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Acta Biochim Biophys
Sin 2006, 38: 22-28
doi:10.1111/j.1745-7270.2006.00124.x
Transiently Expressed Short
Hairpin RNA Targeting 126 kDa Protein of Tobacco Mosaic Virus Interferes with
Virus Infection
Ming-Min ZHAO1,2, De-Rong AN1*, Jian ZHAO2, Guang-Hua HUANG3, Zu-Hua HE4, and Jiang-Ye CHEN3
1 College of Plant
Protection, Northwest Science and Technology University of Agriculture and
Forestry, Yangling 712100, China;
2 College of Agriculture, Yangtze University, Jingzhou 434025,
China;
3 State Key Laboratory of
Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai
200031, China;
4 State Key Laboratory of
Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai
200032, China
Received: August 4,
2005
Accepted: October
21, 2005
This study was
supported by the grants from the Ministry of Science and Technology of China
(No. 100C26216101344) and the State Key Laboratory of Molecular Biology,
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences
*Corresponding
author: Tel, 86-29-87092728; Fax, 86-29-87092401; E-mail,
Abstract RNA interference (RNAi) silences gene
expression by guiding mRNA degradation in a sequence-specific fashion. Small interfering
RNA (siRNA), an intermediate of the RNAi pathway, has been shown to be very
effective in inhibiting virus infection in mammalian cells and cultured plant
cells. Here, we report that Agrobacterium tumefaciens-mediated transient
expression of short hairpin RNA (shRNA) could inhibit tobacco mosaic virus
(TMV) RNA accumulation by targeting the gene encoding the
replication-associated 126 kDa protein in intact plant tissue. Our results
indicate that transiently expressed shRNA efficiently interfered with TMV
infection. The interference observed is sequence-specific, and time- and
site-dependent. Transiently expressed shRNA corresponding to the TMV 126 kDa
protein gene did not inhibit cucumber mosaic virus (CMV), an unrelated
tobamovirus. In order to interfere with TMV accumulation in tobacco leaves, it
is essential for the shRNA constructs to be infiltrated into the same leaves as
TMV inoculation. Our results support the view that RNAi opens the door for
novel therapeutic procedures against virus diseases. We propose that a
combination of the RNAi technique and Agrobacterium-mediated transient
expression could be employed as a potent antiviral treatment in plants.
Key words tobacco mosaic virus; 126 kDa protein; RNA interference;
short hairpin RNA
RNA silencing or interference (RNAi) is a sequence-specific,
post-transcriptional process of mRNA degradation, which is initiated by
double-stranded RNA (dsRNA) or hairpin RNA molecules. RNAi was initially discovered
in plants and subsequently in nematodes [1,2]. Following the initial
identification of RNAi [2], small interfering RNA (siRNA) was identified in Drosophila
and Caenorhabditis elegans [3,4]. Long dsRNA molecules are first processed
by the endonuclease Dicer into 21–25 nt siRNA. The resulting siRNA, as part of a
multiprotein RNA-inducing silencing complex, is targeted to the complementary
target RNA, which is then cleaved. In plants, RNAi is referred to as post-transcriptional gene
silencing and thought to be involved in a natural line of defense against viral
infection [5]. The RNA genome of the invading virus or any homologous RNA is
targeted and eliminated in a sequence-specific manner when the antiviral
mechanism is activated. Virus-induced gene silencing has been demonstrated for
a number of RNA and DNA viruses [6-8] with the production of the virus-specific
siRNA except potato virus X-infected plants [3].Recently, it was shown that siRNA of 21 nt in size, an intermediate
of the RNA-interference pathway, is effective in the inhibition of viral
infection and modulation of viral replication in a variety of mammalian systems
[9–16]
and cultured plant cells [17]. Introduction of siRNA into mammalian cells can
suppress the expression of a specific endogenous gene and target a number of
viruses [9,13]. Moreover, human T cells transfected with lentiviral siRNA
vectors targeting the HIV-1 co-receptor CCR5 displayed a reduction of CCR5
expression and a significant reduction in the number of HIV-1 infected cells
[18]. These findings indicated that siRNA could be useful in antiviral
strategies as a tool for gene therapy. In plants, it has been demonstrated that
siRNA-mediated suppression of gene expression can occur in cultured plant
cells, and siRNA can interfere with and suppress the accumulation of a
nuclear-replicated DNA virus [17]. However, the utility of siRNA transcripts in
non-transgenic plants has not been reported. The Agrobacterium-mediated
transient expression system enabled gene expression within a short period of
time without the requirement for regenerating transgenic plants [19].Tobacco mosaic virus (TMV) has infected a wide variety of
economically important crops worldwide. This virus has a single-strand RNA
genome. Of the several gene products encoded by the virus, the
replication-associated 126 kDa and 183 kDa proteins are indispensable for viral
RNA replication.Here, we expand on previous findings on siRNA in animals by
assessing the potential of short hairpin RNA (shRNA)-mediated inhibition of TMV
replication using the Agrobacterium-mediated transient expression
system. We envisage the use of shRNA to target the 126 kDa protein gene, which
could be a valuable strategy to counter TMV.
