2. 98B.M.Rothetal./VirusResearch102(2004)97–108
Table 1
Plant viral suppressors of RNA silencing
Genus Virus Suppressor Evidence Reference
Carmovirus Turnip crinkle virus (TCV) CP TCV infection does not reverse silencing. In agro-coinfiltration
assay, CP blocks sense and antisense induced local silencing and
prevents systemic silencing.
Qu et al. (2003) and Thomas et al. (2003)
Closterovirus Beet yellows virus (BYV) p21 Suppresses inverted repeat (IR) induced local silencing in
agro-coinfiltration assay. BYV p21 corresponds to BYSV p22.
Reed et al. (2003)
Beet yellow stunt virus (BYSV) p22
Cucumovirus Cucumber mosaic virus (CMV) 2b Infection with CMV or with PVX-2b vector blocks silencing.
Interferes with systemic signal (see text).
Li et al. (2002), see text
Tomato aspermy virus (TAV)
Furovirus Beet necrotic yellow vein virus (BNYVV) P14 Agro-coinfiltration assay with sense induced silencing. BNYVV
P14 corresponds to PCV P15.
Dunoyer et al. (2002)
Geminivirus African cassava mosaic virus (ACMV) AC2 Infection with ACMV, PVX-AC2, or PVX-C2 reverses silencing.
Blocks sense induced silencing in agro-coinfiltration assay. AC2
and C2 are homologs.
Dong et al. (2003), Voinnet et al. (1999)
and van Wezel et al. (2002)Tomato yellow leaf curl virus-China (TYLCV-C) C2
Hordeivirus Barley stripe mosaic virus (BSMV) ␥b RNA mediated cross protection between PVX-GFP and TMV-GFP
vectors is eliminated when ␥b is expressed from the PVX vector.
Yelina et al. (2002)
Poa semilatent virus (PSLV)
Pecluvirus Peanut clump virus (PCV) P15 PCV infection blocks silencing. p15 blocks local and delays
systemic sense-induced silencing in agro-coinfiltration assay.
Dunoyer et al. (2002)
Polerovirus Beet western yellows virus (BWYV) PO BWYV PO suppresses local but not systemic sense-induced
silencing in agro-coinfiltration assay. CABYV PO tested only on
local silencing.
Pfeffer et al. (2002)
Cucurbit aphid-borne yellows virus (CABYV)
Potexvirus Potato virus X (PVX) p25 PVX infection does not suppress silencing. In agro-coinfiltration,
p25 blocks systemic but not always local silencing (see text).
See text
Potyvirus Potato virus Y (PVY) HC-Pro Evidence from multiple types of assay. Does not block systemic
silencing in stable expression grafting assay, but does in
agro-coinfiltration assay (see text).
See text
Tobacco etch virus (TEV)
Sobemovirus Rice yellow mottle virus (RYMV) P1 Infection with PVX-P1 viral vector reverses silencing. Voinnet et al. (1999)
Tenuivirusa Rice hoja blanca virus (RHBV) NS3 Agro-coinfiltration assay of sense induced local silencing. Bucher et al. (2003)
Tombusvirus Tomato bushy stunt virus (TBSV) P19 Limited activity in reversal of silencing; strong activity in
agro-coinfiltration (see text). AMCV (artichoke mottled crinkle
virus) P19 also works as a suppressor.
Voinnet et al. (2003), Qu and Morris
(2002) and Takeda et al. (2002), see textCymbidium ringspot virus (CymRSV)
Tospovirusa Tomato spotted wilt virus (TSWV) NSs TSWV infection reverses silencing. In agro-coinfiltration, NSs
suppressed sense, but not IR, induced local and systemic silencing.
Bucher et al. (2003) and Takeda et al. (2002)
a Tospoviruses and tenuiviruses replicate in their insect vectors and in plants.
3. B.M. Roth et al. / Virus Research 102 (2004) 97–108 99
Fig. 1. RNA silencing pathway.
Zamore et al., 2000) and probably involves interactions
with other proteins, including an argonaute-like protein,
a dsRNA binding protein, and an RNA helicase (Tabara
et al., 2002). There are four Dicer-like (DCL) homologs
in Arabidopsis; however, the specific enzyme responsible
for siRNA production in plants has not yet been identified.
