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Gene silencing through the increased degradation of mRNA appears to represent a novel cellular pathway that is functional in a broad range of organisms. Recent work has established a role for RNA silencing in host antiviral defense and transposon silencing, suggesting a potential application in plant functional genomics.
Addresses
Molecular Virology Laboratory, Institute of Molecular Agrobiology, National University of Singapore, 1 Research Link, Singapore 117604; e-mail: [email protected]
Current Opinion in Biotechnology2000, 11:152–156 0958-1669/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
CMV cucumber mosaic cucumovirus
Cmv2b CMV 2b protein
HC-Pro potyviral helper component proteinase
mut-7 mutator-7
PTGS post-transcriptional gene silencing
PVX potato virus X
qde quelling deficient rde RNAi deficient
RdRP RNA-dependent RNA polymerase
RMVR RNA-mediated virus resistance
RNAi RNA interference
Tav2b 2b protein of tomato aspermy cucumovirus
TGS transcriptional gene silencing
VIGS virus-induced gene silencing
Introduction
The first reports that a transgene introduced into plants would lead to silencing (co-suppression) of both the trans-gene and its homologous endogenous trans-gene were made 10 years ago [1,2]. Transgene-induced gene silencing has since been demonstrated in several other organisms [3]. Based on the known features, transgene-induced silencing effects have been divided into transcriptional and post-transcriptional gene silencing (TGS and PTGS, respectively) [4,5]. Both TGS and PTGS are nucleotide sequence homology dependent; however, genes silenced transcriptionally are homologous in promoter regions, whereas genes targeted for PTGS share homology in tran-scribed regions. Furthermore, TGS silences genes at the level of transcription in the nucleus. In contrast, PTGS has no apparent effect on transcription of the target gene but promotes a rapid and specific turnover of RNA transcripts in the cytoplasm.
PTGS has been studied extensively in plants [4] and in the fungus Neurospora(quelling) [6] and has also been shown to occur in flies [7], protozoans [8] and mice [9]. Recent work has revealed that PTGS shares many key features with both RNA-mediated virus resistance (RMVR) in plants [10] and RNA interference (RNAi) in a number of organisms, including nematodes, flies, trypanosomes, planaria, hydra
and zebrafish [3,11•]. As previously suggested [12], it is per-haps more accurate to refer to these phenomena collectively as ‘RNA silencing’ because the inactivation of genes in each example is achieved through a targeted degradation of RNA. This short review, limited to publica-tions since December 1998, will focus on recent developments in the area of RNA silencing, paying partic-ular attention to the natural biological functions of RNA silencing and a potential application of RNA silencing to plant functional genomics.
RNA silencing as a natural antiviral defense
in plants
Pioneering work by Dougherty and co-workers [13] provid-ed the first link between RNA silencing and viruses in plants by showing that virus infection is able to trigger RNA silencing of a homologous virus-derived transgene in trans-genic tobacco. The activation of silencing is accompanied by recovery of the host from the initially virulent infection so that the new growth is both symptom and virus free and is highly resistant to a secondary challenge by the same virus. This type of RMVR, demonstrated conclusively for a number of dicot plant species with a variety of viruses [10], is also functional in monocot species [14–16].
Classical literature shows that some virus–host interactions naturally lead to host recovery. The natural recovery responses induced by a nepovirus, with a single-stranded RNA genome [17], and a caulimovirus, with a double-stranded DNA genome [18], were shown to be similar to RMVR found in transgenic plants. Additionally, Ratcliff
et al.[19••] have shown that the recovery of Nicotiana
ben-thamiana from infection by a tobravirus expressing green fluorescent protein was also associated with RMVR activa-tion in the recovered leaves. The authors further demonstrated that the induced resistance mechanism is functionally equivalent to PTGS because it could also tar-get homologous mRNA transiently transcribed from the T-DNA of an Agrobacteriumstrain delivered by leaf infiltra-tion [19••]. It appears, therefore, that higher plants contain a novel cellular mechanism to recognize and degrade, with a high specificity, the RNAs of invading viruses.
Recovery is not a usual plant response to virus infection. Thus, can RNA silencing be considered as representative of a generalized plant antiviral defense? In addressing this question, it is significant that upon potato virus X (PVX) infection N. benthamianaplants exhibit the RNA silencing response despite the fact that the infected plants do not recover [19••]. This finding indicates that the ongoing silencing of viral RNAs may occur in many other persistent and successful virus infections and that active viral RNA silencing may not be sufficient to lead to a recovery. Clues to the additional viral determinant(s) required to enable
RNA silencing
the recovery response to occur may come from comparing features of viruses that do and do not induce recovery in a particular host species. Insights into the molecular control of host recovery may also be obtained by isolating and characterizing spontaneous non-recovery mutants of virus-es that normally induce recovery in a host.
