Previous Article | Next Article 
Journal of Virology, December 2001, p. 12288-12297, Vol. 75, No. 24
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.24.12288-12297.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Double-Stranded RNA-Mediated Interference with
Plant Virus Infection
F.
Tenllado and
J. R.
Díaz-Ruíz*
Departamento de Biología de Plantas,
Centro de Investigaciones Biológicas, CSIC, Velázquez
144, 28006 Madrid, Spain
Received 21 May 2001/Accepted 18 September 2001
 |
ABSTRACT |
Double-stranded RNA (dsRNA) has been shown to play a key role as an
inducer of different interference phenomena occurring in both the plant
and animal kingdoms. Here, we show that dsRNA derived from viral
sequences can interfere with virus infection in a sequence-specific
manner by directly delivering dsRNA to leaf cells either by mechanical
inoculation or via an Agrobacterium-mediated transient-expression assay. We have successfully interfered with the
infection of plants by three viruses belonging to the tobamovirus, potyvirus, and alfamovirus groups, demonstrating the reliability of the
approach. We suggest that the effect mediated by dsRNA in plant virus
infection resembles the analogous phenomenon of RNA interference
observed in animals. The interference observed is sequence specific, is
dose dependent, and is triggered by dsRNA but not single-stranded RNA.
Our results support the view that a dsRNA intermediate in virus
replication acts as efficient initiator of posttranscriptional gene
silencing (PTGS) in natural virus infections, triggering the initiation
step of PTGS that targets viral RNA for degradation.
| |
INTRODUCTION |
Gene sequences derived from
different plant viruses have been introduced into a wide variety of
plant species to produce transgenic plants protected against
virus infection. In a number of cases, it is known that the mechanism
of resistance is a posttranscriptional, RNA-mediated process that
targets both the viral RNA and the transgene mRNA for degradation in a
sequence-specific manner (11, 19). RNA-mediated virus
resistance is a manifestation of posttranscriptional gene silencing
(PTGS), a more general phenomenon which was first described as a
coordinated and reciprocal inactivation of host gene and transgenes
encoding the same sense RNA (reviewed in references 33 and
41). More recently, three components in the dynamics of
PTGS have been proposed: initiation, propagation of a systemic silencing signal, and maintenance (27, 31). For the last
step, a nuclear component sharing sequence homology with the target mRNA is absolutely required (8, 28). PTGS also takes place in nontransgenic plants as a natural defense mechanism against virus
infection. According to this idea, following the onset of virus
replication, viral RNA or a derivative would be perceived as a
pathogenic agent by the host, triggering a process that could be
responsible for the progressive slowdown in virus accumulation observed
at late stages in the infection process of some viruses (7, 29,
30). In this scenario, viruses counteract the host response by
encoding suppressors of PTGS in their genomes, which seems to be a
widespread strategy used by RNA and DNA viruses of plants
(42).
RNA interference (RNAi) was originally observed in Caenorhabditis
elegans, where injection of double-stranded RNA (dsRNA) leads to
PTGS of homologous sequences (13). dsRNA blocks specific gene expression even when expressed by bacteria fed to the worms (38) or transcribed from transgenes carrying an internal
inverted repeat (36). Recently, RNAi has been reported in
a wide variety of animals, and in these cases the organism
exhibits gene-specific phenocopies of loss-of-function mutations
(2, 14). A similar phenomenon in Neurospora
crassa is termed quelling (5). Considering that the
interference process occurs posttranscriptionally and involves mRNA
degradation (22, 26), RNAi, quelling, and PTGS in plants
have been proposed to be related phenomena that could play an important
biological role in protecting the organism's genome against foreign
nucleic acids. Moreover, the analysis of mutants defective in those
processes has revealed the involvement of similar gene products in
different organisms (6, 9, 12, 23, 34).
A growing body of evidence suggests that plants, animals, and yeasts
share related mechanisms of specific degradation of RNAs in which
double-stranded forms of RNA are involved (5, 33). It has
been shown that PTGS in plants can be triggered at high efficiency by
the presence of an inverted repeat in the transcribed region of a
transgene (4, 15, 18). Moreover, tobacco plants transformed with constructs that produce RNAs capable of duplex formation induce virus immunity or gene silencing with almost 100%
efficiency when targeted against virus or endogenous genes (35,
43). Globally, strong evidence supports a key role for dsRNA as
an inducer of PTGS in both the plant and animal kingdoms. However,
there is not yet a direct probe of the formation of dsRNA in vivo in
transgenic plants expressing palindromic sequences. Here, we expanded
previous findings on RNAi in animals by using dsRNA to specifically
interfere with viral sequences in plants. We show that exogenously
applied dsRNA can act as a trigger of RNA-mediated virus resistance and
elicit a local response in nontransgenic plants. To assess the
potential of dsRNA-mediated interference in plant virus infections, we
used pepper mild mottle virus (PMMoV), tobacco etch virus (TEV), and
alfalfa mosaic virus (AMV). These three viruses belong to distinct
taxonomic groups of positive-strand RNA viruses
(21). dsRNA-mediated interference in plants
recapitulates many of the features of RNAi in animals: the interference
observed is sequence specific and dose dependent, is triggered by dsRNA but not single-stranded RNA, and seems to require a minimum length of dsRNA.
| |
MATERIALS AND METHODS |
RNA synthesis and inoculation.
For the production of dsRNA,
sense and antisense RNAs were synthesized in vitro from the
corresponding DNA plasmids by using T3 and T7 RNA polymerase. Sense and
antisense RNA strands (2.5 µM) in 25 mM sodium phosphate (pH 7) were
heated at 95°C for 3 min and then cooled and annealed at 37°C for
30 min. Formation of dsRNA was confirmed by testing a shift in gel
mobility of the annealed material compared to each single-stranded RNA
and by resistance to RNase A under high-salt conditions. For PMMoV
dsRNAs, fragments corresponding to positions 3411 to 4388, 5086 to
5682, and 3454 to 3769 in the PMMoV sequence (1) were
subcloned into pT3T7 (Boehringer Mannheim Biochemicals), yielding after
transcription and annealing 977-bp (54-kDa-protein segment),
596-bp (30-kDa-protein segment), and 315-bp (one-third of a
54-kDa-protein segment) dsRNAs, respectively. For TEV dsRNA, a fragment
corresponding to positions 845 to 2328 in the TEV sequence
(10) was subcloned into pBluescript SK(
) (Stratagene),
yielding a 1,483-bp dsRNA (TEV-HC dsRNA). For AMV dsRNA, a fragment
corresponding to positions 369 to 1493 in the AMV RNA 3 sequence
(24) was subcloned into pBluescript SK(), yielding a
1,124-bp dsRNA (AMV-3 dsRNA). Two nonviral dsRNAs were synthesized
corresponding to the SstI fragment (300 bp) from the
Nicotiana plumbaginifolia Cab-E gene and the
AsuII-BamHI fragment (413 bp) from the binary
plant transformation vector pGSJ780A cloned in pT3T7 (37).
