Virus-induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato.

Abstract

Virus-induced gene silencing (VIGS) is one of the commonly used RNA silencing methods in plant functional genomics. It is widely known that VIGS can occur for about 3 weeks. A few reports show that duration of VIGS can be prolonged for up to 3 months. Increasing the duration of endogenous gene silencing and developing a method for nonintegration-based persistent VIGS in progeny seedlings will widen the application of VIGS. We used three marker genes that provoke visible phenotypes in plants upon silencing to study persistence and transmittance of VIGS to progeny in two plant species, Nicotiana benthamiana and tomato. We used a Tobacco rattle virus (TRV)-based VIGS vector and showed that the duration of gene silencing by VIGS can occur for more than 2 years and that TRV is necessary for longer duration VIGS. Also, inoculation of TRV-VIGS constructs by both Agrodrench and leaf infiltration greatly increased the effectiveness and duration of VIGS. Our results also showed transmittance of VIGS to progeny seedlings via seeds. A longer silencing period will facilitate detailed study of target genes in plant development and stress tolerance. Further, the transmittance of VIGS to progeny will be useful in studying the effect of gene silencing in young seedlings. Our results provide a new dimension for the application of VIGS in plants.

Full text

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Virus-induced gene silencing can persist for more than
2 years and also be transmitted to progeny seedlings in
Nicotiana benthamiana and tomato
Muthappa Senthil-Kumar and Kirankumar S. Mysore*
Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK, USA
Received 20 July 2010;
revised 3 December 2010;
accepted 6 December 2010.
*Correspondence (fax 580 224 6692;
e-mail ksmysore@noble.org)
Keywords: functional genomics,
RNA silencing, Nicotiana bentha-
miana, tomato, Solanaceae, TRV.
Summary
Virus-induced gene silencing (VIGS) is one of the commonly used RNA silencing methods in
plant functional genomics. It is widely known that VIGS can occur for about 3 weeks. A few
reports show that duration of VIGS can be prolonged for up to 3 months. Increasing the
duration of endogenous gene silencing and developing a method for nonintegration-based
persistent VIGS in progeny seedlings will widen the application of VIGS. We used three mar-
ker genes that provoke visible phenotypes in plants upon silencing to study persistence and
transmittance of VIGS to progeny in two plant species, Nicotiana benthamiana and tomato.
We used a Tobacco rattle virus (TRV)-based VIGS vector and showed that the duration of
gene silencing by VIGS can occur for more than 2 years and that TRV is necessary for longer
duration VIGS. Also, inoculation of TRV-VIGS constructs by both Agrodrench and leaf infiltra-
tion greatly increased the effectiveness and duration of VIGS. Our results also showed trans-
mittance of VIGS to progeny seedlings via seeds. A longer silencing period will facilitate
detailed study of target genes in plant development and stress tolerance. Further, the trans-
mittance of VIGS to progeny will be useful in studying the effect of gene silencing in young
seedlings. Our results provide a new dimension for the application of VIGS in plants.
Introduction
Virus-induced gene silencing (VIGS) is widely used to silence the
target gene of interest in certain plants by exploiting natural
plant defence mechanisms (Lu et al., 2003; Robertson, 2004;
Voinnet, 2001). VIGS involves delivery of a fragment of plant
gene (intended to be silenced) into plant cells via a recombinant
virus. The plant defence mechanism silences both the targeted
endogenous plant gene and the virus through post-transcrip-
tional gene silencing [PTGS, reviewed in (Robertson, 2004;
Voinnet, 2001)]. VIGS entails generation of double-stranded
RNA (dsRNA) during virus replication and production of small
interfering RNA (siRNA) with a 21–23 nucleotide length through
a plant DICER like nuclease. These siRNAs bind to RNA-induced
silencing complex (RISC) and target this complex to find a
homology containing mRNA for cleavage. Such fragmented
mRNAs are later destroyed by cellular nucleases causing sup-
pression of target gene expression [reviewed in (Robertson,
2004; Watson et al., 2005)].
The Tobacco rattle virus (TRV)-based VIGS, vector is most
widely used for gene silencing in Solanaceae plant species
[reviewed in (Senthil-Kumar et al., 2008)]. TRV is a positive-
strand RNA virus with a bipartite genome [RNA1 and RNA2;
(MacFarlane, 1999)]. RNA1 encodes an RNA-dependent RNA
polymerase and a movement protein. RNA2 encodes a coat
protein (CP) and two nonstructural proteins from the sub-
genomic RNAs. TRV can move systemically in many plants and,
unlike some other viruses, does not encode a strong silencing
suppressor (Ratcliff et al., 2001). Both RNA1 and RNA2 are
neededfor infectionand production
(MacFarlane, 1999). TRV can infect roots and move to aerial
of virusparticles
plant parts and provoke VIGS (Liu et al., 2002a; MacFarlane,
1999; Ryu et al., 2004). TRV is also known to infect meristem
tissue (Liu et al., 2002b; Ratcliff et al., 2001). A 29-kDa protein
from RNA2 is involved in cell-to-cell movement. Also, a cysteine-
rich protein encoded by TRV has been implicated in seed trans-
mission (Liu et al., 2002a; MacFarlane, 1999). For VIGS, both
RNA1 and RNA2 were modified and cloned individually between
the T-DNA borders of an Agrobacterium binary vector (Liu et al.,
2002b). These TRV-VIGS vectors are mainly delivered into plants
by Agrobacterium-mediated transient expression using leaf
infiltration and Agrodrench methods (Ryu et al., 2004). TRV-
mediated VIGS has been used to silence genes in all plant parts
namely root, leaf, stem, flower and fruit in a wide range of
plant species [reviewed in Senthil-Kumar et al. (2008)]. VIGS
was shown to be a transient phenomenon (Liu et al., 2002b; Lu
et al., 2003), and stable inheritance of virus-mediated PTGS is
not reported. In fact one study showed that TRV-mediated VIGS
is not transmitted to plant progenies (Wege et al., 2007). The
ability to generate gene knockdown phenotypes without having
to genetically manipulate the plant genome is one of the advan-
tages of VIGS compared to other gene suppression methods like
mutagenesis and RNA interference (RNAi).
It is a common belief that effective VIGS occurs only for
3 weeks (Ryu et al., 2004). Efficiency of VIGS has been shown
to decrease after 1 month and plants start to recover from
silencing (Ratcliff et al., 2001). Such a recovery can be either
transient or stable and the extent of recovery phenotype can be
partial or complete (Hiriart et al., 2003; Ratcliff et al., 2001).
Persistence of VIGS for longer duration is influenced by age of
the plant, viral titre, and environmental conditions that favour
