Download Virus-mediated reprogramming of gene expression in plants John A

181
Virus-mediated reprogramming of gene expression in plants
John A Lindbo, Wayne P Fitzmaurice and Guy della-Cioppa*
Plant viruses have made many significant contributions to plant
biology over the years: they have provided plant researchers
with functional promoters, transient expression systems and,
most recently, with critical insights into the phenomenon of
posttranscriptional gene silencing. Plant virus expression
vectors have the ability to either overexpress genes or
suppress gene expression in plants. Whereas the ‘rules’ for
gene expression are generally understood conceptually, the
mechanisms for the induction of gene silencing are less well
understood. Recent advances in the understanding of both the
biological role and the mode of action of posttranscriptional
gene silencing will affect both the design and the use of plant
viral vectors and transgenic plants for either geneoverexpression or gene-silencing applications.
Addresses
Large Scale Biology Corporation, 3333 Vaca Valley Parkway,
Vacaville, California 95688, USA
*e-mail: [email protected]
overexpression of genes and for suppressing gene expression, and present some considerations on the future of
viral-vector development.
Gene expression using plant viral vectors
Viral vectors have been generated from a number of different viruses. Each vector has its own particular
advantages and disadvantages as determined by its biology.
Plant viral vectors based upon tobacco mosaic virus
(TMV) or potato virus X (PVX), which are rod-shaped
viruses that express genes from subgenomic mRNAs, are
particularly useful [1] [P1]. These vectors offer the most
flexibility in terms of the size of foreign sequence to be
inserted, their ability to direct foreign proteins to extracellular or sub-cellular locations, and the quantity of
foreign proteins produced in plants. In fact, TMV-based
expression vectors are currently being used to express
commercially relevant quantities of proteins for pharmaceutical applications [2].
Current Opinion in Plant Biology 2001, 4:181–185
1369-5266/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Abbreviations
CP
capsid protein
cRNA
complementary RNA
PDR
pathogen-derived resistance
PTGS
posttranscriptional gene silencing
PVX
potato virus X
RdRP
RNA-dependent RNA polymerase
RNAi
RNA interference
TEV
tobacco etch potyvirus
TMV
tobacco mosaic virus
VIGS
virus-induced gene silencing
Introduction
The explosion of information from genome sequencing
projects has ushered in new fields such as bioinformatics,
structural and functional genomics, transcript profiling,
and proteomics. The ultimate goal of these technologies,
and a current bottleneck, is to connect the sequence of
each gene with a biological phenotype or activity. The US
National Science Foundation proposal to ‘understand the
functions of all plant genes’ by 2010, for example, is an
attempt to bridge this gap in our knowledge. Once this
connection is made, researchers will have a wealth of
information and raw materials for generating genetic and
biochemical diversity in plant systems by expressing or
suppressing genes of interest. To take full advantage
of these opportunities, however, researchers need tools to
rapidly and reliably express or suppress genes of interest
in plants. Plant viruses are providing these tools. We
believe that viruses will continue to have an important
impact on the future of biological research, especially in
light of newly emerging genomics technologies. In
this review, we discuss the use of viral vectors for the
Viral vector transfection systems enable the overproduction of proteins and metabolites in amounts that
could be lethal or deleterious to the regenerating plant if
introduced by traditional plant transformation methods.
Plant viruses can thus be used for complex metabolic
pathway engineering involving the overexpression of
genes encoding enzymes [3,4]. For example, overexpression of phytoene synthase in Nicotiana benthamiana
plants led to a ten-fold increase in phytoene accumulation in plants [3]. Similarly, overexpression of a
capsanthin-capsorubin synthase gene from a Capsicum
species led to the production of capsanthin, a carotenoid
that is not normally found in N. benthamiana. Up to 36%
of the total carotenoid pool in these plants was directed
toward the production of capsanthin. This ability to
readily express non-endogenous genes opens up a wide
range of possibilities that cannot be matched by gene
knockout technologies alone.