All plasmids were linearized with appropriate restriction enzymes and
used as templates for in vitro transcription reactions to generate
sense and antisense RNAs. An exact cDNA copy of the PMMoV
54-kDa-protein gene (nt 3499 to 4908) was produced by PCR using primers
corresponding to the relevant positions of the 54-kDa-protein gene as
previously described (37). This cDNA product was tested
for its interfering activity on PMMoV infection.
In the experiments with PMMoV, the standard inoculum was 10 µg of
purified virus per ml (1). The plasmid pTEV-7D (a generous gift from J. C. Carrington, Washington State University),
containing a full-length clone of TEV, was linearized and transcribed
in vitro with SP6 RNA polymerase as described previously
(10). Transcription with T7 RNA polymerase of full-length
clones of AMV RNA 1 (pUT17A), RNA 2 (pUT27A), and RNA 3 (pAL3) (kindly
provided by J. F. Bol, Leiden University) was performed as
described previously (25). Inoculation mixtures were made
by adding 5 µl of each dsRNA to an equal volume of purified virus
(PMMoV) or to 10 µl of viral transcripts (TEV and AMV). With AMV, 10 µg of purified AMV coat protein (CP) was added to the inoculation
mixture. Inoculation of plants was done on two fully expanded leaves of
at least two plants per assay by gently rubbing the leaf surface with
the inoculum using Carborundum as an abrasive (21). For
comparisons of sense RNA, antisense RNA, dsRNA, and cDNA effects on
virus infection, equal molar concentrations of each molecule were used.
The inoculated plants were kept in growth chambers with a 16-h
light-8-h dark cycle at 25°C, and the development of symptoms of
viral infection in systemic hosts was monitored for the duration of
their life cycles. For local lesion hosts, inoculated leaves were
photographed 5 days after inoculation.
Analysis of viral RNA in plants.
Total RNA was extracted
from inoculated leaves between 6 and 10 days postinoculation (dpi) and
from upper leaves 6 to 21 dpi as previously described
(20). RNA samples (1 to 5 µg) were electrophoresed on 1 to 1.2% agarose formaldehyde gels and transferred to Hybond-N membranes. Ethidium bromide staining of the agarose gels prior to
blotting was done to confirm integrity of the RNA and loading of
similar amounts of RNA. Northern blot hybridization was carried out
with digoxigenin (DIG)-labeled riboprobes (Boehringer Mannheim Biochemicals) as described previously (25). Virus-specific
riboprobes were used to detect the respective viruses. PMMoV RNA was
detected with a probe complementary to PMMoV nucleotides (nt) 3411 to
4388, which was transcribed from pT3T7/54 kDa (37). TEV
RNA was detected with a probe complementary to TEV nt 845 to 2328, which was transcribed from pBluescript SK()/HC (a gift from C. Llave). AMV RNAs 3 and 4 were detected with a probe complementary to nt
369 to 1493 of AMV RNA 3, which was transcribed from pBluescript
SK()/AMV-3 (see above).
Agrobacterium tumefaciens-mediated transient
expression.
The region of PMMoV RNA encoding the 54-kDa
protein and flanking regions (nt 3411 to 5016) were inserted in either
sense or antisense orientation between the 35S promoter of cauliflower mosaic virus and the transcriptional terminator of TL-DNA
gene 7 in binary vector pGSJ780A as previously described
(37). These constructs were introduced into A. tumefaciens strain LBA 4404 by direct transformation. Recombinant
A. tumefaciens was grown overnight at 28°C in tubes
containing 10 ml of Luria-Bertani medium supplemented with 50 µg of
rifampin and 40 µg of streptomycin per ml. Cells were precipitated
and resuspended to a final concentration corresponding to an optical
density at 600 nm of 0.5 in a solution containing 10 mM
MgCl2, 10 mM MES (morpholinepropanesulfonic acid, pH 5.6), and 150 µM acetosyringone. Cultures were incubated at 28°C
for 2 to 3 h before infiltration. Two leaves per plant were infiltrated in their entirety with a 1-ml syringe without a needle, and
the whole plant was covered with a transparent plastic bag for 2 days.
For the coinfiltration of Agrobacterium cultures carrying the PMMoV 54-kDa-protein construct in sense and antisense orientations, equal volumes of both cultures were mixed before infiltration.
| |
RESULTS |
dsRNA causes specific inhibition of PMMoV infection.
As
mentioned above, dsRNA can confer extreme virus resistance when sense
and antisense virus-derived transgenes are simultaneously expressed in
a plant. Therefore, we investigated whether direct delivery by
mechanical inoculation of dsRNA together with the virus could interfere
with infection in a sequence-specific manner. Sense RNA and antisense
RNA corresponding to part of the readthrough domain of the replicase
gene of PMMoV were transcribed in vitro and annealed to each other to
produce the dsRNA (54-kDa-protein dsRNA). The dsRNA and the
single-stranded RNAs with either sense or antisense orientation were
each tested for their ability to inhibit local lesion development
elicited by PMMoV in Nicotiana tabacum cv. Xanthi nc, a
hypersensitive host. Half-leaves were inoculated with virus alone, and
opposite halves were inoculated with virus plus either sense RNA,
antisense RNA, dsRNA (each at 0.62 µM, final concentration), or in
vitro transcription buffer. Figure 1
shows a representative outcome taken as an example of several
experiments carried out with RNAs produced in different transcription
reactions and summarized in Table 1.
Infectivity was completely blocked by coinoculation with a 977-bp
dsRNA that includes part of the replicase gene of PMMoV, whereas the
opposite half of the leaf inoculated only with an equivalent amount of PMMoV displayed more than 50 local lesions (Fig. 1A and Table 1).
Neither antisense RNA nor sense RNA derived from the same region of
PMMoV affected local lesion formation by the virus (Fig. 1B and Table
1). Furthermore, coinoculation with TEV dsRNA (see below), a dsRNA of
viral origin but unrelated to PMMoV, did not have any effect on PMMoV
infectivity (Fig. 1C and Table 1). We carried out experiments similar
to those described above but using Capsicum chinense instead
of N. tabacum. PMMoV induces a hypersensitive reaction on
this pepper indicator plant that was completely prevented by the
presence of 54-kDa-protein dsRNA in the inoculum (Table 1). Thus, PMMoV
infection was specifically inhibited by its cognate dsRNA in two
different local lesion hosts belonging to different genera.
View larger version (87K):
[in this window]
[in a new window]
|
FIG. 1.