virus multiplication (Fu et al., 2006; Tuttle et al., 2008).
ª 2011 The Authors
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd
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However, the exact mechanism for persistence of VIGS has not
been fully understood. Maintenance of effective VIGS for longer
duration is desirable for both forward and reverse genetics
studies.
In this manuscript, we provide experimental evidence to show
that TRV-mediated VIGS can be maintained for more than
2 years in N. benthamiana and tomato plants. Combined inocu-
lation of VIGS vectors by both Agrodrench and leaf infiltration
methods induce effective gene silencing for longer period. Fur-
ther, we report the transmittance of VIGS to progeny seedlings.
Results from this study will help researchers adapt VIGS as a
tool to study gene function in plant development and plant
responses to stress.
Results
VIGS in N. benthamiana and tomato persisted for
24 months and beyond
To assess the persistence of TRV-VIGS for longer duration, we
targeted three different genes namely PDS, ChlH and CpTF-TuB.
Agrobacterium strains containing these TRV-VIGS constructs
were inoculated into N. benthamiana and tomato plants by
Agrodrench and⁄or leaf infiltration. Silencing of NbPDS and
NbChlH in N. benthamiana resulted in photo-bleaching and yel-
lowing phenotypes, respectively, while the silencing of NbCpEF-
TuB resulted in pale yellow (mosaic) leaves (Figure 1a). Similarly,
two tomato species Solanum lycopersicum and S. pennellii
plants silenced with the SlPDS gene also showed clear photo-
bleaching (Figure S1a). Various parameters including frequency,
efficiency and effectiveness of gene silencing as defined earlier
(Senthil-Kumar et al., 2007) were measured from the time of
initiation of silencing till 24 months postinoculation (mpi).
Frequency of gene silencing
Frequency of gene silencing was calculated by counting the
number of plants that showed silencing phenotype as described
earlier (Senthil-Kumar et al., 2007) and in Experimental proce-
dures section. At 24 mpi, the frequency of gene silencing was
more than 80% for all the three genes that were targeted in
N. benthamiana (Figure 1a). Silencing was observed in all
N. benthamiana plant parts ranging from leaf, stem, flower and
capsule (Figure 1b,c). The silencing phenotype was observed
more in leaves and stem with more than 70% primary and
secondary branches showing
(Figure 1c). However, visible silencing phenotype in reproductive
organs like flowers and capsules was relatively low (Figure 1b).
In tomato, S. pennellii showed highest frequency of gene silenc-
ing (>90%) at 24 mpi compared to S. lycopersicum which
showed only 78% (Figure S1a) and silencing phenotype was
clearly visible on stem, flowers and fruits (Figure S1b).
visiblesilencing phenotype
Efficiency of gene silencing
Silencing of genes used in this study showed varying levels of
reduction in total chlorophyll content ranging from 30% to
95%. Not all leaves of a plant silenced for a gene showed visi-
ble silencing phenotype. Hence, as previously shown (Senthil-
Kumar et al., 2007), we assessed efficiency of gene silencing by
estimating chlorophyll content in both leaves that showed
silencing phenotype and green leaves of N. benthamiana and
tomato. At the end of 24 mpi, the leaves showing silenced phe-
notype for NbPDS and NbChlH had more than 95% efficiency
of gene silencing. However, the green leaves from the same
plants showed efficiency of <40%. The leaves showing silencing
phenotype from NbCpEF-TuB-silenced plants showed about
40% efficiency of silencing, and the leaves without silencing
phenotype showed levels similar to control plants (Figure 1d).
Less reduction in chlorophyll in NbCpEF-TuB-silenced plants may
be because of the nature of gene-silencing phenotype, as plants
silenced with this gene produced only mosaic or pale yellow
leaves. Similarly, the efficiency of gene silencing was more than
95% in the leaves showing photo-bleaching phenotype for
SlPDS gene in both species of tomato (Figure S1c).
Endogenous transcript levels of PDS, ChlH and CpEF-TuB
genes were quantified by real-time PCR in N. benthamiana at
24 mpi (Figure 1e). Leaves that showed visible silencing pheno-
type had more than 50% reduction in their respective endo-
genous target gene transcript levels. The transcript reduction
was <40% in leaves without visible silencing phenotype.
Effectiveness of gene silencing
Effectiveness of gene silencing was calculated by counting the
number of leaves that showed silencing phenotype within a
plant as described earlier (Senthil-Kumar et al., 2007) and in
Experimental procedures section. Nicotiana benthamiana plants
inoculated with both Agrodrench and leaf infiltration methods
showed more than 50% effectiveness of gene silencing at
24 mpi (Figure 2). A similar trend was observed in tomato
plants (Figure S1d). These tomato plants also had reduced
endogenous transcript levels (Figure S1e).
Taken together, our data clearly indicated that VIGS can
effectively persist both in N. benthamiana and in tomato for
more than 24 months. Plants silenced with these genes are still
maintained in our green house facility and they continue to
show silencing phenotype, indicating that the gene silencing
can continue beyond 24 mpi, possibly till the death of the
plant.
Agrodrench coupled with leaf infiltration showed
higher effectiveness of gene silencing for longer
duration
To study the influence of TRV titre levels and environmental
influence on the effectiveness and duration of gene silencing,
we inoculated TRV::NbPDS into N. benthamiana plants by Agro-
drench and leaf infiltration methods individually and in combi-
nation. The plants were maintained under two different
environmental conditions as described later. We hypothesised
that combining Agrodrench and leaf infiltration would provide
higher TRV inoculum levels in plants. Agrodrench inoculation
results in TRV infection and multiplication in root tissues and
results in the spread to aerial plant parts (Ryu et al., 2004). Leaf
infiltration supplies TRV inoculum mainly to the inoculated
leaves and systemic spread occurs from these leaves.
Comparison of effectiveness of gene silencing among
different inoculation procedures
We observed the effectiveness of gene silencing of PDS gene in
N. benthamiana plants until 24 mpi. With all the three methods
(described earlier), the plants silenced for PDS gene showed
more than 90% effectiveness of gene silencing by 1 mpi and
gradually declined thereafter (Figure 2). The plants inoculated
with combined Agrodrench and leaf infiltration method consis-
tently showed higher effectiveness of gene silencing compared
to other methods until 24 mpi. The decline in effectiveness of
gene silencing was much faster in the plants inoculated by leaf
ª 2011 The Authors
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 797–806
Muthappa Senthil-Kumar and Kirankumar S. Mysore
798

