene
strongly diminish polerovirus RNA accumulation levels during
infection (34, 46), supporting the idea that P0 at least partially
counteracts the host’s PTGS-like virus defense mechanism. In
view of the low accumulation levels of P0 in plants infected
with wild-type BWYV, we expected that alteration of the viral
RNA sequence to improve P0 expression (by optimization of
the P0 initiation codon context) would augment progeny viral
RNA accumulation relative to the wild type. This proved not to
be the case. Instead, plants infiltrated with pBin-BW5⬘opt con-
tained less progeny viral RNA than plants infiltrated in parallel
with wild-type virus (pBin-BW) and, even more significantly,
variants containing modifications in the P0 initiation codon
were abundant in the mutant virus population (Fig. 7B). In two
pBin-BW5⬘opt-infected plants that were subject to further anal-
ysis, an ACG, GTG, or ATA codon had replaced the P0 ATG
with high frequency. These non-ATG triplets can initiate trans-
lation in plants, but with only 5 to 15% the efficiency of an
ATG (10). In a third plant, the P0 initiation codon underwent
mutation with high frequency and an additional mutation cre-
ating a termination codon occurred just downstream.
At first thought, it may seem surprising that there is selection
in the pBin-BW5⬘opt progeny for sequence variants bearing
mutations that target the P0 initiation codon (or the adjacent
downstream codon) rather than for mutations that restore the
poor initiation codon context characteristic of the wild type. It
should be pointed out, however, that reversion to the wild-type
context would require mutations at two positions (⫺3 and ⫹4)
and, furthermore, the necessary substitutions involve nucleo-
tide transversions, which are known to occur with lower fre-
quency than transitions during viral RNA replication (see ref-
erence 6 and references therein). The observed P0 initiation
codon mutations, on the other hand, involve only one base
change, and in most cases the change is a transition.
Why overexpression of P0 is unfavorable to virus multipli-
cation has not yet been established, but it should be noted that
enhanced initiation of P0 translation on viral RNA, attained by
decreasing the number of ribosomes available to scan down-
stream, will lower production of P1 and P1-2, which would be
BWYV P0-MEDIATED PTGS SUPPRESSION 6823
expected to result in diminished virus replication rates. An-
other possibility is that high-level expression of P0 during the
infection perturbs host cell metabolism in a manner which is
deleterious for virus multiplication. Further experimentation
will be aimed at understanding how P0 expression levels are
regulated during a normal virus infection.
ACKNOWLEDGMENTS
S. Pfeffer and P. Dunoyer contributed equally to this work.
We thank Olivier Voinnet and Christiane Fritsch for numerous
helpful discussions, David Baulcombe for furnishing the GFP-trans-
genic plant line and constructs, and Frank van der Wilk for a clone
containing the PLRV P0 gene. Philippe Hammann was responsible for
DNA sequence analysis and Daniele Scheidecker provided technical
assistance.
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