Materials and Methods
Plasmid construction
pBI121, the base vector for all constructs, contains an enhanced 35S
promoter from cauliflower mosaic virus, a b-glucuronidase gene, and a
35S terminator.pBI121 was used to generate short, unimolecular RNA transcripts
which serve as shRNA. To design target-specific shRNA against the 126 kDa
protein gene, we selected the sequence of the type AA (N21, N presents any
nucleotide) from the coding sequence of 126 kDa mRNA. Sequences from
nucleotides 1519–1538 and 2129–2148 relating to the transcription start site were suitable for the
design of a specific shRNA directed against the TMV 126 kDa protein gene. The
selected shRNA sequences were submitted to the BLAST search engine to ensure
the specificity to the target mRNA. Oligonucleotides contained both the 19 nt
sense and 19 nt antisense strands separated by a 9 nt short spacer. The
oligonucleotides used are shown in Table 1. BamHI, HindIII
and SstI sequences were added at the 5‘ and 3‘ end of the
oligonucleotides, so that the annealed oligonucleotides could be easily cloned
into the pBI121 vector and the positive clone could be identified by the
HindIII digestion (Fig. 1). These oligonucleotides, which form
double-stranded DNA after annealing, were cloned into BamHI/SstI-digested
pBI121. The resulting transcript is predicted to fold back on itself to form a
19 bp shRNA, which is quickly cleaved by the endonuclease Dicer in the cell to
produce a functional siRNA.
Infiltration of plants with Agrobacterium
tumefaciens
Agrobacterium tumefaciens infiltration
assay was performed as described previously [20]. The constructs were
introduced into the A. tumefaciens strain EHA105 by direct
transformation. Recombinant A. tumefaciens was grown overnight at 28 ?C
in tubes containing 5 ml of Luria-Bertani medium supplemented with 50 mg/ml kanamycin.
The cells were collected by centrifugation and resuspended to a final
concentration of A600=0.8 in a solution containing 10 mM
MgCl2, 10 mM 2-morpholinopropane sulfonic acid (pH 5.6), and 150 mM
acetosyringone. The cell suspension was incubated at 28 ?C for 2–3 h before
infiltration. Using a 5 ml syringe, A. tumefaciens cell cultures
carrying the pBI/shRNA constructs were injected into the leaves of healthy Nicotiana
tabacum tobacco plants (obtained from the Institute of Phytopathology,
Northwest Science and Technology University of Agriculture and Forestry,
Yangling, China) through an incision made by a
pinhead. Two leaves of each plant were infiltrated in the entirety and the
whole plant was covered with a transparent plastic bag for 2 d.
Virus inoculation
TMV was inoculated on N. tabacum plants after infiltration
with pBI/shRNA constructs as described previously [21]. TMV particles were
isolated from systemically infected N. tabacum plants and
purified by polyethylene glycol precipitation. Standard inoculation was
performed using 10 mg/ml purified viruses as the inoculum. The inoculation was performed
on two fully expanded leaves of the tobacco plant that were infiltrated with A.
tumefaciens by rubbing the leaf surface with the inoculum, using
carborundum as an abrasive. The inoculated plants were kept in a growth chamber
at 25 ?C with 16 h of light and 8 h of darkness [20].
Analysis of viral RNA in
tobacco
Total RNA was extracted from tobacco leaves (0.1 g) using the Trizol
reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s
instructions. The RNA samples (approximately 20 mg) were separated on 1%
agarose formaldehyde gel, using a buffer consisting of 20 mM
3-(N-morpholino)propane sulfonic acid, 5 mM NaAc, 1 mM ethylene diamine
tetraacetic acid (pH 7.0), and transferred to Hybond-N membranes (Amersham,
Amersham, UK), which were then subjected to ultraviolet cross-linking. The RNA
blots were pre-hybridized in Church buffer at 65 ?C for 1 h. Radiolabeled
probes for the open reading frame of TMV 126 kDa gene were made by a random
priming reaction in the presence of [a–32P]dATP, and
used to detect the RNA. Hybridization was performed overnight in a rotating
incubator at 65 ?C, and this was followed by four washes (20 min each) in 2?standard saline citrate buffer and 0.2% (W/V) sodium
dodecyl sulfate at 65 ?C, 65 ?C, 60 ?C and 50 ?C, respectively. The blots were
scanned using a phosphorimager Storm860 (Amersham Bioscience, Uppsala, USA).