Suppression of RNA silencing and viral diseases
An independent line of evidence that supports RNA silencing as a natural antiviral defense in plants came from studies of synergistic viral disease, in which a mixed infec-tion of two viruses produces a more severe disease than either virus alone. The systematic analysis by Vance and co-workers [20,21] of the classical synergism between PVX and potyviruses led to the identification of the potyviral helper component proteinase (HC-Pro) as the synergy determinant and a broad-range virulence enhancer. Subsequent studies from three groups established that HC-Pro is able to suppress PTGS [22–24], providing a direct link between the ability to suppress RNA silencing and the severity of viral diseases.The 2b protein encoded by cucumber mosaic cucumovirus (CMV) was similarly identified as a suppressor of PTGS in
N. benthamiana [23]. That the CMV 2b protein (Cmv2b) functions as a suppressor of host defense was first proposed in 1995 based on the finding that Cmv2b is essential for the development of CMV disease symptoms in its hosts [25]. Previous analyses also indicated that the Cmv2bgene repre-sents a newly evolved gene as compared to the other four CMV genes [25,26], suggesting that the Cmv2bgene is a viral adaptation to the RNA silencing antiviral defense in plants.
The 2b protein of tomato aspermy cucumovirus (Tav2b) induced a synergistic disease in seven host species when it was expressed from the CMV genome in the place of Cmv2b [27]. This is probably because Tav2b is a more effi-cient suppressor of PTGS than Cmv2b, as demonstrated in
N. benthamiana, for example, by the fact that suppression of a transgene RNA silencing by Tav2b was at least three days earlier than that achieved by Cmv2b [28•]. In a relat-ed host species (N. tabacum), however, Tav2b, but not Cmv2b, triggered a typical hypersensitive virus resistance [28•], characterized by the formation of necrotic lesions and the transcriptional induction of pathogenesis-related protein genes [29]. Thus, plant hosts may combat the sup-pression of RMVR by invoking another independent resistance mechanism. The challenge now is to understand how the recognition of one viral protein by two indepen-dent antiviral mechanisms is regulated in plants.
Voinnet et al.[30•] found recently that eight of 12 newly tested viruses were able to suppress PTGS inN. benthami-ana, suggesting that PTGS suppression is widely used as a counter-defensive strategy by plant viruses. Three new viral PTGS suppressors were identified in this work and, as for Cmv2b/Tav2b and HC-Pro, each was originally char-acterized as a virulence determinant for its respective
virus. In addition, PVX recombinants expressing mutants of Cmv2b defective in silencing suppression were much less virulent than the Cmv2b-expressing PVX in infected plants [31•]. Taken together, these data support a central role for the viral suppression of RNA silencing in the induction of viral diseases in plants [23].
Viral suppressors are also valuable tools for probing the RNA silencing pathway in plants. Comparative analysis revealed that Cmv2b and HC-Pro suppress different stages of PTGS: HC-Pro targets RNA silencing in tissues where silencing is already established, whereas Cmv2b only sup-presses the initiation of PTGS in newly emerged leaves [23,32]. A third type of viral suppressor was reported recently, which only blocks silencing in the veins of newly emerged leaves [30•]. Recent work has demonstrated that Cmv2b contains a functional arginine-rich nuclear localiza-tion signal and that the nuclear accumulalocaliza-tion of the protein was required for efficient PTGS suppression [31•]. This finding suggested the intriguing possibility that RNA silencing, which may target RNA exclusively in the cyto-plasm, can be blocked in the cell nucleus.
Targeted inactivation of endogenous genes by
RNA silencing
The intimate interaction between viruses and RNA silenc-ing in plants is besilenc-ing exploited to create a novel high-throughput technology for functional genomics in plants [33]. This technology, termed virus-induced gene silencing (VIGS), is based on the observation that gene expression in plants can be suppressed in a sequence-spe-cific manner by infection with virus vectors carrying fragments of the host genes to be silenced [33]. A number of nuclear endogenous genes and transgenes have been targeted successfully by VIGS [33,34•], although it is not clear if the endogenous mRNA is involved in the trigger-ing and/or maintenance of RNA silenctrigger-ing. An alternative approach, referred as transgenic VIGS, that produces a sta-ble and heritasta-ble silenced phenotype is based on a transgene comprising a cDNA of replicating PVX RNA, to which a fragment of host gene is inserted [35•]. In a simi-lar manner to transgenic VIGS, efficient RNA silencing was also obtained in transgenic plants by transgene con-structs with inverted repeats in the transcribed regions [36] or by the simultaneous expression of sense and antisense RNAs [37]. Because of the transformation step, however, transgenic VIGS is more difficult to be adapted to a large-scale genome-wide screen than conventional VIGS, with which one can establish the relationship from gene to phe-notype within a few weeks.
