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109
Fast forward genetics based on virus-induced gene silencing
David C Baulcombe
Gene expression in plants can be suppressed in a sequencespecific manner by infection with virus vectors carrying
fragments of host genes. Recent developments have revealed
that the mechanism of this gene silencing is based on an
RNA-mediated defence against viruses. It has also emerged
that a related mechanism is involved in the post-transcriptional
silencing that accounts for between line variation in transgene
expression and cosuppresion of transgenes and endogenous
genes. The technology of virus-induced gene silencing is
being refined and adapted as a high throughput procedure for
functional genomics in plants.
Figure 1
Addresses
The Sainsbury Laboratory, John Innes Centre, Norwich, NR4 7UH, UK;
e-mail: [email protected]
Current Opinion in Plant Biology 1999, 2:109–113
http://biomednet.com/elecref/1369526600200109
© Elsevier Science Ltd ISSN 1369-5266
Abbreviations
PDS
phytoene desaturase
PTGS
post-transcriptional gene silencing
PVX
potato virus X
RMD
RNA-mediated defense
TBRV
tomato blackring virus
TMV
tobacco mosaic virus
VIGS
virus-induced gene silencing
Introduction
A limiting step in genetics and molecular biology is the
ability to relate genes to phenotypes and vice versa. In the
shadow of genome projects this limitation is more conspicuous than ever. Here, I describe a novel approach to
bridging this gap between genetics and molecular biology.
This approach is based on recent discoveries concerning
plant viruses and gene silencing and is still under development. The early results, however, provide encouragement
that virus-based technology will be a useful complement to
existing functional genomics tools.
The most straightforward iteration of this approach is
referred to as virus-induced gene silencing (VIGS) and
employs virus vectors carrying elements from the exons of
plant host genes. Analyses in Nicotiana species have shown
that the symptoms on these plants are phenocopies of mutations in the corresponding host genes. For example, with
tobacco mosaic virus (TMV) [1] and potato virus X (PVX)
vectors [2•] carrying a fragment of phytoene desaturase
(PDS) mRNA, the upper leaves of the infected plant develop a spectacular bleached effect (Figure 1). The cause of
these symptoms is a decline in the level of the endogenous
PDS mRNA leading to low levels of phytoene desaturase, a
block in carotenoid production and, consequently, the
absence of protection against photobleaching. In other exam-
Current Opinion in Plant Biology
Nicotiana benthamiana plant exhibiting virus-induced gene silencing of
phytoene desaturase. The plant was inoculated three weeks earlier with
a PVX vector construct carrying a 400 nucleotide fragment of phytoene
desaturase cDNA. The bleached symptoms contrast with the normal
mild mosaic symptoms associated with PVX infection on this species.
ples VIGS has been targeted against a chelatase gene
required for chlorophyll synthesis [3•] and against various
transgenes [2•,3•,4••,5].
The obvious attraction of VIGS for analysis of gene function is speed and ease of adaptation to high throughput
systems. If the partial sequence of a given gene is
known, a virus vector construct can be assembled within
two days. This construct may be infectious directly as in
vitro transcripts of cDNA clones [6] or as DNA, if the
virus has a DNA genome. Alternatively the cDNA of an
RNA virus can be fused to a promoter that is active in
plant cells. The cDNA of these constructs is infectious
as directly inoculated DNA [7] or by employing an
A. tumefaciens Ti plasmid vector to transfer the DNA into
plant cells [8].
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Genome studies and molecular genetics
There are two lines of supporting evidence for this model.
First there is direct evidence that both RNA nepoviruses
[11••] and DNA caulimoviruses can induce RMD [12,13••].
Initially the plants infected with these viruses exhibit
symptoms and the viruses accumulate to a high level.
However, the upper leaves of these plants emerging after
systemic infection have recovered. They are symptom free
and have only low levels of viral RNA. This recovery can be
attributed to RMD because, in the nepovirus infected
plants, there is RNA sequence-dependent resistance
against viruses inoculated to these upper leaves [11••]. In
the caulimovirus infected plants the recovery is probably
due to RMD as there is post-transcriptional suppression of
viral RNA in the symptom-free leaves.