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(a)
[83.3±16.6]
[87.5±12.5]
[100±0]
Vector control
TRV::NbCpEF-TuB
TRV::NbChlH
TRV::NbPDS
(d)
100
80
120
Efficiency of gene silencing
(b)
[6±0.7]
[44.3±14.4][70.1±5.1]
a
a
0
20
40
60
(c)
[77.8±10.8]
[72.6±7] [88.4±2.4]
c
b
bc
bc
bc
Vector
control
TRV::NbPDS
TRV::NbChlH
TRV::NbCpE
F-TuB
TRV::NbPDS
TRV::NbChlH
TRV::NbCpE
F-TuB
White or
yellow or pale
green leaf
Green leaf
Reduction in chlorophyll (%) over WT
(e)
Treatment
Silencing
Silencing
phenotype
Presence (or)
absence of TRV
(TRV2 CP)
Presence (or)
absence of Agro
(Atu0792)
Endogenous
transcript
reduction (%)
Wild-
type
Green Green
Vector
control
TRV::NbPDS
TRV::NbChlH
TRV::NbCpEF-TuBPlasmid
Green Pale green
control
control
TRV::NbCp
EF-TuB
TRV::Nb
ChlH
TRV::Nb
PDS
Vector
control
Green White GreenYellow
–++++++++
(100 %) (100 %) (87.50 %) (75%) (100 %)(85.75 %)(100 %)(100 %)
–
–
––––––+
(100 %)(100 %) (100 %) (100 %)(100 %) (100 %)(100 %)(100 %)
00
22.6
±4.5
51.6
±0.1
15.9
±0.1
73.1
±0.01
31.9
±4.7
58.9
±0.33
NA
Figure 1 Persistence of virus-induced gene silencing (VIGS) in N. benthamiana. Twenty-one-day-old seedlings were inoculated with Agrobacterium
harbouring TRV::NbPDS or TRV::NbChlH or TRV::NbCpEF-TuB by both Agrodrench and leaf infiltration. Photographs of plants showing photo-bleaching
(TRV::NbPDS), yellowing (TRV::NbChlH) or mosaic leaf phenotype (TRV::NbCpEF-TuB) were taken at 24 months postinoculation (mpi). The inset picture
shows representative leaf from respective gene-silenced plants (a). Values in parenthesis indicate frequency of gene silencing (a). Photographs of repre-
sentative flower & adjacent plant parts (b) and secondary branch (c) with silencing are shown. Values in parenthesis show the frequency of silencing in
respective organs (b, c). Extent of leaf tissue chlorophyll reduction was assessed both from leaves with silencing phenotype and no-phenotype. T-test
(LSD) was performed using GLM procedure of SAS software (P = 0.01). Letters above each bar indicate the significance and bars with the same letters
are not significantly different (d). Presence or absence of TRV2 and Agrobacterium was assessed both in leaves with silencing phenotype and
no-phenotype and the results were tabulated (e). Values in parenthesis represent percentage of leaves showing presence (+) or absence ()) of TRV2 or
Agrobacterium (e). The endogenous transcript levels of respective genes in their gene-silenced plants were quantified by real-time quantitative PCR (e).
All the observations were performed at 24 mpi. Two biologically independent experiments were performed with at least five replicate plants for each
gene. Error bars in graph and ±values indicate standard error values. PDS, phytoene desaturase; ChlH, Mg-protoporphyrin chelatase; CpEF-TuB, chloro-
plast elongation factor TuB; Atu, Agrobacterium tumefaciens chromosomal gene #0792; vector control, virus vector control plants (TRV::GFP); TRV,
tobacco rattle virus; WT, wild-type; NA, not applicable.
ª 2011 The Authors
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Persistence and inheritance of VIGS in plants 799