Results
shRNA-directed interference in
TMV infection
To investigate shRNA-directed interference in TMV infection in
systemic hosts, two leaves of N. tabacum plants were agro-infiltrated
with cultures of A. tumefaciens carrying pBI/shRNA1519, pBI/shRNA2129,
or pBI/shRNA1519m–. An empty vector (pBI121)
was used as a negative control. At 4 d post-infiltration, TMV particles were
directly inoculated onto the entire infiltrated leaf. In several independent
experiments, all plants infiltrated with pBI/shRNA1519m– and pBI121 displayed disease symptoms in upper leaves at 4 d
post-inoculation (dpi), whereas 28 of 34 (approximately 83%) plants that were
agro-infiltrated with the pBI/shRNA1519 construct were free of viral symptoms.
A similar proportion (approximately 85%) of the plants agro-infiltrated with
pBI/shRNA2129 were free of symptoms (Fig. 2).To confirm shRNA-directed interference with TMV infection, we
performed Northern blot hybridization to detect the accumulation of TMV RNA in
the upper leaves of N. tabacum plants. Consistent with the lack of viral
symptoms, TMV RNA levels in the tobacco leaves were significantly reduced when
infiltrated with the pBI/shRNA1519 and pBI/shRNA2129 constructs. In contrast,
viral RNA was abundant in plants infiltrated with A. tumefaciens
containing the empty vector pBI121 and pBI/shRNA1519m– (Fig. 3). The specific inhibition of TMV infection by the TMV-derived shRNA
constructs was confirmed by inoculation with cucumber mosaic virus (CMV), an
unrelated tobamovirus. CMV was inoculated on leaves that had been infiltrated
with pBI/shRNA1519 or the empty vector pBI121. As expected, the symptoms caused
by CMV on the leaves infiltrated with the shRNA construct had no apparent
difference from those infiltrated with the empty vector (data not shown). Thus,
transient expression of TMV shRNA did not interfere with CMV, indicating that
the interference is sequence-specific. To confirm the aforementioned results, the pBI/shRNA constructs and
empty vector were agro-infiltrated into Nicotiana glutinosa plants, a
hypersensitive host, followed by TMV inoculation: on each leaf, one half was
infiltrated with A. tumefaciens cultures containing pBI/shRNA1519,
pBI/shRNA2129 or pBI/shRNA1519m– construct or pBI121, and the other half
was infiltrated with pBI121 alone; the leaf was then inoculated with TMV. The
number of lesions on leaves infiltrated with pBI121-versus-pBI/shRNA constructs
are summarized in Table 2. Similar numbers of local lesions were observed
in the halves of the leaves infiltrated with pBI121 or pBI/shRNA1519m–. No visible or only a few local lesions were observed in the halves
of two leaves infiltrated with pBI/shRNA1519 or pBI/shRNA2129 respectively in
six independent assays (Fig. 4). These findings indicated that
infectivity was blocked by infiltration with pBI/shRNA1519 or pBI/shRNA2129,
whereas the opposite half of the leaves infiltrated with pBI121 and
pBI/shRNA1519m– were susceptible to TMV
infection.
Time and site dependence of
shRNA-mediated interference
A time-course experiment was performed to determine when the
inhibition of TMV would take place and how long it would last after delivery of
pBI/shRNA constructs into plant cells. TMV was inoculated on N. tabacum
plants simultaneously or at 1–7 d after being infiltrated with the pBI/shRNA1519 construct in the
same leaves. The results showed that there was a delay of ?3 d between agro-infiltration with pBI/shRNA1519 and occurrence of
TMV resistance in the agro-infiltrated leaves. Empty vector-infiltrated plants
showed no viral protection at any time point of the interval tested and
displayed systemic symptoms at 5 dpi. Northern blot assay confirmed the results
observed from tobacco leaves (Fig. 5). We detected a dramatic reduction
of TMV RNA in leaves that had been infiltrated with pBI/shRNA1519. TMV
infectivity was almost abolished when plants were infiltrated with the
pBI/shRNA1519 construct 3, 4, 5, 6, or 7 d before virus inoculation.Next, we performed an experiment to
determine whether transient shRNA expression on lower leaves could trigger a
systemic anti-viral response in upper parts of the plants. Lower leaves were
infiltrated with pBI/shRNA constructs or pBI121. After 4 d, upper leaves were
inoculated with TMV. At 5 dpi, all plants displayed systemic symptoms
regardless of whether they had been infiltrated with shRNA constructs or the
empty vector (data not shown). This result indicated that shRNA-mediated
interference of TMV infection has a localized effect and does not spread
systemically. To achieve TMV resistance, it was necessary that the shRNA
constructs were infiltrated into the same leaves where TMV was to be
inoculated.