Specific interference with PMMoV infection by dsRNA in a
hypersensitive host. Response of N. tabacum cv. Xanthi
nc to PMMoV (5 µg/ml) alone (left halves of leaves) or to a
combination of PMMoV plus either 54-kDa-protein dsRNA (A),
54-kDa-protein antisense (As) RNA (B), TEV-HC dsRNA (C), or in vitro
transcription buffer (D) (right halves of the leaves). Leaves were
photographed at 5 dpi. Similar numbers of local lesions (arrowheads)
were observed in both halves of the leaves in panels B, C, and D. No
visible local response was observed in the half-leaf inoculated with
PMMoV plus 54-kDa-protein dsRNA.
|
|
To evaluate the capability of dsRNA to interfere with PMMoV
infection in a systemic host,
Nicotiana benthamiana
plants were
inoculated with mixtures of PMMoV and one of the
transcription
products derived from PMMoV used above. In addition, a
cDNA PCR
product corresponding to the region of the virus transcribed
in
the in vitro reactions was included. By 7 dpi, most plants were
susceptible to virus infection. Interestingly, only plants coinoculated
with PMMoV plus 54-kDa-protein dsRNA were protected against infection,
since they did not display disease symptoms. Consistent with the
above
results, Northern blot analysis of total RNA extracted from
inoculated
or systemic leaves at 7 dpi showed that plants coinoculated
with either
sense RNA, antisense RNA, or DNA molecules homologous
to the virus
accumulated PMMoV positive-strand RNA at levels that
were comparable to
those of the control plant (Fig.
2A).
However,
when dsRNA was present in the inoculum, PMMoV multiplication
was
apparently blocked in the inoculated leaves (Fig.
2A, lane 2)
and
the virus did not accumulate to detectable levels in upper
leaves (lane
8), even after extended exposure of the autoradiogram.
Furthermore,
biologically active virus was not detected in homogenates
of the upper
leaves of these plants when used to back-inoculate
the local lesion
host,
N. tabacum cv. Xanthi nc. In addition,
there was no
evidence of accumulation of PMMoV negative-strand
RNA in these samples
(data not shown). In total, more than 10
independent assays with 22 plants have been done, corroborating
the interference with PMMoV
infection by 54-kDa-protein dsRNA.
However, in a low percentage (about
18%) of individuals, the virus
overcame the protection conferred by
dsRNA and plants displayed
disease symptoms with a delay of 1 to 3 weeks compared to the
control plants. PMMoV RNA accumulated in the
uppermost leaves
of these plants at moderate levels (Fig.
2A, lane 13).
The remaining
plants were free of symptoms until their life cycles were
completed,
and viral RNA did not accumulate at detectable levels up to
3
weeks postinoculation (Fig.
2A, lane 12), nor was virus infectivity
recovered by back-inoculation up to 40 dpi.
View larger version (79K):
[in this window]
[in a new window]
|
FIG. 2.
dsRNA-mediated interference with PMMoV infection in a
systemic host. (A) Specificity of interference with PMMoV infection by
dsRNA. Northern blot analysis of total RNA extracted from inoculated
(lanes 1 to 6) or uppermost systemic (lanes 7 to 13) leaves of
N. benthamiana. Plants were mock inoculated or were
inoculated with PMMoV (5 µg/ml) alone (), with 54-kDa-protein dsRNA
alone, or with PMMoV plus either 54-kDa-protein dsRNA, sense (S)
54-kDa-protein RNA, antisense (As) 54-kDa-protein RNA, or
54-kDa-protein cDNA, as indicated. Leaf tissues were harvested at 7 dpi, except for the samples in lanes 12 and 13, which were harvested at
21 dpi. The samples in lanes 2, 8, and 12 were taken from the same
plant, which did not display symptoms of infection until its life cycle
was completed. The sample in lane 13 was taken from another individual
showing disease symptoms at 21 dpi. The 54-kDa-protein dsRNA used in
the inoculum was run in lane 15 for comparison. (B) Time course
analysis of dsRNA stability on plant leaves. The 54-kDa-protein dsRNA
(10 µl, 0.62 µM) was inoculated on fully expanded leaves of
N. benthamiana (three- to four-leaf stage). After the
inoculated leaves had been washed with Triton X-100 (0.05%) for 30 min, RNA was extracted at the indicated times. Mock, RNA extracted from
a mock-inoculated plant. (C) Interference with PMMoV infection by
homologous dsRNAs. RNA was extracted from upper leaf tissue of plants
inoculated with PMMoV (5 µg/ml) alone () or with PMMoV plus the
indicated RNAs. (D) Interference seems to require a minimum length of
dsRNA. RNA was extracted from inoculated (lanes 1 and 4) or upper
(lanes 2, 3, 5, and 6) leaf tissue of plants infected with PMMoV (5 µg/ml) alone or with PMMoV plus 1/3 54-kDa-protein dsRNA at 7 (lanes
1, 2, 4 and 5) or 12 dpi (lanes 3 and 6). The 1/3 54-kDa-protein dsRNA
used in the inoculum was run in lane 7 for comparison. Similar amounts
(1 µg) of RNA samples were fractionated by 1% agarose gel
electrophoresis in all panels, and a DIG-labeled 54-kDa-protein RNA was
used as a probe. The positions of PMMoV RNA and RNA species derived
from partially denatured input dsRNA are indicated. Ethidium bromide
staining of 25S rRNA was used as a loading control for the RNA blots
(bottom panels).
|
|
Consistently, hybridization bands corresponding to what seems to be
partially denatured 54-kDa-protein dsRNA were observed
in RNA
preparations obtained from the inoculated leaves of plants
challenged
with virus plus 54-kDa-protein dsRNA (Fig.
2A, lane
2) or with
54-kDa-protein dsRNA alone (lane 14), as judged by
comparison with the
behavior on gels of the dsRNA preparation
used as the inoculum (Fig.
2A, lane 15). Furthermore, the same
hybridization pattern was observed
when RNA samples were treated
with RNase A, which will degrade any
ssRNA, and when a 54-kDa-protein
RNA probe specific for the negative
strand was used (data not
shown). Several experiments have been done in
order to determine
the origin and location of these dsRNA molecules.
They include
an extended wash step of the inoculated leaves just before
RNA
extraction and an analysis of the stability of these molecules
after delivery into leaves. The data support the idea that most
of the
input dsRNA was relatively stable and persisted in the
leaf, probably
inside the leaf cells, at detectable levels at
least 7 days after
inoculation (Fig.
2B).