The siRNAs produced from a fully double-stranded RNA
substrate by Dicer have distinctive characteristics: they
represent both polarities and have two nucleotide 3 over-
hangs with 5 phosphate and 3 hydroxyl groups (Elbashir
et al., 2001a,b). In another ATP-dependent step (Nykanen
et al., 2001), the siRNAs are denatured and incorporated
into a multi-subunit endonuclease silencing complex called
RNA-induced silencing complex (RISC; Hammond et al.,
2000). Within the activated RISC, single-stranded siRNAs
act as guides to bring the complex into contact with com-
plementary mRNAs and thereby cause their degradation
(Bernstein et al., 2001; Elbashir et al., 2001a; Hammond
et al., 2000, 2001; Zamore et al., 2000).
One fascinating aspect of RNA silencing is that it is
non-cell-autonomous, and this feature may reflect the antivi-
ral nature of the process (for a recent review, see Mlotshwa
et al., 2002b). Virus infection usually starts with entry via
a small wound. The virus replicates in the initially infected
cell and then moves into adjacent cells, spreading from cell
to cell until it enters the vascular system, which allows rapid
movement to distant parts of the plant. In response, the host
plant initiates RNA silencing against the viral RNA and pro-
duces the mobile silencing signal. The mobile signal moves
along the same route the virus takes. Thus, the plant and the
virus enter a race. If the virus moves ahead of the signal, it
can establish infection when it enters the distant cells. How-
ever, if the mobile silencing signal gets there first, the virus
will enter the distant cell only to find itself targeted by RNA
silencing, and the infection will fail to become systemic.
One major class of viral suppressors comprises proteins that
block systemic silencing, suggesting the co-evolution of de-
fense and counter-defense between the host plant and the
invading virus at the level of systemic spread.
3. Functional assays used to identify suppressors of
RNA silencing
Three major approaches have been widely used to iden-
tify plant viral suppressors of RNA silencing: (1) transient
expression assays, (2) the reversal of silencing assay, and (3)
stable expression assays. These assays are described below
and shown in cartoon form in Figs. 2 through 4.
3.1. Transient expression assays—Agrobacterium
co-infiltration
This approach provides a rapid and easy test of sup-
pressor activity and is currently the technique most com-
monly used to identify viral suppressors (Llave et al., 2000;
Voinnet et al., 2000). The method makes use of a commonly
used bacterial pathogen of plants, Agrobacterium tumefa-
ciens. The Agrobacterium serves two purposes: one strain
is used to induce RNA silencing of a reporter gene (usually
green fluorescent protein (GFP)), and another strain is used
to express the candidate suppressor. The overall strategy is
to co-infiltrate mixtures of the two bacterial strains (one act-
ing to induce silencing, the other to suppress it) into a plant
leaf and then examine the infiltrated patch over time for si-
lencing of the reporter (Fig. 2A). Nicotiana benthamiana is
well suited to these assays because the leaves are easily in-
filtrated and produce high quantities of protein in response
to agro-infiltration. Non-transgenic N. benthamiana as well
as N. benthamiana expressing the reporter gene can be used.
In a typical experiment with Agrobacterium expressing GFP
as the inducer, the infiltrated patch initially expresses high
levels of GFP and glows bright green under ultraviolet (UV)
light (Fig. 3A, first panel). However, in three to five days,
the procedure triggers local RNA silencing, and the patch
4. 100 B.M. Roth et al. / Virus Research 102 (2004) 97–108
Fig. 2. Cartoon guide to transient expression assays. (A) Assay for suppressors of local silencing. (B) Assay for suppressors of systemic silencing.
Fig. 3. Agrobacterium-induced systemic silencing (A) and cartoon guide to reversal of silencing assay (B).
5. B.M. Roth et al. / Virus Research 102 (2004) 97–108 101
becomes a dirty red color under UV light due to a mixture
of green from residual GFP and red from chlorophyll in the
leaves (Fig. 3A, second panel). If the candidate suppressor
expressed from the co-infiltrated Agrobacterium interferes
with RNA silencing, the patch will remain bright green: if it
does not, the patch will turn red (Fig. 2A). Variations of this
technique include using Agrobacterium expressing inverted
repeat (IR) or viral cDNA constructs to induce silencing
in combination with or in place of Agrobacterium express-
ing a sense gene construct (Johansen and Carrington, 2001;
Voinnet et al., 2000).
Another variation of the transient expression approach has
been widely used to investigate the effect of silencing sup-
pressors on the mobile silencing signal (Fig. 2B). For this
purpose, infiltration is performed on a transgenic N. ben-
thamiana line that expresses GFP (line 16C, Ruiz et al.,
1998). Early experiments using an Agrobacterium strain ex-
pressing a sense GFP construct as the inducer of silencing
demonstrated that local induction of GFP silencing produced
a mobile silencing signal that moved out of the infiltrated
spot, along veins, and into surrounding tissues, leaving a trail
of red marking its path (Fig. 3A, third panel) and eventually
silencing GFP throughout the whole plant (Fig. 3A, fourth
panel) (Voinnet and Baulcombe, 1997). Thus, the ability
of suppressors to interfere with systemic silencing can be
tested in this system by co-infiltrating the inducer together
with a putative suppressor of silencing and observing long
enough to determine whether the plant becomes systemi-
cally silenced (Fig. 2B).