Because of their speed and flexibility in terms of protein
expression, viral vectors are also ideal for gene-shuffling [5]
or molecular-breeding applications. Gene-shuffling and
molecular-breeding technologies rely on the in vitro generation of thousands of mutants or chimeric genes, followed
by the expression of the mutant genes and, finally, screening
for or selection of variants with traits of interest. Plant viral
vectors are uniquely suited to function in such an experimental system. Gene libraries can be cloned into plant
viruses, and individual library members can be expressed
systemically throughout the plants in a short time frame.
Using this strategy, it should be possible to create many
novel or improved enzyme activities in plants, such as
thermally activated cellulases and ligninases to facilitate
paper and pulp production and so on.
182
Physiology and metabolism
Gene suppression in plants using viral vectors
Although viral vectors were originally designed to serve as
overexpression tools, plant viruses also have the ability to
‘silence’ or suppress gene expression in plants by activating
a sequence-specific RNA degradation activity known as
posttranscriptional gene silencing (PTGS). To understand
the potential utility of viral-based gene-suppression
strategies, it is necessary to provide some background
information on the phenomenon of PTGS and to resolve
how the biology of PTGS is affecting the use of viral vectors in plants. Many excellent reviews on PTGS have been
published [6,7,8•]). Hence, we will simply present a brief
background here that describes the relationships among
the plant-associated phenomena of PTGS, pathogenderived resistance (PDR), virus-induced gene silencing
(VIGS) [9] and co-suppression, and the phenomena of
quelling [10] and RNA interference (RNAi) [11], which do
not occur in plants.
Co-suppression, PDR, PTGS, and VIGS,
quelling and RNAi
Early in transgenic plant research, the puzzling phenomenon known as co-suppression, or gene silencing, was
described [12]. In attempts to overexpress plant genes,
researchers constructed trans-genes under the control of
strong, constitutive promoters. Some, but not all, of the
transgenic plants generated in this way displayed an
unusual phenotype in which expression from both the
transgene and the homologous endogenous gene was
suppressed. This unexpected phenomenon was termed
co-suppression or gene silencing. Many examples of
co-suppression were caused by a posttranscriptional event
that resulted in reduced steady-state mRNA levels of the
‘silenced gene’ [13].
During this same period, researchers were generating
virus-resistant transgenic plants by applying the principles
of PDR. PDR is an elegant hypothesis which proposes that
pathogen resistance genes can be readily derived from a
pathogen’s own genetic material [14][P2]. For example,
PDR theorized that transgenic host cells overexpressing
viral gene products in a normal or dysfunctional state could
perturb the pathogen’s normal life cycle leading to virus
resistance. Many examples of virus-resistant transgenic
plants were generated using this strategy. Curiously, in
some of the virus-resistant transgenic plants studied, the
expression of virus-derived transgene products did not
correlate with the degree of virus resistance.
In the early 1990s, studies by William Dougherty and
colleagues on transgenic tobacco plants expressing various
mutant forms of the capsid protein (CP) gene sequence of
tobacco etch potyvirus (TEV) provided a crucial link
among PDR, co-suppression, PTGS and VIGS [15].
Molecular analysis of TEV-resistant transgenic plants
demonstrated that only the virus-derived transgene RNA
was needed to confer apparent TEV immunity; no expression of a viral protein was required. This highly
virus-resistant state was associated with low steady-state
levels of the TEV CP transgene mRNA. Nuclear run-off
experiments indicated that these low steady-state levels
were caused by a cytoplasmically functioning sequencespecific RNA degrading activity, which was specifically
directed toward the TEV CP RNA sequence. This activity
is now commonly referred to as PTGS. Because of the similarities between this example of PDR and co-suppression,
it was proposed that a PTGS activity was responsible for
many other examples of PDR and for some examples of
co-suppression [16]. PTGS is also thought to be responsible
for certain examples of cross-protection between viruses
or viroids, and even for the so-called ‘anti-sense’ suppression of some genes [16]. In the TEV CP transgenic plant
system, it was observed that PTGS is a pre-existing
state in some transgenic plant lines, whereas in other
transgenic plant lines PTGS is only activated after the
plant is infected with a virus carrying sequences that are
homologous to the transgene. The ability of a virus to
induce PTGS is now often referred to as virus-induced
gene silencing (VIGS).