Virus accumulation (particles/cell)
Figure 2
Activation of RMD
Time
Current Opinion in Plant Biology
Typical time course of virus accumulation in an infected cell. The graph
illustrates schematically the kinetics of virus accumulation in an
infected cell. There is an initial phase of rapid replication and virus
accumulation that progresses through to the final period when
accumulation has been arrested. The phases shown would take place
over a period of a few days, depending on the type of virus.
After inoculation of the vector there is a delay of only 7–21
days before VIGS-related symptoms develop in the upper
leaves of the infected plant. The relationship from gene to
phenotype, therefore, can be established within a few weeks.
In genomics jargon this strategy is referred to as reverse
genetics. VIGS can also be used in a forward genetics strategy although the resources required are substantial — it
would be necessary to construct cDNA libraries in virus vector constructs and to systematically analyse large numbers of
infected plants.
In the later sections of this article I assess the potential,
the limitations and the practicalities of VIGS both for
forward and for reverse genetics. Because the development of VIGS technology is influenced by knowledge,
however, I first review the current views about the
underlying mechanism.
The mechanism of VIGS
It is likely that VIGS is caused by an RNA-mediated
defense (RMD) mechanism in plants against viruses
[9,10]. According to this idea there is an as yet uncharacterised surveillance system in plants that can specifically
recognise viral nucleic acids and give sequence specificity
to RMD. Normally, with wild-type isolates of plant viruses, it is thought that this mechanism is activated as the
virus begins to accumulate in the plant (Figure 2).
Eventually, as a result of RMD, virus accumulation slows
down and eventually stops.
In the situations when a genetically modified virus has
similarity to a gene in the host plant, the RMD would target both the viral RNA and the corresponding endogenous
mRNA. VIGS would result from the targeting of the
endogenous mRNA
The second supporting evidence for RMD is indirect and
is based on analyses of plants exhibiting post-transcriptional gene silencing (PTGS) of transgenes. PTGS
involves sequence-specific turnover of RNA and is one of
the reasons for between-line variation in transgene expression levels [14]. Lines in which a transgene is expressed at
a low level include those in which PTGS is active and targeted against the transgene RNA. High level expressors
are those in which PTGS is either weak or inactive.
The link with RMD is from the finding that PTGS is suppressed by virus-encoded proteins that are also suppressors of
anti-viral defense in the host [15••–18••]. Presumably the
suppressed anti-viral defense is the putative RMD and the
suppressed PTGS was activated when the transgene or its
RNA was detected by the RMD surveillance system. The
suppressors of PTGS would be part of a counter defense system that allows viruses to accumulate after activation of RMD
[19•]. With some viruses the final steady state level of virus
accumulation would be determined by the effectiveness of
the suppressors. In other instances the virus may use evasion
instead of suppression as a strategy to counteract RMD.
Analysis of PTGS has revealed that there is a signal of
gene silencing that can move systemically in plants
[20••,21••,22•,23•]. This signal can be rationalised in terms
of RMD as it would allow the plant to prevent spread of
the virus. If the signal moves at the same time or ahead of
the virus it would prime RMD in the cells at, or just
beyond the infection front. Consequently the RMD would
be activated more rapidly than in the initially infected cells
and the spread of the virus would be delayed.
A final point about PTGS concerns DNA methylation.
There is an association of DNA methylation and PTGS in
several experimental systems [4••,24–27] and evidence
that the methylation may be targeted to transgenes by
RNA [4••,28]. These observations are difficult to reconcile
with the proposed similarity of PTGS and RMD as most
plant viruses have RNA genomes. Perhaps the DNA
methylation data are an indicator that the underlying
mechanism of PTGS is involved not only in anti-viral
defense. It could be, for example, that antiviral defense is
Fast forward genetics based on virus-induced gene silencing Baulcombe
related to the mechanisms used to protect against retroposons and other forms of mobile DNA in plants [29].
Development of VIGS technology
This relationship of VIGS, PTGS and RMD has important
implications for further development of VIGS technology. It
means, for example, that VIGS would be weak if the virus
vector encodes a suppressor of PTGS and that the most
effective VIGS vectors will be those that use evasion rather
than suppression as a strategy to overcome RMD. Vectors
based on potyviruses, therefore, would be poor VIGS vectors
because they produce strong suppressors of PTGS
[15••,16••,18••]. Cucumber mosaic virus would be unsuitable
for the same reason [16••,17••]. Conversely, PVX and tomato
blackring virus (TBRV) would be suitable vectors for VIGS.