3.2. Reversal of silencing assay
This versatile assay can be used to first identify candi-
date viruses that may suppress silencing as a major line of
counter-defense (Fig. 3B; Brigneti et al., 1998). A modifi-
cation of the same technique can then be used to identify
the specific viral gene product that suppresses silencing. The
overall strategy is to infect a silenced plant with the candi-
date virus and determine whether the silenced phenotype is
reversed (Fig. 3B). The most common version of this tech-
nique uses the GFP-expressing transgenic N. benthamiana
line 16C described above. Soon after germination (at about
the four-leaf stage), the plant is infiltrated with Agrobac-
terium expressing GFP, which triggers local silencing and
then systemic silencing as described above. Ultimately, the
plant becomes completely silenced for GFP (red in UV light)
(Fig. 3A, fourth panel). At this point, the plant is inocu-
lated with the virus being tested for suppressor activity. If
virus infection allows the plant to express GFP, the infect-
ing virus likely encodes a suppressor of silencing (Voinnet
et al., 1999). Individual genes can be assayed for suppres-
sor activity using a modification of the reversal of silencing
technique that employs potato virus X (PVX). PVX is an ef-
ficient vector for systemic expression of heterologous genes
in N. benthamiana and does not, itself, encode a suppres-
sor of silencing that works in the reversal of silencing assay.
Thus, candidate suppressors from heterologous viruses can
be tested in the reversal of silencing assay by expressing
them from a PVX vector (Fig. 3B). Many viral suppressors
have been identified by expression from PVX in this manner
(Table 1; Voinnet et al., 1999).
3.3. Stable expression assays
In this approach, a stable transgenic line expressing a can-
didate suppressor of silencing (usually initially identified in
one of the previous two assays) is crossed to a series of
well-characterized transgenic lines silenced for a reporter
gene (Fig. 4A; Anandalakshmi et al., 1998; Kasschau and
Carrington, 1998). An advantage of this approach is that
it offers the opportunity to examine the effect of the sup-
pressor on different well-defined types of transgene-induced
RNA silencing, thus, providing information about the mech-
anism of suppression. The stable expression assays are also
well suited to investigate the role of suppressors in sys-
temic silencing using grafting (Fig. 4B; Guo and Ding, 2002;
Mallory et al., 2001, 2003). In these assays, the ability of a
plant to send a mobile silencing signal is assayed by graft-
ing an expressing line onto the top of it (the bottom plant
is called the rootstock and the expressing plant grafted onto
the top is called the scion) (Palauqui et al., 1997). If the sup-
pressor of silencing in the rootstock blocks either the pro-
duction or movement of the systemic silencing signal, then
the transgene in the scion will continue to be expressed. If
a suppressor does not block the systemic silencing signal
in either of these ways, then the transgene in the scion will
become silenced (Fig. 4B).
4. Mechanism of suppression
The assays discussed above have proven extremely use-
ful as rapid and sensitive methods to identify suppressors
of silencing. They have also enabled rudimentary charac-
terization of suppressor mechanism, but conflicting results
from different assays have made it difficult to draw firm con-
clusions for many suppressors. Interestingly, the currently
known suppressors share no obvious similarities at either
the nucleic acid or the protein level, perhaps reflecting dif-
ferences at the mechanistic level as well. At present, two
major classes of suppressor action have been identified.
4.1. Suppressors that affect small RNA metabolism
Many suppressors reduce the accumulation of siRNAs,
raising the possibility that silencing is blocked at the step
at which Dicer processes the dsRNA that triggers silencing.