Discussion
RNAi technology has emerged very rapidly as a revolutionary tool for
experimental biology in a variety of organisms [22]. Using RNAi, a number of
interesting disease-related genes have been targeted highlighting the potential
of this gene silencing approach as a therapeutic platform. In plants, Tenlladdo
et al. has shown that A. tumefaciens-mediated transient
expression of homologous hairpin RNA blocked multiplication and spread of a
rapidly replicating plant virus in a sequence-dependent manner in non-transgenic
plants [20]. Recently, it was demonstrated that the use of RNAi was very
efficient in cultured plant cells [17]. Here, we established and used an
agro-infiltration system in intact tissue to facilitate rapid analysis of
transiently expressed shRNA-mediated interference on plant virus infection. Our results showed that transient expression of shRNA specifically
and efficiently inhibited TMV infection. Plant leaves inoculated with
plant sap extracted from pBI/shRNA1519 and pBI/shRNA2129-infiltrated plants
displayed none and few local lesions respectively and showed specific
interference with TMV infection (data not shown). The CMV infection and
pBI/shRNA1519m– infiltration experiments
showed that virus infection was dependent on a high level of sequence identity
between shRNA and the target RNA. Plants infiltrated with pBI/shRNA constructs
were unable to protect against CMV infection. Further evidence for
sequence-dependent resistance came from the observation that plants infiltrated
with A. tumefaciens carrying pBI/shRNA1519m– exhibited
the same susceptibility to TMV as the control. As the virus appears to
replicate exclusively in the cytoplasm [23], we expect that transiently
expressed shRNA specifically degrades the TMV RNA in the cytoplasm. We propose
that transiently expressed shRNA might also serve as the primer for
RNA-dependent RNA polymerase to synthesize dsRNA using TMV mRNA as the
template, thereby amplifying the interfering effects.Next, we designed studies to examine whether transient expression of
shRNA could induce TMV RNA degradation at different times and different sites
after delivery of pBI/shRNA constructs to tobacco leaves. Our data showed that,
for a significant interfering effect on TMV infection to occur, an interval of
3 d or longer is required between agro-infiltration with pBI/shRNA constructs
and virus inoculation. This delayed effect is presumably due to the time
required for A. tumefaciens to transfer the T-DNA into plant cells
(maximum at 48 h post-infiltration) [24] and for the construct to be expressed.
Similarly, introduction of shRNA constructs and the virus into the same leaves
seemed indispensable for interference, as the untreated upper leaves of the
agro-infiltrated plants were highly susceptible to virus infection. This
suggests that transiently expressed shRNA does not move into the distant organs
to trigger RNAi. Until recently, it has remained unclear how silencing signals
propagate and what natural (non-transgenic) role the signal plays. It was
demonstrated that siRNA induced gene silencing in a “transitive
manner” in cultured plant cells [17]. The targeting of siRNAs to one
sequence in a gene resulted in degradation of the entire or most of the mRNA to
short polynucleotides outside the siRNA-targeted region. In conclusion, our results suggest that transiently expressed shRNA
corresponding to the TMV genome is a potent and specific inducer of RNA
degradation in intact plant tissue, which can result in efficient inhibition of
viral replication. Furthermore, our results indicate that agro-infiltration of
RNAi constructs into living plants could be used as an efficient way to study
virus replication and holds potential as an antiviral treatment in plants. We
believe that shRNA-mediated interference with virus infection offers a
potentially powerful tool for inhibiting replication at different stages in the
virus life cycle, and this interference can be achieved by targeting both viral
and cellular genes in plants.
Acknowledgements
We are grateful to Dr. Eugene I. SAVENKOV (Department of Plant
Biology, Genetic Centre, SLU, S-75007 Uppsala, Sweden) for providing the HC-Pro
gene. We would like to thank Dr. Francisco TENLLADO (Departamento de Biologia
de Plantas, Centro de Investigaciones Biologicas, Madrid, Spain) for the
generous gift of the plasmid pBI121, and Professor Qun LI (Institute of Plant
Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences, Shanghai, China) for providing the EHA105 strain, as well
as providing assistance with plant preparation and helpful discussion. We also
thank all members of the laboratory of Professor Jiang-Ye CHEN (Institute of
Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai, China) for their helpful discussion. We
thank the innovative group at the Plant Protection College, Northwest Science
and Technology University of Agriculture and Forestry (Yangling, China) for
their help.