Neither of these viruses is able to reverse PTGS in transgenic plants [16••,17••] and it can be assumed that they do
not produce suppressors or that their suppressors are weak.
TBRV is a seed and pollen transmitted virus as a result of its
ability to infect the progenitor cells of the pollen and eggs in
the meristems [30]. This ability to penetrate the meristem
means that TBRV would be a particularly effective vector
for VIGS. Most plant viruses, including PVX, are excluded
from the meristematic zone of infected plants [31] and
would not be able to silence genes required for meristem
identity and early in leaf and flower development.
Unfortunately, as yet, there are no full length cDNAs of
TBRV or related viruses that could be developed as VIGS
vectors with the ability to target these important genes.
There are several other ways in which understanding of
PTGS could facilitate development of VIGS technology.
For example, it may be possible to enhance effectiveness
of VIGS vectors by engineering increased potential to produce double stranded RNA. Double stranded RNA has
been implicated as an initiator of PTGS in plant systems
[32•,33••,34•] and of a similar gene silencing phenomenon
in nematodes [35,36•].
It may also be possible to enhance VIGS by modification
of the host plant. For example, it might be expected that
VIGS would be stronger in the background of host gene
mutations that enhance gene silencing (egs) [37]. The
products of these genes could be repressors of RMD. It
might also be expected that VIGS would be stronger in
plants that over-express genes required for RMD than in a
wild-type host. The genes that are required for gene
silencing can be recognised by analysis of the suppressed
gene silencing (sgs) [38•] phenotype of mutant alleles.
Transgenic VIGS
In the applications of VIGS, as described above, the activator of gene silencing is the viral RNA in the infected
plant cells. The silencing effect is systemic because the
virus vector is able to spread through the infected plant. In
transgenic VIGS the aim is to produce an RNA that can be
recognised by the RMD surveillance system. As in VIGS,
111
this RNA would include an element from a host sequence
so that the RMD silences the corresponding gene. This
RNA would not need to be infectious, however, because it
would be delivered into cells by expression of a transgene.
An advantage of transgenic VIGS over ‘conventional’
PTGS using sense or antisense transgenes should be consistency. The transgenic VIGS constructs would be
designed to produce an RNA that is detected by the RMD
surveillance system and it would be expected that all, or at
least most, of the lines would exhibit silencing of the target gene. In PTGS it is likely that the transgene RNA is
only recognised by the RMD surveillance system as a
result of sequence rearrangements, DNA methylation of
chromatin structures that are associated with the transgene
after its introduction into the plant genome. As a result, the
degree of PTGS varies greatly between lines.
Transgenic VIGS is less amenable than infectious VIGS to
high throughput applications because of the transformation
step. In plants that can be transformed with high efficiency,
however, it is quite feasible to produce the tens of thousands of transformed lines that would be necessary to
survey the gene function in a typical plant. Once produced,
the seed from these lines would be a permanent resource
that could be used in many different functional screens.
The version of transgenic VIGS corresponding most closely to infectious VIGS employs amplicons. Amplicons are
transgenes comprising a promoter and terminator directing
transcription of a modified viral vector RNA in the plant
cell [39]. The modifications may include deletion or mutation of viral genes required for spread of the virus in the
infected plant and of any other functions that are secondary
to replication. Expression of the amplicon in the plant cell
could activate RMD and, if there are host sequences in the
amplicon construct, PTGS of the corresponding host gene.
There are several instances of amplicon transgenes although
they are not always referred to as such. A PVX amplicon produced consistent silencing as expected [39]. Similarly, a
petunia chalcone synthase gene could be targeted by a gemini virus amplicon with a chalcone synthase insert [40] and a
brome mosaic virus amplicon conferred virus resistance that
was probably due to RMD [41]. The phenotype of amplicons derived from cucumber mosaic virus [42] and a severe
strain of TMV, however, did not appear to involve gene
silencing [43]. The differences between these amplicons
may be due to the suppressors of RMD encoded in the corresponding viral genomes — genomes producing little or no
silencing may produce strong suppressors of RMD, whereas
good silencers may be those that do not encode suppressors
or from which the suppressor genes have been deleted.