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infiltration alone, while the plants inoculated with Agrodrench
alone showed intermediate trend. A similar trend was observed
in N. benthamiana plants silenced for ChlH and CpEF-TuB genes
(Figure S2a,b,c). However, at least few leaves showed silencing
all through the duration across all three inoculation methods.
Taken together, our data suggest that plants inoculated by both
Agrodrench and leaf infiltration showed higher effectiveness of
gene silencing for a longer period, while the reversal of gene
silencing phenotype was higher in plants inoculated by leaf infil-
tration alone.
VIGS and environmental effects
Previous reports (Fu et al., 2006; Tuttle et al., 2008) showed
that temperature is a key player in influencing the silencing
phenotype development in plants. To study the influence of
temperature on persistence of gene silencing, one batch of
plants inoculated with TRV::NbPDS by Agrodrench alone, leaf
infiltration alone or combined inoculation methods was grown
at 18–20 ?C and the other batch at 28–30 ?C. The plants
grown at 18–20 ?C showed profuse growth and provoked clear
photo-bleaching phenotypes in plants infected by all three types
of inoculation methods and silencing continued for 24 mpi and
beyond. However, the plants grown at 28–30 ?C showed only
mild gene silencing phenotype with a few white streaks on
leaves and had comparatively reduced plant growth. Photo-
bleaching phenotype lasted for a month in these plants and
then recovered back to green leaves. Our results showed that
TRV itself got eliminated in most of recovered green leaves of
these plants (data not shown). The frequency of gene silencing
was drastically reduced at 1 mpi when the plants were main-
tained at high temperature. At 3 mpi, only 10% of plants
showedvariegated photo-bleaching
Similarly, the effectiveness of gene silencing was also reduced
at 1 mpi (Figure 3b), and at 3 mpi, only few white streaks were
observed in few leaves. These results suggested that lower
phenotype(Figure 3a).
temperature is important for maintenance of gene silencing for
longer duration. Hence, all our data described in this manu-
script were obtained from plants maintained at 18–20 ?C.
Agrobacterium-mediated transient gene expression and virus
multiplication are also known to be influenced by temperature
(Fu et al., 2006; Ryu et al., 2004; Tuttle et al., 2008).
Plant vigour
PDS gene-silenced N. benthamiana plants produced an average
of about 1600 broad leaves per plant (n = 5) during first year,
and during the second year, this was reduced to an average of
900 leaves per plant. During the second year, the newly
emerged leaves were tiny and wrinkled. A similar trend was
seen in mock inoculated control plants, TRV::NbChlH and
TRV::NbCpEF-TuB plants (Figure S2a). Also, development of
healthier leaves took longer time compared to younger plants
and number of new branches decreased over period. This
indicates that plant vigour also plays a role in influencing effec-
tiveness of VIGS (Figure S2a,b). Therefore, an optimum environ-
ment that can balance both effective virus multiplication and
plant vigour is necessary to maintain higher silencing for a
longer period.
80
100
120
1.0 mpi
a
12 mpi24 mpi
bb
b
ab
00
20
40
60
c
c
d
d
Agrodrench + Leaf
inoculation
Agrodrench Leaf inoculation
Effectiveness of gene silencing (%)
Figure 2 Effectiveness of virus-induced gene silencing (VIGS) for
TRV::NbPDS construct in N. benthamiana. Twenty-one-day-old seedlings
were inoculated with Agrobacterium cultures harbouring TRV::NbPDS by
the indicated types of inoculation. These plants were maintained at
22 ?C. Later, at the indicated time duration, the effectiveness of gene
silencing was assessed. Each inoculation method had 10 plants. Values
presented in the graph are average of two independent experiments.
Error bars in the graph represent standard error values. Single factor
ANOVA was performed (P = 0.05), and letters above each bar indicate the
significance. Bars with the same letters are not significantly different
from each other. mpi, months post inoculation.
100
120
22oC
28oC
(a)
a
a
40
60
80
b
100
0
20
3 mpi1 mpi
Frequency of gene silencing (%)
(b)
c
60
80
a
b
0
20
40
Effectiveness of gene silencing (%)
22oC 28oC
Figure 3 Influence of environmental temperature on TRV::NbPDS medi-
ated virus-induced gene silencing (VIGS) in N. benthamiana. Twenty-
one-day-old seedlings were inoculated with Agrobacterium cultures
harbouring TRV::NbPDS by syringe infiltration of leaves. One batch of
plants (n = 10) were maintained at 22 ?C and another batch (n = 10) at
28 ?C. Frequency of gene silencing was assessed at 1 month postinocu-
lation (mpi) and 3 mpi (a). Effectiveness of gene silencing was assessed
at 1 mpi (b). Values presented in the graphs were average of two inde-
pendent experiments. Error bars in the graph represent standard error
values. Single factor ANOVA was performed (P = 0.05), and letters above
each bar indicate the significance. Bars with the same letters are not
significantly different from each other.
ª 2011 The Authors
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 797–806
Muthappa Senthil-Kumar and Kirankumar S. Mysore
800