Mechanism of PTGS
The phenomena of RNAi in animals and quelling in fungi
are functional equivalents of PTGS in plants. Genetic and
biochemical analyses of PTGS, RNAi and quelling have
demonstrated that the original PTGS model proposed by
Dougherty and colleagues was remarkably accurate [16,17].
This model proposed that plants have a way of detecting
‘foreign’ RNAs that have accumulated to an unacceptable
level. After detection, the plant mounts an active response
specifically targeting such RNAs for degradation. It was
proposed that the specificity of PTGS is due to a nucleicacid component of the system, and that the enigmatic
endogenous RNA-dependent RNA polymerase (RdRP)
activity, known to exist in plants could use specific RNAs as
templates for the production of short complementary RNA
(cRNA) molecules. These cRNAs could diffuse through
the cytoplasm, anneal to their complementary sequence
and serve as a guide for an RNA-degrading enzyme activity.
Among the genes now known to be involved in PTGS-like
activities are genes encoding proteins with similarities to an
RdRP ([18•,19], a RecQ Q-like DNA helicase [20] and an
RNaseD-like protein [21••].
It has been proposed that the RNAs that initiate PTGS
responses in plants are in some way ‘aberrant’ [6,8•], perhaps due to premature termination of transcription for
example. The production of such ‘aberrant’ transcripts
may or may not be related to the methylation state of the
transgene [8•]. Recently, it has been demonstrated that
double-stranded RNAs (dsRNAs) in particular are potent
inducers of PTGS. In cells in which PTGS is active, small
RNAs of 21–23 nucleotides in length, which correspond to
both sense and antisense strands of the RNA targetted by
PTGS, accumulate [22•]. These small RNAs are consistent
with the Dougherty model and may target the RNA
sequence to be degraded [23••,24••,25].
Virus-mediated reprogramming of gene expression in plants Lindbo, Fitzmaurice, della-Cioppa
Biological roles of PTGS: PTGS and viruses
Although PTGS was first identified in transgenic plant systems, it is unlikely that PTGS evolved to help plants
combat transgenesis events in nature. A more plausible
biological role is that PTGS is a plant virus-defense strategy,
though PTGS is likely to have additional biological roles
[26•]. Several lines of evidence suggest that plants detect
viral RNAs and, as a defense mechanism, initiate a PTGSlike response to selectively degrade foreign viral RNAs.
First, in naturally occurring plant–virus systems in which
the plant ‘recovers’ from a viral infection, a PTGS-like
activity is displayed in the ‘recovered’ plant tissue [27,28].
In addition, many viruses express suppressors of
PTGS from their genomes [29-32,33•,34,35•]. Second, a
‘cross-protection’-like phenomenon between normally
compatible viruses exists when the different viruses
carry a common nucleotide sequence [36•]. Third,
genetic mutants of Arabidopsis that interfere with PTGS
are ‘hyper’susceptible to infection by some viruses
[18•,37•,38•]. Fourth, double-stranded RNA is a very
potent inducer of PTGS [39,40•,41], and most plant viruses
have RNA genomes that replicate via a double-stranded
RNA intermediate. Fifth, PTGS that is initiated in a few
cells can migrate over both short (i.e. cell-to-cell) and long
(i.e. leaf-to-leaf) distances in a pattern similar to that of the
short- and long-distance movements of plant viruses
[42,43•,44]. To spread to multiple cells, the putative PTGS
nucleic-acid signal is hypothesized to undergo amplification and translocation steps. The ability of the PTGS
signal to travel both short and long distances within the
plant would allow the plant to protect uninfected cells
from the advancing virus.
Gene suppression from viral vectors
Numerous examples of virus-induced PTGS (i.e. VIGS) of
either trans- or endo-genes have been reported [45•].
Interestingly, viral vectors carrying even partial gene fragments, in either sense or anti-sense orientation, can induce
gene silencing [3]. This is an important feature for
genomics applications, which makes it possible to obtain a
gene-knockout phenotype even without having access to
the entire gene sequence. It should also be possible to
simultaneously silence multiple genes from a single viral
vector by expressing a chimeric gene sequence.