Some alternative approaches to PTGS in transgenic plants
may also be considered as transgenic ‘pseudoVIGS’. The
success of these approaches depends on the ability of non
viral transgenes to produce the virus-like RNAs that activate
112
Genome studies and molecular genetics
RMD. The gene silencing from simultaneous expression of
sense and antisense RNAs [33••] or from transgene constructs with inverted repeats in the transcribed regions [32•]
could both be considered in this ‘pseudoVIGS’ category .
Acknowledgements
I am grateful to the Gatsby Charitable Foundation for supporting work in
the Sainsbury Laboratory and to my colleagues for many stimulating
discussions about the role and mechanisms of gene silencing in plants.
References and recommended reading
VIGS versus other techniques of functional
genomics
Conventional approaches to forward genetics generally
involve insertional mutagenesis either by T-DNA [44] or
transposable elements [45,46]. These are powerful
approaches that have led to many advances in gene discovery; however, there are several complications with
insertional mutagenesis of genes required for basic cell function or early development. In these instances, because the
insertional mutation often results in complete inactivation of
the target gene, the mutant phenotype will be embryo lethal
and difficult to interpret in terms of the cellular or biochemical role of the gene product. Conversely, if the mutant gene
is one of a family it is likely that there may be no phenotype
of an insertional mutant because the functional homologues
may compensate for the loss of function at the mutant locus.
VIGS and related PTGS-based approaches may circumvent
these problems. For example, with multigene families, the
gene silencing mechanism will target not only the precise
homologue of the gene in the VIGS vector; it will also target
partial homologues that vary by up to 10 or 20%. The chlorotic and dwarf phenotypes that result from PTGS of tobacco
ribulose bisphosphate carboxylase (rubisco) is a good illustration of how gene silencing can target a multigene family
[47,48]. It is unlikely that a phenotype would result from
insertional mutagenesis of rubisco because it is encoded in a
multigene family. The precise mismatch between different
tobacco rubisco genes is not known but, by comparison with
other solanaceous plants, it is likely to be up to 14% [49].
When VIGS is targeted against lethal genes the phenotype
is death, as in insertional mutagenesis, but the dying
process may be more informative than the embryo lethal
phenotype of insertional mutants. Because the VIGS is
applied to mature plants and because there may be only
partial suppression of the target gene, the dying process
may be informative about the role of the corresponding
gene products. It might be expected, for example, that
death due to a block in lipid biosynthesis would be clearly
different from death due to suppression of DNA synthesis
or proteins required for respiration
Conclusions
VIGS vectors have been developed from the genomes of
TMV, PVX and TGMV for applications in Nicotiana
species. To develop these vectors for use in other plant
species it will be possible to build on recent discoveries that
VIGS and PTGS are related to antiviral defense in plants
and that viruses produce suppressors of this antiviral
defense. When these vectors are available for Arabidopsis,
maize and other species it will be possible to combine insertional mutagenesis and VIGS to develop a powerful
combined approach to functional surveys of plant genomes.
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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Fast forward genetics based on virus-induced gene silencing Baulcombe
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[15 ,17••,18••] provide indirect but compelling evidence that RMD is a general defense mechanism against plant viruses and that viruses have evolved
counter-defense strategies.
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counter-defense strategies.
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••
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counter-defense strategies.
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•
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21. Voinnet O, Baulcombe DC: Systemic signalling in gene silencing.
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With [20••] the first report that gene silencing is associated with systemic
signalling. The experiments involved localised introduction of green fluorescent protein (GFP) DNA into a GFP transgenic plant and demonstrated that
local events could initiate systemic gene silencing. The systemic signal has
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22. Voinnet O, Vain P, Angell S, Baulcombe DC: Systemic spread of
•
sequence-specific transgene RNA degradation is initiated by
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•
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•
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Fast forward genetics based on virus-induced gene silencing
David C Baulcombe
Gene expression in plants can be suppressed in a sequencespecific manner by infection with virus vectors carrying
fragments of host genes. Recent developments have revealed
that the mechanism of this gene silencing is based on an
RNA-mediated defence against viruses. It has also emerged
that a related mechanism is involved in the post-transcriptional
silencing that accounts for between line variation in transgene
expression and cosuppresion of transgenes and endogenous
genes. The technology of virus-induced gene silencing is
being refined and adapted as a high throughput procedure for
functional genomics in plants.