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Presence of virus is necessary for persistence of VIGS
for longer duration
To know the reason for higher levels of gene silencing for a
longer duration in N. benthamiana plants inoculated by Agro-
drench combined with leaf infiltration, we looked for the pres-
ence of Agrobacterium (used for Agrodrench) in root surfaces
and the rhizosphere. Washates from root surface and soil sam-
ples were plated on LB medium with appropriate antibiotics and
no bacterial growth was observed (Figure S3a). However, plates
without antibiotics showed bacterial growth. Colony PCR of
these bacterial colonies with Agrobacterium Atu gene-specific
primers did not show amplification, indicating that these colo-
nies were not Agrobacterium (Figure S3b). As the possibility of
the presence of Agrobacterium in soil or root surface is ruled
out, we further looked for presence of TRV in leaf tissues. One
of the previous studies (Valentine et al., 2004) showed that TRV
constructs can be maintained for relatively long time in certain
root cells. Hence, another possibility in our case could be the
stable integration of T-DNA containing TRV1- and TRV2-VIGS
constructs in some primary root cells acting as a TRV source. We
isolated genomic DNA from the primary root tissues of these
plants and performed PCR analysis. The results showed that an
average of about 1.5% of root samples tested showed the
amplification of NOS terminator present in the VIGS-vector
(Figure S4a,b). Most likely this could be because of integrated
T-DNA, but it is not feasible to do T-DNA integration assay on
few root cells to confirm this possibility. Taken together, these
results showed that continuous presence of TRV is needed for
VIGS to hold effective gene silencing for a longer period. This
explains the reason for persistence of effective gene silencing
for longer duration in plants inoculated by Agrodrench. The
enhanced effects in plants inoculated by both Agrodrench and
leaf infiltration may be caused by additive effect of virus titre.
The spread in gene silencing could be because of presence of
diffusible silencing signal [e.g. siRNA, (Voinnet, 2001)] or because
of TRV itself. To determine which, we tested the presence of TRV
in N. benthamiana leaves that were showing silencing phenotype
and also green leaves at 24 mpi. Using RT-PCR, we were able to
amplify TRV2-CP gene both from leaves showing silencing pheno-
type and from green leaves indicating the presence of TRV
(Figure 1e). However, we could not detect the virus in some
green leaves, and this could be because of recovery of plant tis-
sues from virus infection. Similarly, gene-silenced tomato leaves
also had TRV. However, in tomato, the percentage of leaves
showing TRV in the photo-bleached leaves was much less com-
pared to N. benthamiana (Figure S1e). Further, leaf sap from
plants previously displaying silencing phenotypes effectively trig-
gered VIGS in N. benthamiana (Figure S5a) and produced typical
virus-specific lesions in Chenopodium amaranticolor (Figure S5b).
This indicates that virus is present with insert (fragment of PDS
and ChlH) in both green leaves and also leaves showing silencing
phenotype. Taken together, these results showed that VIGS
persists for longer duration in N. benthamiana and tomato plants
by continuous presence of TRV.
Transmittance of VIGS to N. benthamiana and tomato
progeny seedlings
Seeds collected from TRV::NbPDS and TRV::NbChlH-infected
plants at 24 mpi were germinated on MS medium. Contrary to
seeds from stable RNA interference (RNAi) Arabidopsis plants
silenced with PDS gene (Tao et al., 2005), seeds from our VIGS
study germinated well (more than 94% germination). Germi-
nated seedlings did not show any characteristic virus symptoms,
but some seedlings showed clear gene-silenced phenotype. The
frequency of gene silencing was about 1.0% (Figure 4a), and
effectiveness of gene silencing was more than 95% in the seed-
lings showing silencing phenotype (Figure 4b). Further, the
progeny seedlings showing silencing phenotype had more than
85% reduction in their respective endogenous transcript levels
(Figure 4b). We also observed transmittance of VIGS to next
generation in tomato (Figure S6a,b). Presence of TRV was
observed in all the seedlings that showed gene silencing pheno-
type (Figure 4c,d), and the sap from these seedlings provoked
silencing in wild-type N. benthamiana (Figure 4d) and lesions in
C. amaranticolor (Figure 4e). Similarly, the green progeny seed-
lings also had virus with insert, and the sap from these seedlings
also provoked silencing in N. benthamiana. These results indi-
cated that the transmittance of VIGS to progeny seedlings is
due to transmittance of TRV through the seeds. We found that
approximately 47% of green seedlings that had TRV showed
reduction in endogenous transcript levels of target gene (e.g.
ChlH), even though they did not show any visible phenotype.
This suggests that VIGS can actually occur at higher frequency
in progeny seedlings (Figure S7a). The extent of gene down-reg-
ulation was probably not high enough in these seedlings to see
a visible phenotype. Seedlings (progeny-1) that showed gene
silencing phenotype (yellow plants) were grown, with extra care,
until maturity and seeds were collected (Figure S7b). Seedlings
(progeny-2) germinated from these seeds showed on an average
approximately 10% frequency of visible gene silencing pheno-
type (Figure S7c). This 10-fold increase in visible silencing fre-
quency from progeny-1 to progeny-2 may be an indication that
TRV along with insert is getting adapted to the host, and hence,
VIGS is stabilised over generations. The effectiveness of gene
silencing in progeny-2 ranged from 80% to 90% (Figure S7d),
and this was similar to progeny-1. The progeny-2 plants did not
show any virus symptoms, and their growth was normal
(Figure S7e). We also observed that the inheritance of gene
silencing does not depend on the method of inoculation (Agro-
drench or leaf infiltration). We refer to this method as noninte-
gration-based seed transmissible VIGS. This method, unlike stable
RNAi, does not involve genetic manipulation of plant DNA.
Discussion
VIGS is generally considered a transient assay system. In this
study, we have demonstrated that TRV-based VIGS can last for
more than 2 years. Although influence of environmental factors
on VIGS are known (Fu et al., 2006; Tuttle et al., 2008), the
exact mechanism that contributes to persistence of VIGS for
longer duration is largely unknown. In this study, we proposed
two hypotheses to explain the persistence of VIGS for longer
duration in N. benthamiana and tomato. First, Agrobacterium
may survive in the rhizosphere for a longer period and act as a
source of TRV delivery into root cells. However, our results ruled
out this possibility. Second, we hypothesised continuous pres-
ence of TRV-VIGS vector in the plant tissue. Indeed we
observed continuous presence of TRV in N. benthamiana and
tomato plantsfor 2 years.
the mechanism for persistence of TRV in mature plants. There
are two possible explanations for continuous presence of TRV.
One possibility is that the T-DNAs carrying TRV1 and TRV2 were
stably integrated into plant genomic DNA during transient
Hence,wefurther explored
ª 2011 The Authors
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 797–806
Persistence and inheritance of VIGS in plants 801