Thus, it may be that many suppressors prevent silencing by
blocking production of the siRNAs that provide the sequence
specificity of the process. A second mechanism involving
siRNAs is exemplified by the suppressor P19, which has
been shown to bind siRNAs, perhaps sequestering them and
6. 102 B.M. Roth et al. / Virus Research 102 (2004) 97–108
Fig. 4. Cartoon guide to stable expression assays. (A) Genetic crosses with silenced transgenic lines. (B) Grafting assay for suppression of systemic
silencing.
thereby blocking their function (Silhavy et al., 2002). Inter-
estingly, the effect of suppressors on small RNA metabolism
may extend to other types of small regulatory RNAs. For
example the silencing suppressor HC-Pro affects the accu-
mulation not only of the siRNAs that mediate silencing but
also of endogenous microRNAs (miRNAs), which have been
implicated in development (Kasschau et al., 2003; Mallory
et al., 2002b). Surprisingly, although HC-Pro blocks the ac-
cumulation of siRNAs, it enhances the accumulation of miR-
NAs (Mallory et al., 2002b). Even more interesting, there
is evidence that HC-Pro might block the function of miR-
NAs (Kasschau et al., 2003). It remains to be seen if other
viral suppressors also affect the miRNA pathway, perhaps
using that pathway to turn off expression of genes required
for silencing.
4.2. Suppressors that affect systemic silencing
Systemic silencing can be assayed in either transient ex-
pression experiments or in experiments with stably trans-
formed transgenic lines (Figs. 2B and 4B). Many suppressors
have been demonstrated to block systemic silencing in at
least one of these assays (Table 1). Because of the conflicting
results sometimes obtained with different assays, the most
successful attempts to determine whether a suppressor pri-
marily affects systemic silencing have used a multi-pronged
approach rather than relying on just one type of assay.
For example, although HC-Pro did not block systemic si-
lencing in grafting experiments using stable transgenic lines
(Mallory et al., 2001, 2003), it interfered with systemic si-
lencing in the transient expression assay (Hamilton et al.,
2002). Because HC-Pro is a powerful suppressor of local
silencing in all assays, these results suggest that HC-Pro pri-
marily affects local silencing, but also has a smaller effect
on systemic silencing. In contrast, CMV 2b primarily targets
systemic silencing because it blocks movement of the sig-
nal in many assays, but has a lesser effect on local silencing
(Brigneti et al., 1998; Bucher et al., 2003; Guo and Ding,
2002). It may not be surprising that a primary effect of a
particular suppressor on one aspect of silencing could lead,
perhaps through feedback mechanisms, to secondary effects
on other parts of the pathway, thereby making it appear that
the suppressor works at multiple points.
4.3. Conflicting results using different assays
Why do different assays give different results when look-
ing at effects of viral suppressors on systemic silencing?
The major conflicts are seen with stable expression assays
versus transient ones and probably reflect a number of in-
trinsic differences between the systems. Transient expres-
sion assays and the reversal of silencing assay use Agrobac-
terium to induce silencing. Because Agrobacterium is a plant
pathogen, infiltration likely induces plant defensive and bac-
terial counter-defensive interactions, which might modify
the activity of some viral suppressors. Thus, attempts to
compare suppressor activities or to understand the mecha-
nism of action of the different suppressors are complicated
7. B.M. Roth et al. / Virus Research 102 (2004) 97–108 103
by the largely unknown effects of Agrobacterium on the
system. Similarly, the reversal of silencing assay involves
virus infection and likely induces accompanying defense
and counter-defensive responses that complicate the inter-
pretation of results. Further sources of variation include dif-
ferences in the level and time course of expression of the
silencing suppressor and silencing inducer in the different
systems.
Of the three widely used types of assay (Section 3), stable
expression assays are the ones most free of complications
due to extraneous pathogens. In addition, use of the same
well-characterized silenced transgenic line to test the effects
of different viral suppressors affords a degree of standard-
ization from lab to lab not yet found with transient expres-
sion assays. It is important to use well-characterized lines
to compare suppressor activities because different types of
transgenes trigger silencing in different ways (Vance and
Vaucheret, 2001).
Understanding the basis of the conflicting results given
by the currently widely used assays will likely offer con-
siderable insight into the mechanisms of suppressor action.
Alternative approaches such as yeast two-hybrid, biochemi-
cal, and localization studies (see Section 5) are increasingly
being used to investigate the mechanisms of action of the
different viral suppressors of silencing and will undoubtedly
help clear up the confusion.
5. Better studied suppressors of silencing
5.1. Cucumber mosaic virus (CMV) 2b
The CMV 2b protein was one of the first identified sup-
pressors of RNA silencing and also one of the best studied
from a mechanistic standpoint. The initial indication that
CMV 2b suppressed silencing came from the reversal of
silencing assay in which 2b expressed from PVX could
prevent the initiation of silencing but could not reverse si-
lencing that was already established (Brigneti et al., 1998).