Figure 1
Addresses
The Sainsbury Laboratory, John Innes Centre, Norwich, NR4 7UH, UK;
e-mail: [email protected]
Current Opinion in Plant Biology 1999, 2:109–113
http://biomednet.com/elecref/1369526600200109
© Elsevier Science Ltd ISSN 1369-5266
Abbreviations
PDS
phytoene desaturase
PTGS
post-transcriptional gene silencing
PVX
potato virus X
RMD
RNA-mediated defense
TBRV
tomato blackring virus
TMV
tobacco mosaic virus
VIGS
virus-induced gene silencing
Introduction
A limiting step in genetics and molecular biology is the
ability to relate genes to phenotypes and vice versa. In the
shadow of genome projects this limitation is more conspicuous than ever. Here, I describe a novel approach to
bridging this gap between genetics and molecular biology.
This approach is based on recent discoveries concerning
plant viruses and gene silencing and is still under development. The early results, however, provide encouragement
that virus-based technology will be a useful complement to
existing functional genomics tools.
The most straightforward iteration of this approach is
referred to as virus-induced gene silencing (VIGS) and
employs virus vectors carrying elements from the exons of
plant host genes. Analyses in Nicotiana species have shown
that the symptoms on these plants are phenocopies of mutations in the corresponding host genes. For example, with
tobacco mosaic virus (TMV) [1] and potato virus X (PVX)
vectors [2•] carrying a fragment of phytoene desaturase
(PDS) mRNA, the upper leaves of the infected plant develop a spectacular bleached effect (Figure 1). The cause of
these symptoms is a decline in the level of the endogenous
PDS mRNA leading to low levels of phytoene desaturase, a
block in carotenoid production and, consequently, the
absence of protection against photobleaching. In other exam-
Current Opinion in Plant Biology
Nicotiana benthamiana plant exhibiting virus-induced gene silencing of
phytoene desaturase. The plant was inoculated three weeks earlier with
a PVX vector construct carrying a 400 nucleotide fragment of phytoene
desaturase cDNA. The bleached symptoms contrast with the normal
mild mosaic symptoms associated with PVX infection on this species.
ples VIGS has been targeted against a chelatase gene
required for chlorophyll synthesis [3•] and against various
transgenes [2•,3•,4••,5].
The obvious attraction of VIGS for analysis of gene function is speed and ease of adaptation to high throughput
systems. If the partial sequence of a given gene is
known, a virus vector construct can be assembled within
two days. This construct may be infectious directly as in
vitro transcripts of cDNA clones [6] or as DNA, if the
virus has a DNA genome. Alternatively the cDNA of an
RNA virus can be fused to a promoter that is active in
plant cells. The cDNA of these constructs is infectious
as directly inoculated DNA [7] or by employing an
A. tumefaciens Ti plasmid vector to transfer the DNA into
plant cells [8].
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Genome studies and molecular genetics
There are two lines of supporting evidence for this model.
First there is direct evidence that both RNA nepoviruses
[11••] and DNA caulimoviruses can induce RMD [12,13••].
Initially the plants infected with these viruses exhibit
symptoms and the viruses accumulate to a high level.
However, the upper leaves of these plants emerging after
systemic infection have recovered. They are symptom free
and have only low levels of viral RNA. This recovery can be
attributed to RMD because, in the nepovirus infected
plants, there is RNA sequence-dependent resistance
against viruses inoculated to these upper leaves [11••]. In
the caulimovirus infected plants the recovery is probably
due to RMD as there is post-transcriptional suppression of
viral RNA in the symptom-free leaves.
Virus accumulation (particles/cell)
Figure 2
Activation of RMD
Time
Current Opinion in Plant Biology
Typical time course of virus accumulation in an infected cell. The graph
illustrates schematically the kinetics of virus accumulation in an
infected cell. There is an initial phase of rapid replication and virus
accumulation that progresses through to the final period when
accumulation has been arrested. The phases shown would take place
over a period of a few days, depending on the type of virus.
After inoculation of the vector there is a delay of only 7–21
days before VIGS-related symptoms develop in the upper
leaves of the infected plant. The relationship from gene to
phenotype, therefore, can be established within a few weeks.
In genomics jargon this strategy is referred to as reverse
genetics. VIGS can also be used in a forward genetics strategy although the resources required are substantial — it
would be necessary to construct cDNA libraries in virus vector constructs and to systematically analyse large numbers of
infected plants.