Page 6

expression as previously described (Zottini et al., 2008) and con-
tinue to produce virus carrying full length inserted sequence.
Our preliminary results indicate approximately 1.5% chance of
such integration of T-DNA carrying one of the TRV genomes in
primary root cells. T-DNAs carrying both TRV genomes have to
be integrated to produce a virus particle. The chances of that
will be much <1.5%. Therefore, it is unlikely that this is the
main contribution for the continuous presence of TRV. The
other likely possibility could be that the initial inoculum of TRV
is the actual source of virus that continue to escape silencing,
multiply and spread to newer tissues. Apart from the presence
of TRV, it is also possible that siRNA or other silencing signals
(Li et al., 2006; Palauqui et al., 1997) can account for persis-
tence of gene silencing even without the presence of virus.
However, short-distance movement of the silencing signal of
PDS gene during potato virus-X (movement defective mutant)-
based VIGS in N. benthamiana has produced only a weak and
transient photo-bleaching phenotype (Voinnet, 2001). Hence,
we conclude that the persistence of VIGS for long duration
in our experiment is because of ubiquitous presence of virus.
Initial inoculums of virus acted as source, and further multipli-
cation of virus and escape from targeted VIGS against virus
contributed for continuous presence.
Our results indicate that TRV titre may be decreasing after
3–4 months in some plant species (for example in tomato), thus
decreasing the effectiveness of silencing in the leaf infiltrated
plants (Figure 5). Initiation and systemic spread phases of VIGS
(Benedito et al., 2004; Ruiz et al., 1998) are dependent on the
virus titre in the plant (Hiriart et al., 2003; Liu et al., 2002b;
Ruiz et al., 1998; Voinnet, 2001). Hence, TRV is necessary for
initiation of VIGS in a new cell and for persistence of silencing
(Figure S8a,b,c). Our results suggest that Agrodrench coupled
with leaf inoculation provoke effective silencing. Booster dose
of inoculation is useful to maintain higher level of TRV and thus
VIGS in tomato. Maintenance of environmental conditions are
also important for effective silencing. Decrease in effectiveness
of VIGS over a period of time can be because of loss of plant
vigour or because of the type of gene being silenced (Figures 5
and S2a,b). Hence, adequate plant care to maintain healthy
growth of gene-silenced plants is important.
Persistence of VIGS for longer duration can have significant
impact on functional genomic studies in plants. Uniform VIGS
for entire plant duration can potentially substitute for mutants
and stable RNAi lines. Persistence of VIGS for longer duration
will allow researchers to perform wide range of assays in stud-
ies focused on assessing abiotic and biotic stress–related genes.
For example, the function of a gene imparting stress tolerance
can be assessed by subjecting plants to stress from seedlings to
the terminal growth stage of plants. This is not possible with
the presently available VIGS protocols because of the transient
+
+
+
+
+
+
+
+
+
+
+
+
Vector
control
–
TRV::NbPDS
[1.0±0.32]
Vector control
(a)
TRV::NbChlH
[1.08±0.37]
(b)
TRV::GFP TRV::NbPDS TRV::NbChlH
0
100
0 97.5±0.15
(c)
Effectiveness of gene silencing (%)
Endogenous transcript reduction (%)
96.66±3.3
87.4±3.9
(d)
TRV::NbPDS
[42±4.8] [15.8±9.6]
(75%) (100%)(25%) (100%) (25%)
TRV::NbChlH
(e)
Sap from
white/yellow
Sap from
green
[16.6±7.8]
[8.33±2.7]
Sap from white/yellow
seedlings
Sap from green seedlings
TRV::NbPDS
TRV::NbChlH
TRV::NbPDS
TRV::NbChlH
TRV positive
control
Figure 4 Transmittance of virus-induced gene
silencing (VIGS) to progeny (P1) seedlings in
N. benthamiana. Seeds were collected from the
TRV::NbPDS plants (n = 10) or TRV::NbChlH
plants (n = 10), inoculated by both Agrodrench
and leaf infiltration at 24 months postinocula-
tion. A total of 1027 seeds from TRV::NbPDS
and 972 seeds from TRV::NbChlH plants were
sown on MS medium. Cotyledonary leaves of
seedlings showed typical photo-bleaching or yel-
lowing phenotype (a; also see insert). Frequency
of gene silencing was calculated at 20 days after
sowing (DAS) and values are given in parenthesis
(a). Effectiveness of gene silencing (b, top panel)
and extent of corresponding endogenous
transcript reduction (real-time PCR) at 20 DAS
(b, bottom panel) were calculated for the leaves
with silencing phenotype (white⁄yellow). Also
the presence (+) or absence ()) of TRV both in
seedlings with and without silencing phenotype
was assessed at 20 DAS by RT-PCR (c). Values in
parenthesis indicate percentage of seedlings that
had the virus (c). Further, leaf sap was extracted
from progeny seedlings of TRV::NbPDS (n = 10)
and TRV::NbChlH (n = 15) plants and inoculated
on to lower leaves of 3-week-old wild-type
N. benthamiana plants. Photographs of these
plants were taken at 10 dpi (d). Values in paren-
thesis indicate frequency of silencing (d). Lesion
assay was also performed on Chenopodium
amaranticolor plants and photographs were
taken at 5 dpi (e). ± indicates standard error
values.
ª 2011 The Authors
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 797–806
Muthappa Senthil-Kumar and Kirankumar S. Mysore
802

Page 7

nature of VIGS. In case of biannual or perennial plants, VIGS
can persist for several years possibly till the death of the plant
and this will provide an opportunity to study function of genes
involved in senescence.
We accidentally observed photo-bleaching phenotype in
seedlings emerged from seeds fallen from TRV::NbPDS inocu-
lated N. benthamiana plants (Senthil-Kumar, 2007). We further
analysed this phenomenon and propose possible reasons for
such transmittance of silencing to progeny seedlings. First, seed
transmission of TRV-VIGS constructs is possible. TRV encoded
16 kDa protein has been implicated to have a role in seed
transmission (Liu et al., 2002a; MacFarlane, 1999). About 40%
chance of seed transmission of TRV has been reported in spin-
ach, beet, potato and tobacco (Hoek et al., 2006). TRV is
reported to be present in pollen and fertilised flowers and can
then enter the seed through the megaspore mother cell and
egg, or through the pollen mother cells and premeiotic pollen
(Johansen et al., 1994). A recent study (Marton et al., 2010),
along with our results, showed the presence of TRV in the
progeny seedlings. Hence, we propose that the seed transmit-
tance of TRV is the main cause for transmittance of gene silenc-
ing to progeny. Such epigenetic transmittance of gene silencing
to progeny seedlings should also be possible in other VIGS sys-
tems that involve viruses, like Barley stripe mosaic virus (BSMV),
Apple latent spherical virus, Cucumber mosaic virus (CMV) and
Pea early browning virus, because these viruses are reported to
be seed transmitted under natural conditions (Johansen et al.,
1994) and also feasible in other virus-host combinations. It war-
rants further investigation to understand the exact mechanism
of transmittance of VIGS over generations. Second, possibility
for inheritance of gene silencing in progeny seedlings could be
because of RNA-directed DNA methylation (RdDM)-mediated
promoter methylation of target gene in seeds that lead to tran-
scriptional gene silencing (TGS) (Jones et al., 2001; Robertson,
2004). A recent study showed that CMV-mediated heritable
gene silencing in Petunia and tomato can be induced by target-
ing double-stranded RNA to the endogenous gene promoters
(Kanazawa et al., 2010). We have not tested this possibility, as
the presence of TRV was ubiquitous and correlated with our
silencing results.
In conclusion, our study showed that persistence of TRV-
VIGS for longer duration can be achieved, possibly till death,
in N. benthamiana and tomato. Our study also demonstrated
a possibility for a nonintegration-based persistent seed trans-
missible VIGS. Our results indicate a need for suitable bio-
safety regulation for containment of seeds from VIGS vector
inoculated plants. Inheritance of VIGS will be useful in the
study of genes involved in seed germination, early seedling
emergence and vigour in a plant which is not genetically
manipulated. However, more evidence, through systematic
studies in several crop plants with various viral vectors, are
required to consider seed transmissibility of VIGS to several
generations as an alternative to stable gene silencing methods
like RNAi.
Experimental procedures
Plant material and environmental conditions
Nicotiana benthamiana and tomato (Solanum lycopersicum var.
Glamour and wild-type S. pennellii) plants were used in this
study. S. pennellii seeds were obtained from Tomato Genetics
Resource Center, Davis, CA. All plants were grown in green
house (unless otherwise specified), under the following growth
conditions. Day and night temperatures were 19 ± 2 ?C and
18 ± 2 ?C, respectively. Day length was 14 h with 600 lmol⁄
m2⁄s light intensity. Relative humidity of the green house was
maintained at 65%–70%. We have provided real-time values
(for a representative month) of the green house environmental
conditions (Table S1). Optimal plant care was necessary to main-
tain plants for longer duration. For continuous new growth of
. . ..
.
.
Hypothesized
..
.
...
.
.
.
30
35
40
45
.
..
...
.. ..
Duration of silencing or
age of the plant
(months)
R
R
R
Observed
..
.
.
.
10
15
20
25
..
.
..
.
..
..
Effectiveness of gene silencing
Agrodrench+leaf
Agrodrench+leaf
inoculation
. .
0
5
Leaf inoculation Agrodrench
Figure 5 A model explaining the persistence of TRV-Virus-induced gene silencing (VIGS) in N. benthamiana over a longer period. We propose that
TRV-based VIGS (e.g. PDS silencing) in N. benthamiana can persist until plant death. However, the efficacy of gene silencing varies depending on TRV
titre and age of the plant. Extent of effectiveness of gene silencing can be influenced by method of TRV inoculation as this can control initial TRV titer
in plant cell. X-axis indicates duration of gene silencing or age of the plant (both in months). Y-axis of each triangle shows the effectiveness of gene
silencing (or titer of TRV). Arrows with ‘R’ indicates the recovery of photo-bleaching phenotype (70% or more green leaves per plant). White and
green (light and dark) colour in the triangle indicates photo-bleaching and green leaves, respectively. The phenotypic observations were made until
about 30 months postinoculation and the expected pattern is given for rest of the duration. This model can be applied to VIGS of two other genes
(e.g. ChlH and CpEF-TuB) and also can be extended to other Solanaceae plant species (e.g. tomato).
ª 2011 The Authors
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 797–806
Persistence and inheritance of VIGS in plants 803