The interference with viral infection exhibited by a dsRNA
corresponding to the readthrough region of the gene encoding the
replicase of PMMoV could reflect any kind of inhibitory effect
of this
sequence in particular on virus replication. We investigated
whether
dsRNA generated from a different region of the PMMoV genome
could
specifically block PMMoV infection when delivered simultaneously
with
the virus into the plant. To address this question, a dsRNA
covering a
596-nt segment of the 30-kDa-protein gene of PMMoV
was produced
and its effect on virus infection was compared with
that of
54-kDa-protein dsRNA derived from PMMoV or a nonhomologous,
viral
dsRNA. Like 54-kDa-protein dsRNA, the presence of 30-kDa-protein
dsRNA
in the inoculum prevented expression of viral symptoms on
N. benthamiana plants at times when control plants displayed disease
symptoms. Correspondingly, accumulation of viral RNA was not detectable
in RNA extracted from upper leaf tissue of these plants (Fig.
2C, lanes
2 and 3). However, coinoculation of PMMoV together with
a 1,483-bp
dsRNA corresponding to most of the helper component
gene of TEV (TEV-HC
dsRNA) had no effect on symptom expression,
and PMMoV RNA accumulated
in upper leaves as in control plants
(Fig.
2C, lanes 1 and 4). This
failure of nonhomologous dsRNA
to interfere with PMMoV infection has
also been observed with
different nonviral dsRNA segments of various
lengths, precluding
any effect concerning molar stoichiometry on the
lack of interference
observed with nonhomologous dsRNA (see Materials
and Methods;
also data not shown). Thus, we found interference with
PMMoV infection
only when dsRNA molecules shared sequence identity with
the virus.
At present, it is not known how effective the protection
conferred
by dsRNA is when the challenging virus and the protective
molecules
share a lower degree of sequence
homology.
While the 54-kDa- and 30-kDa-protein dsRNAs (977 and 596 nt,
respectively) were effective in protecting plants against PMMoV
infection, a smaller dsRNA covering approximately one-third (315
nt)
the length of 54-kDa-protein dsRNA (1/3 54-kDa-protein dsRNA)
had a
marginal effect. The appearance of viral symptoms on these
plants was
delayed by 1 to 2 days compared to plants inoculated
only with PMMoV,
and viral RNA accumulated at levels similar to
that of the control
(Fig.
2D). So this result may indicate that
the ability of dsRNA to
specifically interfere with PMMoV infection
could be length
dependent.
To evaluate the protective effect against PMMoV infection at different
times after delivery of dsRNA to plant leaves, 54-kDa-protein
dsRNA and
PMMoV were sequentially inoculated into the same leaves.
It was
not necessary to mix PMMoV and 54-kDa-protein dsRNA in
a tube
just before mechanical coinoculation to observe inhibition
of virus
infection. Although the immediate, sequential inoculation
of virus and
dsRNA was also able to protect plants against infection,
a interval of
24 h (and up to 96 h) between consecutive inoculations,
did
not result in interference with PMMoV infection (data not
shown).
dsRNA inhibits infection by different plant viruses.
To
determine whether the use of dsRNA molecules could be a general
strategy to prevent infection by plant viruses other than PMMoV, we
assessed the effects of specific dsRNA on the infectivity of two
viruses unrelated to the Tobamovirus genus: TEV, which belongs to the family Potyviridae, and AMV, within the
family Bromoviridae (21).
Potyviruses are a positive-strand RNA virus group with a monopartite
genome organization similar to that of the picornavirus
superfamily.
N. tabacum plants were inoculated either with SP6
transcripts of a cDNA clone of TEV alone or with a mixture containing
both TEV RNA transcripts and TEV-HC dsRNA. By 2 weeks postinoculation,
neither localized lesions nor systemic symptoms appeared on the
inoculated or upper leaves of 10 plants inoculated with the mixture,
whereas plants inoculated only with TEV displayed typical disease
symptoms of vein-clearing and etching at 6 dpi. Figure
3A shows
a Northern
blot analysis of total RNA extracted from two representative
plants per
treatment at 6 dpi. TEV RNA accumulated in both the
inoculated leaf
tissue (Fig.
3A, lanes 1 and 2) and the upper
leaf tissue (lanes 5 and
6) of the control plants, whereas viral
RNA levels were below the limit
of Northern blot detection in
plants coinoculated with the virus and
the protective, homologous
dsRNA (lanes 3, 4, 7, and 8), even after a
longer exposure of
the autoradiogram. As before, hybridization bands of
variable
intensity corresponding to TEV-HC dsRNA were observed in the
inoculated
leaves of plants challenged with virus plus dsRNA.
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 3.
Interference of dsRNA with the infection of different
plant viruses. (A) dsRNA-mediated interference with TEV. Northern blot
analysis of total RNA extracted from inoculated (lanes 1 to 4) or
systemic (lanes 5 to 8) leaves of N. tabacum plants
challenged with TEV alone or with TEV plus TEV-HC dsRNA at 6 dpi.
Similar amounts (5 µg) of each RNA sample were fractionated by 1%
agarose gel electrophoresis, and the filter was hybridized with a
DIG-labeled RNA probe specific for TEV. The positions of TEV RNA and
RNA species derived from partially denatured input TEV-HC dsRNA are
indicated. Differences in stability between different RNA samples could
probably account for variations in the intensity of the dsRNA bands.
Ethidium bromide staining of 25S rRNA was used as a loading control for the RNA gel blot (bottom panel). (B)
dsRNA-mediated interference with AMV. Northern blot analysis of total
RNA extracted at 6 dpi from inoculated (lanes 1, 3, and 4) or systemic
(lanes 6 and 7) leaves of N. benthamiana plants
challenged with AMV RNAs 1, 2, and 3 alone () or with this mixture
plus AMV-3 dsRNA. M, RNA extracted from a mock-inoculated plant. In
vitro-transcribed AMV RNAs 3 and 4 (lane 2) and AMV-3 dsRNA (lane 5)
used in the inoculum were run for comparison. Similar amounts (1 µg)
of RNA samples were fractionated by 1.2% agarose gel electrophoresis,
and the filter was hybridized with a DIG-labeled RNA probe specific for
AMV RNA3. The positions of AMV RNAs 3 and 4 and input AMV-3 dsRNA are
indicated. Ethidium bromide staining of 25S rRNA was used as a loading
control for the RNA blot (bottom panel).
|
|
Alfamoviruses are a positive-strand RNA virus group with a multipartite
genome organization similar to that of members of
the Sindbis-like
virus superfamily. A distinctive property of
the alfamoviruses is that
a mixture of the three genomic RNAs
of AMV is not infectious to plants
unless AMV CP is added in the
inoculum (
3). Ten
N. benthamiana plants were inoculated either
with a mixture of T7
transcripts of AMV RNAs 1, 2, and 3 and AMV
CP or with this mixture
plus a dsRNA covering a 1,124-nt segment
of AMV RNA 3 (AMV-3 dsRNA).
Figure
3B shows a Northern blot analysis
of total RNA extracted from
inoculated or systemic leaves of these
plants using a probe specific
for both the genomic RNA 3 and the
subgenomic RNA 4 of AMV. In
vitro-transcribed RNAs 3 and 4 and
AMV-3 dsRNA used in the inoculum
were loaded as controls (Fig.