Virus-induced gene silencing has been shown for a variety
of plant genes expressed from a variety of different vectors,
including vectors based on TMV, PVX, cauliflower mosaic
virus (CaMV), tobacco rattle virus (TRV) [36•] and gemini
viruses. Because some viruses express gene products that
interfere with the plant's gene-silencing mechanism, it can
be appreciated that some vectors will be more efficient
than others at inducing silencing. A recent review [45•] has
discussed the use of viruses as gene-silencing tools. In this
review, Baulcombe notes that some viral vectors are more
effective at inducing (systemic) gene silencing than others.
Such viruses are able to induce a ‘recovery’-type response
in some plant hosts, whereas the symptoms caused by
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other viruses persist. Recent reports examining virus–host
systems in which the plant ‘recovers’ from an infection
(i.e. in which the virus-induced symptoms are transient)
have linked this phenomenon to sequence-specific RNA
degrading activity [27,28,46]. In these host–virus systems,
the host readily targets the RNAs from the viruses for
silencing, causing the symptoms to ‘disappear’. Vectors
based on such virus systems may be particularly efficient at
inducing the gene silencing of vectored genes.
Conclusions and future prospects
Given that PTGS seems to be a common virus control
strategy employed by plants, it is interesting to propose different strategies that viruses might employ to counter this
defense. The strategy that a virus uses to combat the PTGS
response will affect its utility as a gene-overexpression or
gene-suppression vector. Some of the possibilities include:
avoiding detection and therefore avoiding activation of
PTGS, outrunning the PTGS signal or activity, protecting
viral sequences from recognition and/or degradation by
PTGS, and expressing gene products that interfere with one
or more stages of the PTGS protective mechanism.
As mentioned previously, virus-encoded suppressors of
gene silencing have already been identified from some
virus groups. Analysis of these suppressors of silencing will
prove crucial to our understanding of the scope of activities
involved in PTGS regulation, and have already facilitated
the identification of host proteins that are involved in the
release of gene silencing of transgenes [47••]. More reliable
gene silencing or overexpression may be obtained in the
future by modifying our current approaches with transgene
and transient expression vectors to include known negative or positive modifiers of PTGS activities.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Chapman S, Kavanagh T, Baulcombe D: Potato virus X as a vector
for gene expression in plants. Plant J 1992, 2:549-557.
2.
McCormick AA, Kumagai MH, Hanley K, Turpen TH, Hakim I, Grill LK,
Tuse D, Levy S, Levy R: Rapid production of specific vaccines for
lymphoma by expression of the tumor-derived single-chain Fv
epitopes in tobacco plants. Proc Natl Acad Sci USA 1999,
96:703-708.
3.
Kumagai MH, Donson J, della-Cioppa G, Harvey D, Hanley K,
Grill LK: Cytoplasmic inhibition of carotenoid biosynthesis with
virus-derived RNA. Proc Natl Acad Sci USA 1995, 92:1679-1683.
4.
Kumagai MH, Keller Y, Bouvier F, Clary D, Camara B: Functional
integration of non-native carotenoids into chloroplasts by
viral-derived expression of capsanthin-capsorubin synthase in
Nicotiana benthamiana. Plant J 1998, 14:305-315.
5.
Stemmer WP: DNA shuffling by random fragmentation and
reassembly: in vitro recombination for molecular evolution. Proc
Natl Acad Sci USA 1994, 91:10747-10751.
6.
Sijen T, Kooter JM: Post-transcriptional gene-silencing: RNAs on
the attack or on the defense? Bioessays 2000, 22:520-531.
7.
Marathe R, Anandalakshmi R, Smith TH, Pruss GJ, Vance VB:
RNA viruses as inducers, suppressors and targets of
post-transcriptional gene silencing. Plant Mol Biol 2000, 43:295-306.
184
Physiology and metabolism
8. Waterhouse PM, Smith NA, Wang MB: Virus resistance and gene
•
silencing: killing the messenger. Trends Plant Sci 1999, 4:452-457.