That early result raised the possibility that 2b might block
systemic silencing. Subsequently, stable expression assays
and grafting experiments provided an elegant demonstration
that 2b blocks the movement of the systemic silencing signal
(Guo and Ding, 2002). The stable expression assays made
use of a well-characterized silenced tobacco line called
6b5 (Elmayan and Vaucheret, 1996), which is silenced for
the reporter gene GUS and is a model for sense-transgene
induced RNA silencing. The 6b5-GUS locus triggers si-
lencing even when it is hemizygous, produces high levels
of GUS siRNAs, and is highly effective in the production
and transmission of a systemic silencing signal as demon-
strated by grafting experiments. A genetic cross between
line 6b5 and a stable transgenic tobacco line expressing
CMV 2b established that 2b could partially suppress local
sense-transgene induced silencing: offspring of the cross
accumulated GUS mRNA, but GUS siRNA accumulation,
although reduced, was not eliminated. However, grafting
experiments definitively demonstrated that CMV 2b blocks
the movement of the systemic silencing signal. In the first
protocol, 6b5 rootstocks suppressed for silencing by CMV
2b did not silence grafted GUS-expressing scions, showing
that expression of CMV 2b in the rootstocks prevented
production or transmission of the systemic silencing signal
(Fig. 5A, first panel). In the second protocol, a small spacer
of transgenic tobacco expressing CMV 2b, grafted between
a GUS-silenced 6b5 rootstock and a GUS-expressing scion,
blocked systemic silencing (Fig. 5A, second panel). Thus,
CMV 2b prevents transmission of the systemic silencing
signal in a stable expression assay.
The experiments described above provide compelling ev-
idence that the mechanism of action of CMV 2b, at least in
part, is to block systemic silencing. Guo and Ding (2002)
also report similar conclusions based on Agrobacterium
co-infiltration experiments. Paradoxically, the same type of
co-infiltration experiments in another laboratory produced a
different result: CMV 2b delayed but did not block systemic
silencing (Hamilton et al., 2002). A possible cause of this
discrepancy is that the two labs were using different ratios
of silencing inducer to suppressor in the co-infiltrations
(S.W. Ding, personal communication), suggesting that a
standardized methodology for co-infiltration assays would
help resolve some of the current inconsistencies.
How does CMV 2b protein block the movement of the
mobile silencing signal? The ability of 2b protein to pre-
vent the transmission of the signal suggests that it either se-
questers or inactivates the signal in the phloem stream. One
possibility is that 2b acts directly by binding to the signal.
However, the finding that 2b localizes to the nucleus (Lucy
et al., 2000) suggests that the suppressor acts indirectly, per-
haps by activating one or more processes that subsequently
affect the signal.
5.2. Potyviral helper-component protease (HC-Pro)
HC-Pro was the first identified suppressor of RNA silenc-
ing. The original reports demonstrated that it suppresses both
transgene- and virus-induced silencing (Anandalakshmi
et al., 1998; Kasschau and Carrington, 1998). In contrast to
CMV 2b protein, it is able to reverse established silencing
in the reversal of silencing assay (Brigneti et al., 1998), sug-
gesting that the two suppressors work at different steps in the
silencing pathway. Although HC-Pro alone has suppressor
activity (Anandalakshmi et al., 1998; Brigneti et al., 1998),
it is frequently expressed as the proteinase 1 (P1)/HC-Pro
polyprotein in suppression of silencing studies. The
P1/HC-Pro construct is the N-terminal portion of the natural
viral polyprotein and allows HC-Pro to be proteolytically
processed just as when expressed from the viral genome.
Some evidence suggests that P1 might enhance HC-Pro
activity as a suppressor of silencing (Pruss et al., 1997).
A variety of approaches, including both transient and sta-
ble expression assays, have been used to investigate how
8. 104 B.M. Roth et al. / Virus Research 102 (2004) 97–108
Fig. 5. Cartoon guide to (A) CMV 2b and (B) HC-Pro grafting experiments.
HC-Pro suppresses RNA silencing. Despite some conflicting
results, at least one common finding has emerged: HC-Pro
affects small RNA metabolism. Exactly how HC-Pro exerts
its effect on small RNAs and how this leads to suppression
of silencing are questions that remain to be resolved. The
interaction(s) of HC-Pro and its cellular effector(s) probably
occur in the cytoplasm because HC-Pro is found primarily
in cytoplasm (Mlotshwa et al., 2002a).