In the later sections of this article I assess the potential,
the limitations and the practicalities of VIGS both for
forward and for reverse genetics. Because the development of VIGS technology is influenced by knowledge,
however, I first review the current views about the
underlying mechanism.
The mechanism of VIGS
It is likely that VIGS is caused by an RNA-mediated
defense (RMD) mechanism in plants against viruses
[9,10]. According to this idea there is an as yet uncharacterised surveillance system in plants that can specifically
recognise viral nucleic acids and give sequence specificity
to RMD. Normally, with wild-type isolates of plant viruses, it is thought that this mechanism is activated as the
virus begins to accumulate in the plant (Figure 2).
Eventually, as a result of RMD, virus accumulation slows
down and eventually stops.
In the situations when a genetically modified virus has
similarity to a gene in the host plant, the RMD would target both the viral RNA and the corresponding endogenous
mRNA. VIGS would result from the targeting of the
endogenous mRNA
The second supporting evidence for RMD is indirect and
is based on analyses of plants exhibiting post-transcriptional gene silencing (PTGS) of transgenes. PTGS
involves sequence-specific turnover of RNA and is one of
the reasons for between-line variation in transgene expression levels [14]. Lines in which a transgene is expressed at
a low level include those in which PTGS is active and targeted against the transgene RNA. High level expressors
are those in which PTGS is either weak or inactive.
The link with RMD is from the finding that PTGS is suppressed by virus-encoded proteins that are also suppressors of
anti-viral defense in the host [15••–18••]. Presumably the
suppressed anti-viral defense is the putative RMD and the
suppressed PTGS was activated when the transgene or its
RNA was detected by the RMD surveillance system. The
suppressors of PTGS would be part of a counter defense system that allows viruses to accumulate after activation of RMD
[19•]. With some viruses the final steady state level of virus
accumulation would be determined by the effectiveness of
the suppressors. In other instances the virus may use evasion
instead of suppression as a strategy to counteract RMD.
Analysis of PTGS has revealed that there is a signal of
gene silencing that can move systemically in plants
[20••,21••,22•,23•]. This signal can be rationalised in terms
of RMD as it would allow the plant to prevent spread of
the virus. If the signal moves at the same time or ahead of
the virus it would prime RMD in the cells at, or just
beyond the infection front. Consequently the RMD would
be activated more rapidly than in the initially infected cells
and the spread of the virus would be delayed.
A final point about PTGS concerns DNA methylation.
There is an association of DNA methylation and PTGS in
several experimental systems [4••,24–27] and evidence
that the methylation may be targeted to transgenes by
RNA [4••,28]. These observations are difficult to reconcile
with the proposed similarity of PTGS and RMD as most
plant viruses have RNA genomes. Perhaps the DNA
methylation data are an indicator that the underlying
mechanism of PTGS is involved not only in anti-viral
defense. It could be, for example, that antiviral defense is
Fast forward genetics based on virus-induced gene silencing Baulcombe
related to the mechanisms used to protect against retroposons and other forms of mobile DNA in plants [29].
Development of VIGS technology
This relationship of VIGS, PTGS and RMD has important
implications for further development of VIGS technology. It
means, for example, that VIGS would be weak if the virus
vector encodes a suppressor of PTGS and that the most
effective VIGS vectors will be those that use evasion rather
than suppression as a strategy to overcome RMD. Vectors
based on potyviruses, therefore, would be poor VIGS vectors
because they produce strong suppressors of PTGS
[15••,16••,18••]. Cucumber mosaic virus would be unsuitable
for the same reason [16••,17••]. Conversely, PVX and tomato
blackring virus (TBRV) would be suitable vectors for VIGS.
Neither of these viruses is able to reverse PTGS in transgenic plants [16••,17••] and it can be assumed that they do
not produce suppressors or that their suppressors are weak.
TBRV is a seed and pollen transmitted virus as a result of its
ability to infect the progenitor cells of the pollen and eggs in
the meristems [30]. This ability to penetrate the meristem
means that TBRV would be a particularly effective vector
for VIGS. Most plant viruses, including PVX, are excluded
from the meristematic zone of infected plants [31] and
would not be able to silence genes required for meristem
identity and early in leaf and flower development.
Unfortunately, as yet, there are no full length cDNAs of
TBRV or related viruses that could be developed as VIGS
vectors with the ability to target these important genes.