The authors present multiple models for the induction of PTGS in transgenic plants and discuss the biological role of PGTS as a defense against
virus infection.
9.
Mueller E, Gilbert J, Davenport G, Brigneti G, Baulcombe DC:
Homology-dependent resistance: transgenic virus resistance in
plants related to homology-dependent gene silencing. Plant J 1995,
7:1001-1013.
10. Cogoni C, Macino G: Conservation of transgene-induced
post-transcriptional gene silencing in plants and fungi. Trends
Plant Sci 1997, 2:438-443.
11. Ketting RF, Plasterk RH: A genetic link between co-suppression
and RNA interference in C. elegans. Nature 2000, 404:296-298.
12. Napoli C, Lemieux C, Jorgensen R: Introduction of a chimeric chalcone
synthase gene into petunia results in reversible co-suppression of
homologous genes in trans. Plant Cell 1990, 2:279-289.
13. Metzlaff M, Odell M, Cluster PD, Flavell RB: RNA-mediated RNA
degradation and chalcone synthase a silencing in petunia. Cell 1997,
88:845-854.
14. Sanford JC, Johnston SA: The concept of pathogen derived
resistance: deriving resistance genes from the parasite’s own
genome. J Theor Biol 1985, 113:395-405.
15. Dougherty WG, Lindbo JA, Smith HA, Parks TD, Swaney S,
Proebsting WM: RNA-mediated virus resistance in transgenic
plants: exploitation of a cellular pathway possibly involved in
RNA degradation. Mol Plant Microbe Interact 1994, 7:544-552.
16. Lindbo JA, Silva-Rosales L, Proebsting WM, Dougherty WG:
Induction of a highly specific antiviral state in transgenic plants:
implications for regulation of gene expression and virus
resistance. Plant Cell 1993, 5:1749-1759.
17.
Dougherty WG, Parks TD: Transgenes and gene suppression:
telling us something new? Curr Opin Cell Biol 1995, 7:399-405.
18. Dalmay T, Hamilton A, Rudd S, Angell S, Baulcombe DC: An
•
RNA-dependent RNA polymerase gene in Arabidopsis is required
for posttranscriptional gene silencing mediated by a transgene
but not by a virus. Cell 2000, 101:543-553.
This work provides evidence for the role of an RNA-dependent RNA polymerase in a PTGS response in plants. The authors propose that the host
RdRP is not needed for virus-induced gene silencing because the replicating
RNA virus encodes its own RdRP.
19. Smardon A, Spoerke JM, Stacey SC, Klein ME, Mackin N, Maine EM:
EGO-1 is related to RNA-directed RNA polymerase and functions
in germ-line development and RNA interference in C. elegans.
Curr Biol 2000, 10:169-178. [Published erratum appears in Curr
Biol 2000, 10:R393-R394].
20. Cogoni C, Macino G: Posttranscriptional gene silencing in
Neurospora by a RecQ DNA helicase. Science 1999,
286:2342-2344.
21. Ketting RF, Haverkamp TH, van Luenen HG, Plasterk RH: Mut-7 of
•• C. elegans, required for transposon silencing and RNA
interference, is a homolog of Werner syndrome helicase and
RnaseD. Cell 1999, 99:133-141.
This study identifies multiple C. elegans mutants that are simultaneously defective in RNAi, co-suppression, and transposon silencing. This finding suggests that
these phenomena may be mediated (at least in part) by common molecular mechanisms, possibly involving RNA-directed RNA degradation. The powerful tools of
C. elegans genetics should continue to provide interesting insight into RNAi.
22. Hamilton AJ, Baulcombe DC: A species of small antisense RNA in
•
posttranscriptional gene silencing in plants. Science 1999,
286:950-952.
The authors identify small RNAs (estimated to be about 25 nucleotides in
length) that are homologous to the silenced genes that accumulate in plants
displaying PTGS.
23. Hammond SM, Bernstein E, Beach D, Hannon GJ: An RNA-directed
•• nuclease mediates post-transcriptional gene silencing in
Drosophila cells. Nature 2000, 404:293-296.