Page 8

leaves, starting with 3 months after sowing, plants were pruned
and flowering was allowed only when required. Regular repot-
ting (into bigger pots) with fresh soil medium was performed to
provide adequate soil for root growth. More details about plant
care are described earlier (Ryu et al., 2004; Senthil-Kumar et al.,
2007).
VIGS constructs
pTRV1 and pTRV2 VIGS vectors were obtained from Dr S.P.
Dinesh-Kumar (Yale University, USA). Constructs used in this
study include TRV::PDS (PDS, phytoene desaturase from either
N. benthamiana-NbPDS or tomato-SlPDS), TRV::NbChlH (ChlH,
Mg-protoporphyrin chelatase
TRV::GFP (GFP, green fluorescent protein-a nonplant gene). The
GFP gene was obtained from the jellyfish Aequorea victoria (Ha-
seloff et al., 1997). A 451-bp GFP fragment was amplified
(using primers described in Table S2), from N. benthamiana
16C transgenic plants (Brigneti et al., 1998) and cloned into
pTRV2 VIGS vector. Plants infected with TRV::GFP were consid-
ered as virus vector control. Fragment of PDS gene from
N. benthamiana or tomato cDNA was amplified and cloned into
pTRV2 vector to make respective TRV::PDS constructs (Senthil-
Kumar et al., 2007). A 365-bp ChlH gene fragment was ampli-
fied by RT-PCR (using primers described in Table S2) from
N. benthamiana plants and cloned into pTRV2 to make
TRV::NbChlH construct (Ryu et al., 2004). More details about
these vector constructs are described in earlier studies (Ryu
et al., 2004; Senthil-Kumar et al., 2007). TRV::NbCpEF-TuB
(CpEF-TuB, chloroplast elongation factor TuB, NCBI accession #
GU968906) construct was selected from a N. benthamiana
cDNA library (Anand et al., 2007). Construct details are given in
Figure S9.
from
N. benthamiana) and
VIGS protocol and inoculation methods
The Agrobacterium strain GV2260 containing pTRV1 or pTRV2
derivatives were grown at 28 ?C in LB medium containing
appropriate antibiotics. The cells were harvested from overnight
grown cultures and re-suspended in the infiltration buffer
(10 mM 2-(N-morpholino) ethanesulfonic acid pH 5.5; 200 lM
acetosyringone) to a final absorbance (OD600= 1.0) and incu-
bated for 2 h at room temperature in a shaker. For leaf infiltra-
tion, each Agrobacterium strain containing pTRV1 and pTRV2
derivatives were mixed in 1 : 1 ratio in MES buffer (pH 5.5) and
used for inoculation (OD600= 0.5) (Liu et al., 2002b; Ratcliff
et al., 2001). Agrobacterium tumefaciens strain GV2260 carry-
ing pTRV1 and pTRV2 (or its derivatives) were delivered into
plants by Agrodrench or leaf infiltration or both. Agrodrench
involves inoculation of Agrobacterium cultures carrying VIGS
vector into plant roots. Leaf infiltration was performed by using
a needless syringe (Ryu et al., 2004). Inoculation of 10- to 15-
day-oldtomatoseedlings and
induced effective silencing (unless otherwise specified).
21-day-old
N. benthamiana
Assessing the presence of TRV and Agrobacterium
To assess the presence of TRV, RT-PCR was performed using
TRV2 CP-specific primers. For synthesis of first-strand cDNA,
2 lg of total RNA was reverse transcribed (Omniscript RT kit;
Qiagen Inc, Valencia, CA) using reverse primer of CP. Presence
or absence of Agrobacterium GV2260 in the silenced plant root
surface and rhizosphere was assessed by washing the soil and
root surface with water, collecting the water, and plating on
Luria–Bertani (LB)-agar medium with appropriate antibiotics or
without antibiotics. DNA from colonies that grew on plates
without antibiotics was subject to PCR amplification using
A. tumefaciens gene Atu0792 (NCBI accession # NP_353815)-
specific primers. All primer details are given in Table S2.
Real-time PCR to quantify endogenous gene transcript
levels
To quantify the endogenous transcript levels in the respective
gene-silenced plants, real-time quantitative RT-PCR (qRT-PCR)
was performed. The total RNA was extracted from silenced and
mock infiltrated plants and the first-strand cDNA was synthes-
ised using oligo (dT)15primers. qRT-PCR was performed using
ABI PRISM 7000 (ABI applied biosystems Inc., Foster city, CA)
using SYBR green (ABI). The primers used were designed using
primer express software 2.0 (ABI). As a control for silenced and
virus vector control plants, parallel reaction using N. benthami-
ana elongation factor 1-a (EF2) was performed and the data
obtained were used to normalise respective gene transcripts.
Each sample was run in triplicate and repeated twice from
pooled samples of three independently silenced and virus vector
control plants. Endogenous transcript levels of PDS, ChlH and
CpEF-TuB genes were calculated by following the protocol
as described earlier (Pfaffl, 2001; Senthil-Kumar et al., 2007).
Primer details are given in (Table S2).
Lesion assay to identify presence of TRV
Progeny seedlings were obtained from seeds collected from
N. benthamiana plants (at 24 mpi) inoculated with TRV::NbPDS
or TRV::NbChlH. Leaf sap was extracted from progeny seedlings
by independently grinding them in phosphate buffer (pH 7.2)
along with carborundum (320 grit; Sigma, St. Louis, MO, USA).
The sap was inoculated into leaves of 4-week-old C. amaranti-
color and 3-week-old wild-type N. benthamiana plants by
gentle rubbing and the inoculated plants were observed for
lesions and visible silencing phenotype.
Scoring VIGS phenotype in N. benthamiana and
tomato
Procedures for assessing frequency, effectiveness and efficiency
of gene silencing are described below and the details of the
same are also reported in our earlier manuscript (Senthil-Kumar
et al., 2007). The frequency of gene silencing was calculated by
comparing the number of plants that showed photo-bleaching
or yellowing phenotype with the total number of plants that
were inoculated with TRV constructs. The effectiveness of gene
silencing, in all the plants that showed photo-bleaching or
yellowing phenotype, was calculated by comparing the number
of leaves that showed symptoms with the total number of
leaves in the plant. To calculate plant vigour, leaves were
counted at the end of a scheduled time interval before pruning
the plant.
Formulae used in this study are as follows:
Frequencyofgenesilencingð%Þ
¼Numberofplantsshowingsilencingphenotype
Totalnumberofplantsinoculated
Effectivenessofgenesilencingð%Þ
¼Numberofleavesshowingsilencingphenotypeinaplant
Totalnumberofleavesinaplant
? 100
? 100
ª 2011 The Authors
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 797–806
Muthappa Senthil-Kumar and Kirankumar S. Mysore
804