There are several other ways in which understanding of
PTGS could facilitate development of VIGS technology.
For example, it may be possible to enhance effectiveness
of VIGS vectors by engineering increased potential to produce double stranded RNA. Double stranded RNA has
been implicated as an initiator of PTGS in plant systems
[32•,33••,34•] and of a similar gene silencing phenomenon
in nematodes [35,36•].
It may also be possible to enhance VIGS by modification
of the host plant. For example, it might be expected that
VIGS would be stronger in the background of host gene
mutations that enhance gene silencing (egs) [37]. The
products of these genes could be repressors of RMD. It
might also be expected that VIGS would be stronger in
plants that over-express genes required for RMD than in a
wild-type host. The genes that are required for gene
silencing can be recognised by analysis of the suppressed
gene silencing (sgs) [38•] phenotype of mutant alleles.
Transgenic VIGS
In the applications of VIGS, as described above, the activator of gene silencing is the viral RNA in the infected
plant cells. The silencing effect is systemic because the
virus vector is able to spread through the infected plant. In
transgenic VIGS the aim is to produce an RNA that can be
recognised by the RMD surveillance system. As in VIGS,
111
this RNA would include an element from a host sequence
so that the RMD silences the corresponding gene. This
RNA would not need to be infectious, however, because it
would be delivered into cells by expression of a transgene.
An advantage of transgenic VIGS over ‘conventional’
PTGS using sense or antisense transgenes should be consistency. The transgenic VIGS constructs would be
designed to produce an RNA that is detected by the RMD
surveillance system and it would be expected that all, or at
least most, of the lines would exhibit silencing of the target gene. In PTGS it is likely that the transgene RNA is
only recognised by the RMD surveillance system as a
result of sequence rearrangements, DNA methylation of
chromatin structures that are associated with the transgene
after its introduction into the plant genome. As a result, the
degree of PTGS varies greatly between lines.
Transgenic VIGS is less amenable than infectious VIGS to
high throughput applications because of the transformation
step. In plants that can be transformed with high efficiency,
however, it is quite feasible to produce the tens of thousands of transformed lines that would be necessary to
survey the gene function in a typical plant. Once produced,
the seed from these lines would be a permanent resource
that could be used in many different functional screens.
The version of transgenic VIGS corresponding most closely to infectious VIGS employs amplicons. Amplicons are
transgenes comprising a promoter and terminator directing
transcription of a modified viral vector RNA in the plant
cell [39]. The modifications may include deletion or mutation of viral genes required for spread of the virus in the
infected plant and of any other functions that are secondary
to replication. Expression of the amplicon in the plant cell
could activate RMD and, if there are host sequences in the
amplicon construct, PTGS of the corresponding host gene.
There are several instances of amplicon transgenes although
they are not always referred to as such. A PVX amplicon produced consistent silencing as expected [39]. Similarly, a
petunia chalcone synthase gene could be targeted by a gemini virus amplicon with a chalcone synthase insert [40] and a
brome mosaic virus amplicon conferred virus resistance that
was probably due to RMD [41]. The phenotype of amplicons derived from cucumber mosaic virus [42] and a severe
strain of TMV, however, did not appear to involve gene
silencing [43]. The differences between these amplicons
may be due to the suppressors of RMD encoded in the corresponding viral genomes — genomes producing little or no
silencing may produce strong suppressors of RMD, whereas
good silencers may be those that do not encode suppressors
or from which the suppressor genes have been deleted.
Some alternative approaches to PTGS in transgenic plants
may also be considered as transgenic ‘pseudoVIGS’. The
success of these approaches depends on the ability of non
viral transgenes to produce the virus-like RNAs that activate
112
Genome studies and molecular genetics
RMD. The gene silencing from simultaneous expression of
sense and antisense RNAs [33••] or from transgene constructs with inverted repeats in the transcribed regions [32•]
could both be considered in this ‘pseudoVIGS’ category .
Acknowledgements
I am grateful to the Gatsby Charitable Foundation for supporting work in
the Sainsbury Laboratory and to my colleagues for many stimulating
discussions about the role and mechanisms of gene silencing in plants.