The authors show that ‘loss-of-function’ phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs.
A nuclease activity is identified in Drosophila cells that show RNAi. The
nuclease described contains an essential RNA component. After partial
purification, the sequence-specific nuclease co-fractionates with a discrete
25-nucleotide RNA species that may confer specificity to the enzyme
through homology to the substrate mRNAs.
24. Zamore PD, Tuschl T, Sharp PA, Bartel DP: RNAi: double-stranded
•• RNA directs the ATP-dependent cleavage of mRNA at 21 to 23
nucleotide intervals. Cell 2000, 101:25-33.
This work describes an in vitro system of Drosophila extracts in which a
sequence-specific RNA-degrading activity (i.e. RNAi) can be established. In
this system, RNA is degraded into fragments of 21–23 nucleotides in length.
Such in vitro systems should be useful in dissecting the biochemical functions involved in RNAi.
25. Bass BL: Double-stranded RNA as a template for gene silencing.
Cell 2000, 101:235-238.
26. Matzke MA, Mette MF, Matzke AJM: Transgene silencing by the host
•
genome defense: implications for the evolution of epigenetic
control mechanisms in plants and vertebrates. Plant Mol Biol
2000, 43:401-415.
This review discusses potential biological roles for transcriptional and posttranscriptional gene regulation mechanisms as defense mechanisms against
foreign DNAs or RNAs.
27.
Ratcliff F, Harrison BD, Baulcombe DC: A similarity between viral
defense and gene silencing in plants. Science 1997,
276:1558-1560.
28. Al-Kaff NS, Covey SN, Kreike MM, Page AM, Pinder R, Dale PJ:
Transcriptional and posttranscriptional plant gene silencing in
response to a pathogen. Science 1998, 279:2113-2115.
29. Ding B, Li Q, Nguyen L, Palukaitis P, Lucas WJ: Cucumber mosaic
virus 3a protein potentiates cell-to-cell trafficking of CMV RNA in
tobacco plants. Virology 1995, 207:345-353.
30. Kasschau KD, Carrington JC: A counterdefensive strategy of plant
viruses: suppression of posttranscriptional gene silencing. Cell 1998,
95:461-470.
31. Anandalakshmi R, Pruss GJ, Ge X, Marathe R, Mallory AC, Smith TH,
Vance VB: A viral suppressor of gene silencing in plants. Proc Natl
Acad Sci USA 1998, 95:13079-13084.
32. Beclin C, Berthome R, Palauqui JC, Tepfer M, Vaucheret H: Infection
of tobacco or Arabidopsis plants by CMV counteracts systemic
post-transcriptional silencing of nonviral (trans)genes. Virology
1998, 252:313-317.
•33. Llave C, Kasschau KD, Carrington JC: Virus-encoded suppressor of
posttranscriptional gene silencing targets a maintenance step in
the silencing pathway. Proc Natl Acad Sci USA 2000,
97:13401-13406.
The authors of this paper analyzed the role of suppressors of PTGS using a
rapid Agrobacterium-directed approach and demonstrated that the HC-Pro
(Helper Component Proteinase) gene of potyviruses can interfere with the
maintenance of silencing.
34. Voinnet O, Pinto YM, Baulcombe DC: Suppression of gene
silencing: a general strategy used by diverse DNA and RNA
viruses of plants. Proc Natl Acad Sci USA 1999, 96:14147-14152.
35. Voinnet O, Lederer C, Baulcombe DC: A viral movement protein
•
prevents spread of the gene silencing signal in Nicotiana
benthamiana. Cell 2000, 103:157-167.
This study identifies a virally-encoded protein that interferes with PTGS
signal migration providing further support for the concept that plants combat
virus infections with a PTGS-like response.
36. Ratcliff FG, MacFarlane SA, Baulcombe DC: Gene silencing without
•
DNA: RNA-mediated cross-protection between viruses. Plant Cell
1999, 11:1207-1215.
This work demonstrates that a PTGS-like response may be involved in virus
cross-protection in plants. It also demonstrates that the ‘recovery’ phenotype
induced by tobacco rattle virus in non-transgenic plants shares similarities
with PTGS.