5.2.1. HC-Pro and small RNAs
HC-Pro has been reported to alter the accumulation of
several classes of small RNAs: the siRNAs that direct RNA
degradation during silencing, a novel class of slightly-larger
small RNAs of unknown function, and the endogenous mi-
croRNAs (miRNAs) that have been implicated in regulation
of development. In stable expression assays in tobacco,
HC-Pro has been reported to suppress three classes of
transgene-induced RNA silencing, in each case interfering
with the accumulation of siRNAs (Mallory et al., 2001,
2002b). Similarly, siRNA accumulation is dramatically
reduced during HC-Pro suppression of silencing in tran-
sient expression assays (Johansen and Carrington, 2001;
Llave et al., 2000). In stable expression assays in tobacco,
HC-Pro suppression of IR transgene-induced silencing
or amplicon-transgene-induced silencing—but not sense
transgene-induced silencing—resulted in the accumulation
of a novel class of slightly-larger small RNAs (Mallory
et al., 2002b). Similar slightly-larger small RNAs have also
been observed in transient expression assays (Hamilton
et al., 2002). The function of this class of small RNAs is
not clear; however, they do not appear to act as siRNAs
because their presence is not correlated with RNA degra-
dation (Hamilton et al., 2002; Mallory et al., 2002b). They
correlate with systemic silencing in transient expression
assays (Hamilton et al., 2002) but not in stable expression
assays (Mallory et al., 2002b). Finally and surprisingly,
the accumulation of endogenous miRNAs is increased in
both tobacco and Arabidopsis transgenic lines that express
HC-Pro (Kasschau et al., 2003; Mallory et al., 2002b),
suggesting a more general role in the biogenesis of small
regulatory RNAs. A recent report that HC-Pro enhances
the stability of several miRNA target messages raises the
possibility that HC-Pro affects the function as well as the
biogenesis of small RNAs (Kasschau et al., 2003).
5.2.2. HC-Pro and systemic silencing
In stable expression experiments utilizing grafting,
HC-Pro failed to block systemic silencing (Fig. 5B, first
panel; Mallory et al., 2001, 2003). Three different trans-
genic tobacco lines were used as rootstocks, representing
three different means of inducing silencing: sense trans-
gene, inverted repeat transgene, and amplicon transgene.
In the case of the amplicon-transgene induced silencing,
in which the transgene is the cDNA of a replication com-
petent virus, HC-Pro actually promoted systemic silencing
(Mallory et al., 2003). These results suggest that production
and transmission of the systemic silencing signal are largely
unaffected by HC-Pro, implying that HC-Pro suppression
of silencing occurs downstream of the signal. To test that
hypothesis, the effect of HC-Pro on perception of and/or
response to the silencing signal was tested in additional
grafting experiments (Fig. 5B, second panel; Mallory et al.,
2001). A silenced GUS line previously shown to silence
GUS systemically across a graft junction was used as root-
stock, and the scion was a line that expressed both GUS
and HC-Pro. The scion failed to become silenced for GUS,
9. B.M. Roth et al. / Virus Research 102 (2004) 97–108 105
showing definitively that HC-Pro works downstream of the
systemic silencing signal: The rootstock is known to send
a signal; therefore, in the presence of HC-Pro, the scion
either fails to perceive the signal or fails to respond to it.
Although the grafting experiments described above make
a convincing case that HC-Pro does not block the systemic
silencing signal, results from Agrobacterium co-infiltration
assays suggest that this suppressor does interfere with sys-
temic silencing (Hamilton et al., 2002). There are a num-
ber of possible reasons for obtaining different results with
the two assays. First, the stable transgenic lines express the
P1/HC-Pro polyprotein, whereas the transient assay exper-
iments used a construct that encodes only HC-Pro and has
a methionine substituted for the natural N-terminal amino
acid of the protein. The protein produced in the transient as-
say, therefore, is not identical to HC-Pro produced from the
virus. Thus, it is possible that the difference between the two
assays reflects this small difference in the proteins. Indeed,
earlier experiments expressing either P1/HC-Pro or HC-Pro
(with an N-terminal methionine) from a PVX vector reported
a dramatic difference in the effect on PVX replication (Pruss
et al., 1997). A second possibility is that the result in the
transient assay depends on the ratio of inducer to suppres-
sor, as suggested for CMV 2b protein (see Section 5.1), and
this possibility could be tested by using a range of ratios.
A more exciting possibility is that the discrepancy reflects
an intrinsic difference between the two assays, as discussed
in Section 4.3. Thus, HC-Pro might block systemic silenc-
ing in some circumstances, but not in others, and identify-
ing the important variables will help in our understanding
of HC-Pro suppression of silencing.