References
1 Baulcombe DC. RNA as a target and an
initiator of post-transcriptional gene silencing in transgenic plants. Plant
Mol Bio 1996, 32: 79–88
2 Fire A, Xu S, Montgomery MK, Kostas SA, Driver
SE, Mello CC. Potent and specific genetic interference by double-stranded RNA
in Caenorhabditis elegans. Nature 1998, 391: 806–811
3 Hamilton AJ, Baulcombe DC. A species of small
antisense RNA in posttranscriptional gene silencing in plants. Science 1999,
286: 950–952
4 Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi:
Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23
nucleotide intervals. Cell 2000, 101: 2533
5 Voinnet O. RNA silencing as a plant immune
system against viruses. Trends Genet 2001, 17: 449–459
6 Vance V, Vaucheret H. RNA silencing in plants—defense and
counterdefense. Science 2001, 292: 2277–2280
7 Ratcliff FG, MacFarlane SA, Baulcombe DC. Gene
silencing without DNA. RNA-mediated cross protection between viruses. Plant
Cell 1999, 11: 1207–1216
8 Li H, Li WX, Ding SW. Induction and suppression
of RNA silencing by an animal virus. Science 2002, 296: 1319–1321
9 Gitlin L, Karelsky S, Andino R. Short
interfering RNA confers intracellular antiviral immunity in human cells. Nature
2002, 418: 430–434
10 Jacque JM, Triques K, Stevenson. Modulation
of HIV-1 replication by RNA interference. Nature 2002, 418: 435–438
11 Lee NS, Dohjima T, Bauer G, Li H, Li MJ,
Ehsani A, Salvaterra P et al. Expression of small interfering RNAs
targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 2002, 20:
500–505
12 Novina CD, Murray MF, Dykxhoorn DM, Beresford
PJ, Riess J, Lee SK, Collman RG et al. siRNA-directed inhibition of
HIV-1 infection. Nat Med 2002, 8: 681–686
13 Elbashir SM, Harborth J, Lendeckel W, Yalcin
A., Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference
in cultured mammalian cells. Nature 2001, 411: 494–498
14 Garrus JE, von Schwedler UK, Pornillos OW,
Morham SG, Zavitz KH, Wang HE, Wettstein DA et al. Tsg101 and the vacuolar
protein sorting pathway are essential for HIV-1 budding. Cell 2001, 107: 55–65
15 Paddison PJ, Caudy AA, Bernstein E, Hannon
GJ, Conklin DS. Short hairpin RNAs (shRNAs) induce sequence-specific silencing
in mammalian cells. Genes Dev 2002, 16: 948–958
16 Sui MJ, Tsai LC, Hsia KC, Doudeva LG, Ku WY,
Han GW, Yuan HS. Metal ions and phosphate binding in the H-N-H motif: Crystal
structures of the nuclease domain of CoIE7/Im7 in complex with a phosphate ion
and different divalent metal ions. Protein Sci 2002, 11: 2947–2957
17 Vanitharani R, Chellappan P, Fauquet CM. Short
interfering RNA-mediated interference of gene expression and viral DNA
accumulation in cultured plant cells. Proc Natl Acad Sci USA 2003, 100: 9632–9636
18 Qin XF, An DS, Chen IS, Baltimore D.
Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of
small interfering RNA against CCR5. Proc Natl Acad Sci USA 2003, 100: 183–188
19 Johansen LK, Carrington JC. Silencing on the
spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated
transient expression system. Plant Physiol 2001, 126: 930–938
20 Tenllado F, Diaz-ruiz JR. Double-stranded
RNA-mediated interference with plant virus infection. J Virol 2001, 75: 12288–12297
21 Zhao MM, An DR, Huang GH, He ZH, Chen JY. A
viral protein suppresses siRNA-directed interference in tobacco mosaic virus
infection. Acta Biochim Biophys Sin 2005, 37: 248–253
22 Schutze N. siRNA technology. Mol Cell
Endocrinol 2004, 213: 115119
23 Waterhouse P, Wang MB, Lough T. Gene
silencing as an adaptive defense against viruses. Nature 2001, 411: 834–842
24 Kasschau KD, Carrington JC. Long-distance
movement and replication maintenance functions correlate with silencing
suppression activity of potyviral HC-Pro. Virology 2001, 285: 71–81