3B, lanes 2 and 5, respectively). By 6 dpi, none of the plants
inoculated with the mixture of AMV RNAs plus
AMV-3 dsRNA showed
disease symptoms, whereas all of the plants
inoculated only with
AMV RNAs were susceptible to virus infection,
showing severe stunting
and mosaic on systemically infected leaves. In
agreement with
the observed symptoms, accumulation of AMV RNAs was not
detectable
in plants inoculated with the mixture of AMV RNAs plus dsRNA
(Fig.
3B, lanes 4 and 7), whereas plants inoculated only with AMV RNAs
accumulated RNAs 3 and 4 in both the inoculated (Fig.
3B, lane
3) and
upper (lane 6) leaf
tissue.
Inhibition by dsRNA is dose dependent and acts inside plant
cells.
To obtain a semiquantitative assessment of the relationship
between dsRNA dose and inhibition of virus infection, a series of log
dilutions (100 to 10
4)
were made from the 54-kDa-protein dsRNA preparation (0.8 µg/µl) typically used in the experiments with PMMoV. The dilutions were mixed
in equal volumes with a fixed concentration of PMMoV (10 µg/ml), and
the mixtures were tested for their ability to inhibit PMMoV infection
on N. benthamiana plants. At the highest dose tested
(undiluted dsRNA), the amount of dsRNA corresponds to a 5,240-fold
molar excess of dsRNA over viral RNA. In accord with previous results,
PMMoV accumulation was completely inhibited by a concentration of
54-kDa-protein dsRNA corresponding to undiluted dsRNA preparation,
whereas lowering the dsRNA concentration 10-fold had a slight effect on
viral infection (Fig. 4). Symptom
expression in these plants was delayed by only 3 days compared to that
in plants inoculated with PMMoV alone. PMMoV RNA accumulated at very low levels in the inoculated leaves of these plants at 6 dpi (Fig. 4,
lane 3) (visible after longer exposure of the blot) but reached a
moderate level in upper leaf tissue at 15 dpi (Fig. 4, lane 9). The
dsRNA diluted 100-fold or more showed no effect on virus infection, and
PMMoV RNA accumulated in both the inoculated and upper leaves of these
plants at levels comparable to that of the control plant. Thus, the
ability of 54-kDa-protein dsRNA to provoke inhibition of PMMoV
multiplication was dose dependent.
View larger version (79K):
[in this window]
[in a new window]
|
FIG. 4.
Dose-dependent interference with PMMoV infection by
dsRNA. Northern blot analysis of total RNA extracted from inoculated
(lanes 1 to 6) or uppermost systemic (lanes 7 to 12) leaves of
N. benthamiana at 6 and 15 dpi, respectively. Plants
were inoculated with PMMoV (5 µg/ml) alone () or with PMMoV plus a
series of log dilutions (100 to 104) of the
54-kDa-protein dsRNA, as indicated. Similar amounts (1 µg) of RNA
samples were fractionated by 1% agarose gel electrophoresis, and the
filter was hybridized with a DIG-labeled 54-kDa-protein RNA probe. The
positions of PMMoV RNA and RNA species derived from partially
denatured, input 54-kDa-protein dsRNA are indicated. The minor,
low-molecular-weight bands in lanes 4 and 6 are probably degradation
products of genomic RNA. Ethidium bromide staining of 25S rRNA was used
as a loading control for the RNA blot (bottom panel).
|
|
One possible explanation for the observation that coinoculation of
dsRNA with either viral RNA or virus particles specifically
prevents
infection by different plant viruses is that some kind
of inhibitory
interaction occurs in the mixture before the virus
penetrates into the
cell. We used an
Agrobacterium-mediated transient-expression
assay (agroinfiltration) (
40) to test if dsRNA could
inhibit
virus infection when expressed directly inside plant cells. We
cloned the entire 54-kDa-protein coding region and flanking sequences
of PMMoV (nt 3411 to 5016) in either the sense or antisense orientation
under the control of the cauliflower mosaic virus 35S constitutive
promoter in a binary plasmid vector.
A. tumefaciens cultures
carrying
the sense and antisense 54-kDa-protein RNA-expressing vectors
were mixed in a 1:1 ratio and coinfiltrated into
N. benthamiana leaves in their entirety. For comparative purpose,
plants were
agroinfiltrated with single cultures carrying plasmids
encoding
the sense or the antisense 54-kDa-protein RNA. At 4 days
postinfiltration,
plants were challenged with PMMoV that was directly
inoculated
on the entire infiltrated leaves. In three independent
experiments,
all the plants transiently expressing sense or antisense
54-kDa-protein
RNA displayed disease symptoms in upper leaves at 10 days after
inoculation, whereas plants that were agroinfiltrated with
vectors
expressing the sense-antisense mixture showed no symptoms or
symptoms
that were delayed 1 to 3 weeks compared to the controls.
Figure
5 shows a Northern blot analysis
of the accumulation of PMMoV
RNA in total RNA preparations extracted
from two individuals per
treatment at 15 dpi. PMMoV accumulated at very
low level, if any,
in the inoculated leaves of plants infiltrated with
the mixture
of single-stranded 54-kDa-protein RNAs, which could have
annealed
to each other to form a dsRNA structure inside the plant cell.
There was no signal of PMMoV RNA in upper leaf tissue of these
plants.
In contrast, neither sense nor antisense 54-kDa-protein
RNA expressed
by
Agrobacterium prevented PMMoV accumulation in
either the
inoculated or systemic leaves of the control plants.
Thus, the specific
interference with virus infection exhibited
by homologous dsRNA seems
to operate inside the plant cell and
to not be due to some other
inhibitory effect occurring in vitro.
Furthermore, the
interference effect on PMMoV infection took place
even when a
longer interval (up to 7 days) between agroinfiltration
with
54-kDa-protein dsRNA and virus inoculation was tested. However,
cointroduction of dsRNA and virus into the same leaves seemed
critical,
because dsRNA-agroinfiltrated plants challenged with
PMMoV in upper, noninfiltrated leaves became susceptible to
virus
infection without any apparent delay in symptom expression (data
not shown).
View larger version (53K):
[in this window]
[in a new window]
|
FIG. 5.
Agrobacterium-mediated transient
expression of 54-kDa-protein dsRNA interferes with PMMoV infection.
Leaves of N. benthamiana plants were initially
infiltrated as indicated with A. tumefaciens cultures
carrying either the sense (S) or the antisense (As) 54-kDa-protein
RNA-expressing vector, or a mixture of both cultures. After 4 days, the
agroinfiltrated leaves of these plants were challenge inoculated with
PMMoV or were mock inoculated (M). After another 15 days, accumulation
of PMMoV RNA was assessed on inoculated (lanes 1 to 7) and upper (lanes
8 to 14) leaves of two plants per treatment by Northern blot analysis.