37. Morel JB, Vaucheret H: Post-transcriptional gene silencing
•
mutants. Plant Mol Biol 2000, 43:275-284.
The authors of this paper describe the isolation of Arabidopsis mutants that
suppress gene silencing and belong to three complementation groups. These
mutants, which do not display developmental abnormalities, are likely to have
deficiencies in genes that encode regulators and activators of the PTGS
response. These mutations impede both sense-mediated PTGS and cosuppression events, providing further evidence for the linkage of these phenomena.
38. Mourrain P, Beclin C, Elmayan T, Feuerbach F, Godon C, Morel JB,
•
Jouette D, Lacombe AM, Nikic S, Picault N et al.: Arabidopsis SGS2
and SGS3 genes are required for posttranscriptional gene
silencing and natural virus resistance. Cell 2000, 101:533-542.
The sequences of the Arabidopsis suppressor-of-gene-silencing genes
SGS1 and SGS2 are reported. The genes affected in the QDE-1 quellingdefective mutant of Neurospora, the EGO-1 RNAi mutant of C. elegans and
Virus-mediated reprogramming of gene expression in plants Lindbo, Fitzmaurice, della-Cioppa
the sgs2 PTGS mutant of Arabidopsis all share homologies with the tomato
RdRP gene, establishing a mechanistic link among PTGS, quelling and
RNAi. However, the sgs3 gene encodes a protein of undetermined function
and has no close homologs in either Neurospora crassa or C. elegans.
39. Fire A: RNA-triggered gene silencing. Trends Genet 1999, 15:358-363.
40. Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG,
•
Waterhouse PM: Gene expression — total silencing by intron-spliced
hairpin RNAs. Nature 2000, 407:319-320.
The authors describe a transgene construct designed to efficiently generate
dsRNA in plants. The construct induced PTGS in almost 100% of transgenic
plants generated. This PTGS can be directed toward either endogenous
genes or a viral pathogen.
41. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC:
Potent and specific genetic interference by double-stranded RNA
in Caenorhabditis elegans. Nature 1998, 391:806-811.
42. Palauqui JC, Elmayan T, Pollien JM, Vaucheret H: Systemic acquired
silencing: transgene-specific post-transcriptional silencing is
transmitted by grafting from silenced stocks to non-silenced
scions. EMBO J 1997, 16:4738-4745.
43. Fagard M, Vaucheret H: Systemic silencing signal(s). Plant Mol
•
Biol 2000, 43:285-293.
This is a good discussion on the initiation, propagation and maintenance
stages of PTGS, and of how systemic PTGS resembles the recovery from
virus infection induced by some viruses in non-transgenic plants.
185
44. Voinnet O, Baulcombe DC: Systemic signalling in gene silencing.
Nature 1997, 389:553.
45. Baulcombe DC: Fast forward genetics based on virus-induced
•
gene silencing. Curr Opin Plant Biol 1999, 2:109-113.
This review discusses some of the ways in which different virus groups may
combat a PTGS-like response from plants and describes how certain virus
groups seem to be better inducers of VIGS than others.
46. Covey SN, Al-Kaff NS: Plant DNA viruses and gene silencing. Plant
Mol Biol 2000, 43:307-322.
47.
••
Anandalakshmi R, Marathe R, Ge X, Herr JM Jr, Mau C, Mallory A,
Pruss G, Bowman L, Vance VB: A calmodulin-related protein that
suppresses posttranscriptional gene silencing in plants. Science
2000, 290:142-144.
The authors use a virally-encoded suppressor of gene silencing to help
identify the first plant-encoded suppressor of gene silencing.
Patents
P1. Donson J, Dawson WO, Granthan GL, Turpen TH, Turpen AM,
Garger SJ, Grill LK: Plant viral vectors having heterologous
subgenomic promoters for systemic expression of foreign genes.
US Patent 1994: 5,316,931.
P2. Johnston SA, Sanford JC: Parasite-derived resistance. US Patent
1998: 5,840,481.