Page 9

Estimation of chlorophyll content
Chlorophyll was extracted from 100 mg leaf tissue in ace-
tone:DMSO (1 : 1 v⁄v) mix, and the supernatant was made up
to 1 mL using this mix. The absorbance was recorded at
663 nmand 645 nmusing
Model DU800 (Shimadzu Corporation, Kyoto, Japan). Total
chlorophyll was estimated as described earlier (Senthil-Kumar,
2007; Senthil-Kumar et al., 2007) and expressed as percent
reduction over their corresponding control (wild-type or vector
control plants). As described earlier (Senthil-Kumar et al., 2007),
extent of totalchlorophyllreduction
efficiency of gene silencing.
UV-Visible spectrophotometer
wasconsidered as
Acknowledgements
We thank Ms Janie Gallaway for excellent plant care, Ms Beth-
any Bishop for technical help and Dr Hema Ramanna for help
with TRV lesion assay. We thank Drs Xin Ding, Alex Valentine
and Hema Ramanna for critical reading, and Mrs Pat Weaver-
Meyers for editing the manuscript. VIGS-related projects in
KSM laboratory are supported by the Samuel Roberts Noble
Foundation, National Science Foundation (grant no. 0445799),
Oklahoma Center for the Advancement of Science and Tech-
nology (grant no. PSB09-020) and the U.S.-Israel Binational
Agricultural Research Development Fund (BARD grant no.
IS-3922-06).
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Supporting information
Additional Supporting information may be found in the online
version of this article:
Figure S1 Persistence of VIGS in tomato.
ª 2011 The Authors
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 797–806
Persistence and inheritance of VIGS in plants 805

Page 10

Figure S2 Persistence of VIGS and its relation to plant vigor.
Figure S3 Agrobacterium strain GV2260 containing pTRV1 or
pTRV2 was not present in rhizosphere and primary root surface
of N. benthamiana plants inoculated by both Agrodrench and
leaf infiltration.
Figure S4 PCR amplification of NOS terminator sequences from
N. benthamiana roots to study whether T-DNA integration in
root cells occurred or not.
Figure S5 Inoculation of leaf sap from gene silenced N. benth-
amiana and tomato plants provoked respective gene silencing
in N. benthamiana and produced virus specific lesion in
Chenopodium amaranticolor.
Figure S6 Transmittance of VIGS to progeny seedlings in
tomato.
Figure S7 Transmittance of VIGS to progeny seedlings for two
generations in N. benthamiana.
Figure S8 Booster inoculation of TRV::SlPDS in tomato plants
restored the declined VIGS.
Figure S9 TRV based VIGS constructs used in this study.
Table S1 Details of environmental conditions during plant
growth namely, temperature, relative humidity and light inten-
sity in the greenhouse for one representative month.
Table S2 Details of primers used for real-time quantitative PCR,
detecting TRV & Agrobacterium, assessing T-DNA integration
and pTRV2 plasmid construction.
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ª 2011 The Authors
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 797–806
Muthappa Senthil-Kumar and Kirankumar S. Mysore
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