Similar amounts (1 µg) of RNA samples were fractionated by 1%
agarose gel electrophoresis, and the filter was hybridized with a
DIG-labeled 54-kDa-protein RNA probe. A faint degradation product of
genomic RNA is observed in RNA extracts containing PMMoV RNA. Ethidium
bromide staining of 25S rRNA was used as a loading control for the RNA
blot (bottom panel).
|
|
| |
DISCUSSION |
We have shown that dsRNA derived from viral sequences can
interfere with virus infection in a sequence-specific manner by directly delivering dsRNA to leaf cells by mechanical inoculation. This
approach differs from strategies based on transgenic expression of RNAs
with the potential to form duplexes which confer protection against
potato virus Y through a PTGS mechanism (35, 43). Our
results support the view suggested by others that the dsRNA intermediate in virus replication acts as an efficient initiator of
PTGS in natural virus infections (29, 30). In our study, we targeted three viruses (PMMoV, TEV, and AMV) that represent extremes
in the evolution of positive-strand RNA viruses in plants with the
corresponding dsRNAs. We did not detect any reduction of virus
accumulation in plants inoculated in the presence of either sense
single-stranded RNA, antisense single-stranded RNA, DNA, or
nonhomologous dsRNA. This specificity in both sequence and structure of
the interfering molecule argues against hypothetical artifacts produced
by the inoculation procedure. Also, the interference phenotype was not
restricted to a particular plant species but was expressed in different
host and nonhost plants, demonstrating the reliability of the
phenomenon. Furthermore, we have been able to demonstrate interference
with virus infection by two different strategies: (i) direct,
mechanical inoculation of dsRNA together with the virus and (ii)
delivery of plasmid constructs via Agrobacterium whose
transcription products are predicted to form dsRNA in vivo in a process
separate from virus entry. Both approaches protected plants against
virus infection, making it very unlikely that in vitro interactions
between dsRNA and virus inocula were the cause of the protective
effect. Therefore, we suggest that the effects mediated by dsRNA in
plant virus infection reflect the phenomenon of RNAi, a particular case
of PTGS, observed in animals (2, 14). In support of this
view, the helper component proteinase of potyviruses, a well-known
suppressor of PTGS in plants (42), prevents the
interfering activity on PMMoV infection by dsRNA when both this
proteinase and 54-kDa-protein dsRNA are delivered into plant cells via
Agrobacterium (unpublished data). In addition, preliminary
data on interference with PMMoV infection observed here using RNA
duplexes of different lengths is in agreement with results obtained
with RNAi in C. elegans (13) and
Drosophila (16).
The results obtained in a dilution series of 54-kDa-protein dsRNA
suggest that a 500-fold excess of dsRNA over PMMoV RNA is the threshold
required for limited interference with virus multiplication. Threshold
concentrations of dsRNA required for RNAi have also been reported for
plants (32) and animals (39). In our case, this may reflect a minimum amount of dsRNA required for the onset of
RNAi associated with a highly replicating pathogen. Alternatively, because input dsRNA itself cannot move between cells at detectable levels in the inoculated leaves (unpublished data), this dose effect
probably reflects a minimum amount of dsRNA required to ensure that
most viral RNA penetrates into the cell in combination with dsRNA.
Taking into account the interference of dsRNA with PMMoV infection on a
hypersensitive host, we favor the hypothesis that dsRNA interferes with
virus infection at the cell level in the inoculated leaves. Indeed, in
cases where the virus is able to overcome the protection conferred by
dsRNA in a systemic host, there is a delay in symptom expression from 1 to 3 weeks, suggesting a reduced number of competent infection foci in
the inoculated leaves. In plants, analysis of the dynamics of
virus-induced gene silencing, a particular case of PTGS, revealed that
there are separate initiation and maintenance stages (31).
In our scenario, dsRNA delivered by inoculation or expressed directly
inside plant cells would act as the dsRNA intermediate involved in
viral replication, triggering the initiation step of PTGS, which
targets viral RNA for degradation. As the maintenance step of PTGS
requires a nuclear component (transgene or DNA homologous to the
target) that is absent in our plants, the sequence-specific signal
involved in PTGS does not propagate to upper parts of the plant, and
PTGS would not progress beyond the initiation stage (8,
17). It has been shown that biolistic introduction of 35S
nitrate reductase cDNA constructs induces local but not systemic PTGS
in nontransgenic plants (28). Thus, in our case, once a
few infectious particles penetrate into the cells alone, without dsRNA,
the virus spreads and moves to the upper leaves, the infection
progresses, and the plant become susceptible to the virus. In support
of this lack of a PTGS systemic signal, when dsRNA-agroinfiltrated
plants were challenged with PMMoV in upper, noninfiltrated leaves, they
became susceptible to virus infection.
Although coinoculation and rapid sequential inoculation of dsRNA and
virus produced interference with PMMoV infection, a long interval (24 h) between sequential inoculations resulted in a complete
susceptibility to virus infection. Therefore, it seems likely that some
kind of interaction must occur between the protective dsRNA, or
derivatives, and the viral RNA before dsRNA becomes degraded or
inaccessible to the RNAi machinery. One question arises regarding the
fate of dsRNA delivered on the leaf. It has been reported that in
Drosophila, dsRNA is processed efficiently to 21- to 23-nt
fragments that may be the active agents in initiating gene silencing
(44). In our work, evidence indicating partial persistence
of undegraded, input dsRNA on the leaf for at least 7 days after
inoculation has been obtained. However, initial attempts to determine
whether input dsRNA is cleaved to small RNAs did not yield positive
results. The absence of a nuclear component and/or a replicating virus
in our system could preclude the accumulation of detectable levels of
these RNA species (8).
The direct delivery of dsRNA by mechanical inoculation or via
Agrobacterium adds to the tools available for induction of
RNAi in plants. Our approach provides an alternative to genetic
transformation of plant species with dsRNA-expressing constructs able
to produce interference with endogenous plant genes (4,
18). In principle, it should be possible to silence specific
host sequences by direct delivery of dsRNA with homology to the target
gene, as has been done recently in cereals by a biolistic procedure
(32). In this case, the maintenance stage of PTGS would
operate because of the presence of a nuclear component (endogenous
gene), and the dsRNA-mediated genetic interference would be expressed
in the whole plant. In addition, this strategy would also allow the
study of the initiation stage of PTGS in cases involving transgenic
resistance against virus infection. Our results suggest that homologous
dsRNA could serve as protective molecules against virus infections,
provided that inexpensive and effective means of production and
delivery of adequate interference products onto plant surfaces are
developed. Further research will help to identify the limits and uses
of such strategies.
| |
ACKNOWLEDGMENTS |
We thank J. F. Bol for the generous gift of full-length
clones of AMV. The kind gift of pTEV-7D by J. C. Carrington is
acknowledged. We also thank J. J. López-Moya for his
comments on the manuscript, C. Llave for providing pBluescript
SK()/HC, and D. Hermán for technical assistance.