5.2.3. HC-Pro interacting proteins
If HC-Pro works by interacting with components or regu-
lators of the silencing machinery, identification of plant pro-
teins that interact with HC-Pro should provide clues about
its mechanism of action. Using the yeast two-hybrid sys-
tem, an HC-Pro-interacting protein called regulator of gene
silencing calmodulin-like protein (rgsCaM) was identified
(Anandalakshmi et al., 2000). Like HC-Pro, rgsCaM could
reverse silencing when expressed from PVX in the rever-
sal of silencing assay. In addition, when over-expressed in
stable expression experiments, rgsCaM caused a develop-
mental phenotype typical of HC-Pro-expressing transgenic
lines and interfered with virus induced gene silencing. Be-
cause calmodulins are classic signaling molecules, these re-
sults raise the possibility that HC-Pro suppresses silencing
indirectly via a signal cascade involving rgsCaM.
5.3. Tombusvirus P19
This protein has been an exciting addition to the reper-
toire of plant viral suppressors. Initially described in the re-
versal of silencing assay (Voinnet et al., 1999), it appeared
to be a weak suppressor, only reversing silencing in the re-
gion of veins. Similarly, P19 delayed but did not prevent
virus-induced gene silencing and did not suppress silencing
induced by a defective interfering RNA that accumulates to
high levels (Qiu et al., 2002). In transient expression as-
says, however, P19 is a star, blocking both local and sys-
temic silencing and apparently eliminating all small RNAs
(Hamilton et al., 2002; Silhavy et al., 2002; Takeda et al.,
2002; Voinnet et al., 2003). Interestingly, biochemical stud-
ies have shown that P19 binds siRNAs and that binding de-
pends on characteristics of RNase III products (dsRNAs with
two nucleotide 3 overhangs) (Silhavy et al., 2002). This re-
sult raises the possibility that P19 suppresses silencing by
sequestering siRNAs, thereby preventing their incorporation
into the RISC complex to serve as guides. This is a novel
mechanism among suppressors, and because it theoretically
stems from an intrinsic property of the protein to bind func-
tional siRNAs, it is possible that P19 could interfere with
silencing in a broad range of different plants (and even other
organisms).
5.4. Potato virus X (PVX) p25
Three of five potexviruses tested in the reversal of silenc-
ing assay suppressed RNA silencing, although PVX was
one that did not show suppressor activity (Voinnet et al.,
1999). Paradoxically, however, PVX is the only potexvirus
for which a suppressor of RNA silencing has subsequently
been characterized. In grafting experiments designed to sep-
arate movement of the systemic silencing signal from that
of the virus in virus induced silencing (VIGS), Voinnet et al.
(2000) unexpectedly found that PVX infection failed to pro-
duce systemic silencing independent of the presence of virus,
suggesting that a PVX-encoded protein interferes with pro-
duction or transmission of the systemic signal. Protein p25,
which is required for cell-to-cell movement of potexviruses,
was identified as the culprit in a set of experiments in which
deletion derivatives of replication competent PVX-GFP viral
vector constructs were agro-infiltrated into plants express-
ing GFP. The PVX-GFP constructs were restricted to ini-
tially infiltrated cells because they all lacked coat protein,
which is also required for potexviral movement. Induction
of systemic silencing in these experiments, therefore, de-
pends entirely on the systemic signal. Only viral constructs
from which p25 expression had been eliminated induced
widespread systemic silencing in all plants.
Agro-coinfiltration experiments showed that p25, with-
out any other PVX protein, was sufficient to block sys-
temic silencing. Moreover, p25 blocked systemic silencing
whether silencing was induced by a simple transgene con-
struct (35S-GFP) or by a replication competent PVX-GFP
construct. The effect of p25 on local silencing in these ex-
periments, however, depended on the nature of the construct
used to induce silencing. Although p25 suppressed local si-
lencing in agro-coinfiltration assays with 35S-GFP, it did
not suppress local silencing induced by infiltration of repli-
cation competent PVX-GFP constructs. p25 suppression of
systemic silencing in agro-coinfiltration assays is correlated
10. 106 B.M. Roth et al. / Virus Research 102 (2004) 97–108
with the absence of a slightly-larger class of small RNAs,
possibly indicating the step in the pathway affected by p25
(Hamilton et al., 2002).
Although these transient expression experiments make a
convincing case that PVX p25 blocks systemic silencing,
a different result was obtained in experiments using trans-
genic plants constitutively expressing white clover mosaic
potexvirus (WClMV) p25. Agro-infiltration of a 35S-IR con-
struct systemically silenced these plants (Foster et al., 2002).