References and recommended reading
VIGS versus other techniques of functional
genomics
Conventional approaches to forward genetics generally
involve insertional mutagenesis either by T-DNA [44] or
transposable elements [45,46]. These are powerful
approaches that have led to many advances in gene discovery; however, there are several complications with
insertional mutagenesis of genes required for basic cell function or early development. In these instances, because the
insertional mutation often results in complete inactivation of
the target gene, the mutant phenotype will be embryo lethal
and difficult to interpret in terms of the cellular or biochemical role of the gene product. Conversely, if the mutant gene
is one of a family it is likely that there may be no phenotype
of an insertional mutant because the functional homologues
may compensate for the loss of function at the mutant locus.
VIGS and related PTGS-based approaches may circumvent
these problems. For example, with multigene families, the
gene silencing mechanism will target not only the precise
homologue of the gene in the VIGS vector; it will also target
partial homologues that vary by up to 10 or 20%. The chlorotic and dwarf phenotypes that result from PTGS of tobacco
ribulose bisphosphate carboxylase (rubisco) is a good illustration of how gene silencing can target a multigene family
[47,48]. It is unlikely that a phenotype would result from
insertional mutagenesis of rubisco because it is encoded in a
multigene family. The precise mismatch between different
tobacco rubisco genes is not known but, by comparison with
other solanaceous plants, it is likely to be up to 14% [49].
When VIGS is targeted against lethal genes the phenotype
is death, as in insertional mutagenesis, but the dying
process may be more informative than the embryo lethal
phenotype of insertional mutants. Because the VIGS is
applied to mature plants and because there may be only
partial suppression of the target gene, the dying process
may be informative about the role of the corresponding
gene products. It might be expected, for example, that
death due to a block in lipid biosynthesis would be clearly
different from death due to suppression of DNA synthesis
or proteins required for respiration
Conclusions
VIGS vectors have been developed from the genomes of
TMV, PVX and TGMV for applications in Nicotiana
species. To develop these vectors for use in other plant
species it will be possible to build on recent discoveries that
VIGS and PTGS are related to antiviral defense in plants
and that viruses produce suppressors of this antiviral
defense. When these vectors are available for Arabidopsis,
maize and other species it will be possible to combine insertional mutagenesis and VIGS to develop a powerful
combined approach to functional surveys of plant genomes.
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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virus-induced gene silencing. Plant Cell 1998, 10:937-946.
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Kjemtrup S, Sampson KS, Peele CG, Nguyen LV, Conkling MA,
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Extends and establishes general importance of RNA directed methylation of
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viroid as in the previous work. The paper is also important because it demonstrates VIGS in a legume. Most of the other examples are in Nicotiana species.
4.
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Fast forward genetics based on virus-induced gene silencing Baulcombe
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•• Baulcombe DC: Viral pathogenicity determinants are suppressors
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••
[15 ,17••,18••] provide indirect but compelling evidence that RMD is a general defense mechanism against plant viruses and that viruses have evolved
counter-defense strategies.
Beclin C, Berthome R, Palauqui JC, Tepfer M, Vaucheret H: Infection
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This paper and the others appearing within a few weeks of each other
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counter-defense strategies.
17.
••
18. Anandalakshmi R, Pruss GJ, Ge X, Marathe R, Smith TH, Vance VB: A
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counter-defense strategies.
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•
strategies and counter-strategies. Curr Opin Plant Biol 1998,
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A concise and informative review that puts RMD in the context of the various virus resistance mechanisms deployed in plants and the corresponding
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silenced plants. The systemic signal has not yet been characterised but is
likely to be a nucleic acid.
21. Voinnet O, Baulcombe DC: Systemic signalling in gene silencing.
•• Nature 1997, 389:553.
With [20••] the first report that gene silencing is associated with systemic
signalling. The experiments involved localised introduction of green fluorescent protein (GFP) DNA into a GFP transgenic plant and demonstrated that
local events could initiate systemic gene silencing. The systemic signal has
not yet been characterised but is likely to be a nucleic acid.
22. Voinnet O, Vain P, Angell S, Baulcombe DC: Systemic spread of
•
sequence-specific transgene RNA degradation is initiated by
localised introduction of ectopic promoterless DNA. Cell 1998,
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Further characterises the mechanisms of systemic silencing but does not
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•
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95:9675-9680.
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•
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been isolated and characterised in genes required for silencing. This report
provides the first step to that end. It also describes plant lines that would be
useful for gene over-expression.
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