This work was supported by grants BIO97-0615-C02-01, BIO98-0849,
BIO2000-1605-C02-02, and BIO2000-0914 from Comisión
Interministerial de Ciencia y Tecnología (CICYT) and by grant
07M/0123/2000 from the Dirección General de Investigación
de la Comunidad de Madrid. F.T. is a recipient of a contract from
Consejo Superior de Investigaciones Científicas.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centro de
Investigaciones Biológicas, Velázquez 144, 28006 Madrid,
Spain. Phone: 34-9-1-5611800. Fax: 34-9-1-5627518. E-mail:
jrdiazruiz{at}cib.csic.es.
| |
REFERENCES |
| 1.
|
Alonso, E.,
I. García-Luque,
A. De la Cruz,
B. Wicke,
M. J. Avila-Rincón,
M. T. Serra,
C. Castresana, and J. R. Díaz-Ruíz.
1991.
Nucleotide sequence of the genomic RNA of pepper mild mottle virus, a resistance-breaking tobamovirus in pepper.
J. Gen. Virol.
72:2875-2884[Abstract/Free Full Text].
|
| 2.
|
Bass, B. L.
2000.
Double-stranded RNA as a template for gene silencing.
Cell
101:235-238[CrossRef][Medline].
|
| 3.
|
Bol, J. F.
1999.
Alfalfa mosaic virus and ilarviruses: involvement of coat protein in multiple steps of the replication cycle.
J. Gen. Virol.
80:1089-1102[Medline].
|
| 4.
|
Chuang, C.-F., and E. M. Meyerowitz.
2000.
Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana.
Proc. Natl. Acad. Sci. USA
97:4985-4990[Abstract/Free Full Text].
|
| 5.
|
Cogoni, C., and G. Macino.
1999.
Homology-dependent gene silencing in plants and fungi: a number of variations on the same theme.
Curr. Opin. Microbiol.
2:657-662[CrossRef][Medline].
|
| 6.
|
Cogoni, C., and G. Macino.
1999.
Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase.
Nature
399:166-169[CrossRef][Medline].
|
| 7.
|
Covey, S. N.,
N. S. Al-Kaff,
A. Langara, and D. S. Turner.
1997.
Plants combat infection by gene silencing.
Nature
385:781-782[CrossRef].
|
| 8.
|
Dalmay, T.,
A. Hamilton,
E. Mueller, and D. C. Baulcombe.
2000.
Potato Virus X amplicons in Arabidopsis mediate genetic and epigenetic gene silencing.
Plant Cell
12:369-379[Abstract/Free Full Text].
|
| 9.
|
Dalmay, T.,
A. Hamilton,
S. Rudd,
S. Angell, and D. C. Baulcombe.
2000.
An RNA-dependent RNA polymerase in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus.
Cell
101:543-553[CrossRef][Medline].
|
| 10.
|
Dolja, V. V.,
H. J. McBride, and J. C. Carrington.
1992.
Tagging of plant potyvirus replication and movement by insertion of  -glucuronidase into the viral polyprotein.
Proc. Natl. Acad. Sci. USA
89:10208-10212[Abstract/Free Full Text].
|
| 11.
|
English, J. J.,
E. Mueller, and D. C. Baulcombe.
1996.
Suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes.
Plant Cell
8:179-188[Abstract].
|
| 12.
|
Fagard, M.,
S. Boutet,
J.-B. Morel,
C. Bellini, and H. Vaucheret.
2000.
AGO-1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals.
Proc. Natl. Acad. Sci. USA
97:11650-11654[Abstract/Free Full Text].
|
| 13.
|
Fire, A.,
S. Xu,
M. K. Montgomery,
S. A. Kostas,
S. E. Driver, and C. C. Mello.
1998.
Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
Nature
391:806-811[CrossRef][Medline].
|
| 14.
|
Fire, A.
1999.
RNA-triggered gene silencing.
Trends Genet.
15:358-363[CrossRef][Medline].
|
| 15.
|
Hamilton, A. J.,
S. Brown,
H. Yuanhai,
M. Ishizuka,
A. Lowe,
A.-G. A. Solis, and D. Grierson.
1998.
A transgene with repeat DNA causes high frequency, post-transcriptional suppression of ACC-oxidase gene expression in tomato.
Plant J.
15:737-746[CrossRef].
|
| 16.
|
Hammond, S.,
E. Bernstein,
D. Beach, and G. Hannon.
2000.
An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.
Nature
404:293-296[CrossRef][Medline].
|
| 17.
|
Jones, A. L.,
A. J. Hamilton,
O. Voinnet,
C. L. Thomas,
A. J. Maule, and D. C. Baulcombe.
1999.
RNA-DNA interactions and DNA methylation on post-transcriptional gene silencing.
Plant Cell
11:2291-2302[Abstract/Free Full Text].
|
| 18.
|
Levin, J. Z.,
A. J. Framond,
A. Tuttle,
M. W. Bauer, and P. B. Heifetz.
2000.
Methods of double-stranded RNA-mediated gene inactivation in Arabidopsis and their use to define an essential gene in methionine biosynthesis.
Plant Mol. Biol.
44:759-775[CrossRef][Medline].
|
| 19.
|
Lindbo, J. A.,
L. Silva-Rosales,
W. M. Proebsting, and W. G. Dougherty.
1993.
Induction of a highly specific antiviral state in transgenic plants: implications for regulation of gene expression and virus resistance.
Plant Cell
5:1749-1759[Abstract].
|
| 20.
|
Logemann, J.,
J. Schell, and L. Willmitzer.
1987.
Improved method for the isolation of RNA from plant tissue.
Anal. Biochem.
163:16-20[CrossRef][Medline].
|
| 21.
|
Matthews, R. E. F.
1991.
Plant virology, 3rd ed.
Academic Press, San Diego, Calif.
|
| 22.
|
Montgomery, M. D.,
S. Xu, and A. Fire.
1998.
RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans.
Proc. Natl. Acad. Sci. USA
95:15502-15507[Abstract/Free Full Text].
|
| 23.
|
Mourrain, P.,
C. Beclin,
T. Elmayan,
F. Feuerbach,
C. Godon,
J. B. Morel,
D. Jouette,
A. M. Lacombe,
S. Nikic,
N. Picault,
K. Remoue,
M. Sanial,
T. A. Vo, and H. Vaucheret.
2000.
Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.
Cell
101:533-542[CrossRef][Medline].
|
| 24.
|
Neeleman, L.,
A. van der Kuyl, and J. F. Bol.
1991.