Moreover, the systemic silencing reverted a severe develop-
mental phenotype, indicating that the systemic signal was
able to enter the shoot apical meristem. Thus, WClMV p25
did not prevent systemic silencing in this experimental sys-
tem. Whether the difference in the results obtained with the
PVX and WClMV p25 proteins reflects differences in the
assays or in the activities of the proteins has not yet been
determined.
6. Do all plant viruses have suppressors of silencing?
The discovery that plants have a generalized antiviral de-
fense mechanism triggered by dsRNA has revolutionized
our thinking about plant–virus interactions. In the euphoric
aftermath of the initial identification of plant viral suppres-
sors of silencing, a popular expectation was that most or all
plant viruses would encode a suppressor of RNA silencing.
Although many different viral suppressors have been iden-
tified, the fact that HC-Pro helps so many different viruses
suggests that a lot of viruses do not effectively suppress si-
lencing. Such viruses may have evolved other ways to try
to avoid silencing, such as by replicating within spherules
in the ER (Schwartz et al., 2002), where the dsRNA is hid-
den, or by replicating and moving rapidly enough to outrun
the mobile silencing signal. Furthermore, plants have other
defense mechanisms, and silencing might not be the major
threat for all viruses. Some viruses, therefore, may well have
suppressors of other defense pathways.
7. Suppressors of silencing as tools
The finding that certain viral proteins suppress RNA si-
lencing has provided a new tool for technologies utilizing
genetically modified plants and is, therefore, of practical sig-
nificance. Many biotechnological applications are impaired
by RNA silencing, and suppressors of silencing can be used
to attain consistent, high-level expression of transgenes in
plants (Mallory et al., 2002a; Voinnet et al., 2003). With si-
lencing under control, transgenic plants can be engineered to
produce a range of transgene expression: moderate levels of
expression to produce desired plant traits or very high-level
expression to use the plant as a factory producing pharma-
ceuticals, vaccines or other high-value gene products.
Perhaps more importantly, viral suppressors of silencing
also provide unique tools to understand the mechanism of
RNA silencing. Much of what is currently known about
the RNA silencing pathway comes from elegant in vitro
and genetic studies in organisms other than plants (for a
recent review see Tijsterman et al., 2002). In plants, tra-
ditional genetic approaches have led to the identification
of a number of cellular genes required for RNA silencing
(Dalmay et al., 2000, 2001; Fagard et al., 2000; Mourrain
et al., 2000). Surprisingly, however, all of these genes are
required for sense-, but not IR-transgene induced silencing
(Boutet et al., 2003). The plant viral suppressors, many of
which appear to work downstream of dsRNA, provide a
novel means of entry into parts of the silencing pathway
that are not easily accessible by genetic means. The cur-
rently known suppressors appear to work at different steps
in silencing, thereby providing access to a number of points
in the pathway where silencing can be controlled.
Identifying host proteins that interact with a viral sup-
pressor of RNA silencing is one very promising approach
that is being used to take advantage of viral suppressors
to elucidate the silencing pathway. The yeast two-hybrid
system has been used to find tobacco proteins that in-
teract with HC-Pro, identifying a calmodulin-related pro-
tein called rgsCaM that suppresses RNA silencing when
over-expressed (Anandalakshmi et al., 2000). This result
suggests that a calcium controlled signal transduction path-
way involving rgsCaM is one of the mechanisms regulating
RNA silencing. Intriguingly, in the case of geminiviruses,
yeast two-hybrid studies have identified SNF1 kinase as a
cellular interactor of the tomato golden mosaic virus AL2
protein (Hao et al., 2003). AL2 is a homologue of the
ACMV and TYLCV-C suppressors of silencing. Whether
the interaction of AL2 with SNF1, which is a regulator of
metabolism in response to stress, plays any role in suppres-
sion of silencing is unknown as yet.
The potential of using viral suppressors to help understand
the mechanism of RNA silencing in plants is largely un-
tapped, and these studies promise to be an exciting and fer-
tile area of research. The recent identification of a viral sup-
pressor that works in animal cells (Li et al., 2002) offers the
possibility that such proteins may provide a similar tool to
understand the silencing pathway in other organisms as well.
Note added in proof
The tomato mosaic virus replication protein has recently
been reported to suppress RNA silencing (Kubota, K., Tsuda,
S., Tamai, A., Meshi, T., 2003. Tomato mosaic virus repli-
cation protein suppresses virus-targeted posttranscriptional
gene silencing. J. Virol. 77, 11016–11026).
Acknowledgements
VBV gratefully acknowledges support from the USDA
Competitive Grants Program, NIH, and Dow AgroSciences
LLC.
11. B.M. Roth et al. / Virus Research 102 (2004) 97–108 107
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