Role of alfalfa mosaic virus coat protein in symptom formation.
Virology
181:687-693[CrossRef][Medline].
|
| 25.
|
Neeleman, L., and J. F. Bol.
1999.
cis-acting functions of alfalfa mosaic virus proteins involved in replication and encapsidation.
Virology
254:324-333[CrossRef][Medline].
|
| 26.
|
Ngo, H.,
C. Tschudi,
K. Gull, and E. Ullu.
1998.
Double-stranded RNA induces mRNA degradation in Trypanosoma brucei.
Proc. Natl. Acad. Sci. USA
95:14687-14692[Abstract/Free Full Text].
|
| 27.
|
Palauqui, J.-C., and H. Vaucheret.
1998.
Transgenes are dispensable for the RNA degradation step of cosuppression.
Proc. Natl. Acad. Sci. USA
95:9675-9680[Abstract/Free Full Text].
|
| 28.
|
Palauqui, J.-C., and S. Balzergue.
1999.
Activation of systemic acquired silencing by localised introduction of DNA.
Curr. Biol.
9:59-66[CrossRef][Medline].
|
| 29.
|
Ratcliff, F.,
B. D. Harrison, and D. C. Baulcombe.
1997.
A similarity between viral defense and gene silencing in plants.
Science
276:1558-1560[Abstract/Free Full Text].
|
| 30.
|
Ratcliff, F.,
S. MacFarlane, and D. C. Baulcombe.
1999.
Gene silencing without DNA: RNA-mediated cross protection between viruses.
Plant Cell
11:1207-1215[Abstract/Free Full Text].
|
| 31.
|
Ruiz, M. T.,
O. Voinnet, and D. C. Baulcombe.
1998.
Initiation and maintenance of virus-induced gene silencing.
Plant Cell
10:937-946[Abstract/Free Full Text].
|
| 32.
|
Schweizer, P.,
J. Pokorny,
P. Schulze-Lefert, and R. Dudler.
2000.
Double-stranded RNA interferes with gene function at the single-cell level in cereals.
Plant J.
24:895-903[CrossRef][Medline].
|
| 33.
|
Sijen, T., and J. M. Kooter.
2000.
Post-transcriptional gene-silencing: RNAs on the attack or on the defense?
BioEssays
22:520-531[CrossRef][Medline].
|
| 34.
|
Smardon, A.,
J. M. Spoerke,
S. C. Stacey,
M. E. Klein,
N. Mackin, and E. M. Maine.
2000.
EGO-1 is related to RNA-directed RNA polymerase and function in germ-line development and RNA interference in C. elegans.
Curr. Biol.
10:169-178[CrossRef][Medline].
|
| 35.
|
Smith, N.,
S. Singh,
M.-B. Wang,
P. Stoutjesdijk,
A. Green, and P. Waterhouse.
2000.
Total silencing by intron-spliced hairpin RNAs.
Nature
407:319-320[CrossRef][Medline].
|
| 36.
|
Tavernarakis, N.,
S. L. Wang,
M. Dorovkov,
A. Ryazanov, and M. Driscoll.
2000.
Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes.
Nat. Genet.
24:180-183[CrossRef][Medline].
|
| 37.
|
Tenllado, F.,
I. García-Luque,
M. T. Serra, and J. Díaz-Ruíz.
1995.
Nicotiana benthamiana plants transformed with the 54 kDa region of the pepper mild mottle tobamovirus replicase gene exhibit two types of resistance responses against viral infection.
Virology
211:170-183[CrossRef][Medline].
|
| 38.
|
Timmons, L., and A. Fire.
1998.
Specific interference by ingested dsRNA.
Nature
395:854[CrossRef][Medline].
|
| 39.
|
Tuschl, T.,
P. Zamore,
D. Bartel, and P. Sharp.
1999.
Targeted mRNA degradation by double-stranded RNA in vitro.
Genes Dev.
13:3191-3197[Abstract/Free Full Text].
|
| 40.
|
Vaucheret, H.
1994.
Promoter-dependent trans-inactivation in transgenic tobacco plants: kinetic aspects of gene silencing and gene reactivation.
C. R. Acad. Sci. Ser. III
317:310-323.
|
| 41.
|
Vaucheret, H.,
C. Beclin,
T. Elmayan,
F. Feuerbach,
C. Godon,
J.-B. Morel,
P. Mourrain,
J.-C. Palauqui, and S. Vemhettes.
1998.
Transgene-induced gene silencing in plants.
Plant J.
16:651-659[CrossRef][Medline].
|
| 42.
|
Voinnet, O.,
Y. M. Pinto, and D. C. Baulcombe.
1999.
Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants.
Proc. Natl. Acad. Sci. USA
96:14147-14152[Abstract/Free Full Text].
|
| 43.
|
Waterhouse, P.,
M. Gramham, and M.-B. Wang.
1998.
Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.
Proc. Natl. Acad. Sci. USA
95:13959-13964[Abstract/Free Full Text].
|
| 44.
|
Zamore, P. D.,
T. Tuschl,
P. A. Sharp, and D. P. Bartel.
2000.
RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.
Cell
101:25-33[CrossRef][Medline].
|
Journal of Virology, December 2001, p. 12288-12297, Vol. 75, No. 24
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.24.12288-12297.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Agrawal, N., Dasaradhi, P. V. N., Mohmmed, A., Malhotra, P., Bhatnagar, R. K., Mukherjee, S. K.
(2003). RNA Interference: Biology, Mechanism, and Applications. Microbiol. Mol. Biol. Rev.
67: 657-685
[Abstract]
[Full Text]
-
Vanitharani, R., Chellappan, P., Fauquet, C. M.
(2003). Short interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells. Proc. Natl. Acad. Sci. USA
100: 9632-9636
[Abstract]
[Full Text]
-
Adelman, Z. N., Sanchez-Vargas, I., Travanty, E. A., Carlson, J. O., Beaty, B. J., Blair, C. D., Olson, K. E.
(2002). RNA Silencing of Dengue Virus Type 2 Replication in Transformed C6/36 Mosquito Cells Transcribing an Inverted-Repeat RNA Derived from the Virus Genome. J. Virol.
76: 12925-12933
[Abstract]
[Full Text]
-
Yelina, N. E., Savenkov, E. I., Solovyev, A. G., Morozov, S. Y., Valkonen, J. P. T.
(2002). Long-Distance Movement, Virulence, and RNA Silencing Suppression Controlled by a Single Protein in Hordei- and Potyviruses: Complementary Functions between Virus Families. J. Virol.
76: 12981-12991
[Abstract]
[Full Text]
-
Coburn, G. A., Cullen, B. R.
(2002). Potent and Specific Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference. J. Virol.
76: 9225-9231
[Abstract]
[Full Text]
Top
Abstract
Introduction