transposons [39]. It was reported recently that the produc-tion of promoter transcripts from either a transgene [40•] or a VIGS vector [34•] resulted in transsilencing of the gene driven by the homologous promoter. As promoter sequences are often more variable than coding regions within a multigene family, VIGS can also be used to achieve a more precise gene inactivation by targeting pro-moter sequences [34•].
Cellular components of the RNA silencing
pathway
Genetic screens have identified a number of loci involved in the RNA silencing pathway in Neurospora [41], Arabidopsis [4] and, more recently, in nematodes [42••,43••]. The first cellular component of the pathway cloned was the quelling deficient (qde) 1 gene from
Neurospora[44••]. The product of the qde-1 gene is homol-ogous to an RNA-dependent RNA polymerase (RdRP) isolated from tomato [45]. The involvement of a host-encoded RdRP in PTGS was first proposed in 1993 [13] and several current models include RdRP as a central com-ponent in the RNA silencing pathway [12]. A demonstrated essential role for RdRP indicates that RNA silencing requires novel RNA synthesis.
Although it is not clear what the in vivotemplate for RdRP might be [12], there is a good candidate for the product of the enzyme — the 25-nucleotide antisense RNA species (25-nt aRNA) recently identified in plants, which was associated with four different types of RNA silencing [46••]. The prop-erties of this 25-nt aRNA suggest that it may represent the specificity determinant of RNA silencing. Amplification of the 25-nt aRNA by RdRP, if confirmed in other species, may also explain why only a few molecules of double-stranded RNA per cell are required to produce RNAi [3].
Tabara et al.[42••] recently identified four loci in nema-todes that are required for RNAi. Mutants defective in either RNAi deficient(rde) 1 or rde-4 loci showed no addi-tional defects, whereas rde-2and rde-3mutants exhibited mobilization of the endogenous transposons. Ketting et al.
[43••] independently discovered this link between RNAi and transposon mobilization. Thus, another natural func-tion of RNA silencing may be in host defense to transposon invasion, as suggested earlier [7]. The product of rde-1
belongs to a large gene family of unknown function includ-ing piwi/sting/argonaute/zwille/elF2C [42••]. The sting gene of flies is required for silencing the repetitive germline-spe-cific Stellate locus [47]. The requirement of the mutator-7
(mut-7) gene, isolated by Ketting et al. [43••], for RNA silencing is more obvious as it encodes a protein that is homologous to an RNA-degrading enzyme, RNaseD.
The qde-3gene has recently been cloned [48••]. Its encod-ed protein, QDE3, is a member of the RecQ DNA helicase family [49], suggesting a critical role for DNA–DNA inter-action in the activation of RNA silencing in Neurospora. It is not clear, however, whether a RecQ DNA helicase is
required for RMVR in non-transgenic plants, a phenome-non that does not involve homologous DNA sequences [19••]. Interestingly, in addition to the seven RecQ heli-case domains [49] conserved in the QDE3 protein [48••], Werner syndrome helicase (WRN) also encodes a func-tional RNaseD domain [50], found in the mut-7 protein [43••]. Thus, it is possible that induction of RNA silencing requires the coordinated actions of RecQ helicase (qde-3) and RNaseD (mut-7). In this regard, it is important to determine if the mammalian WRN protein or its homo-logues play any role in RNA silencing [9] and/or complement mutations in the mut-7 and qde-3 loci in heterologous systems.
Conclusions
It appears that RNA silencing, which involves the specific recognition and degradation of RNA molecules, is a con-served mechanism in many organisms. Recent work has clearly demonstrated a role of RNA silencing in transposon silencing in nematodes and as an innate antiviral defense mechanism in higher plants. RNA silencing is effective against plant viruses with either an RNA or DNA genome. In the latter case, the RNA transcripts serve as the target of RNA silencing [18]. Consistent with these findings, many RNA viruses, as well as a geminivirus having a cir-cular single-stranded DNA, were found to encode efficient suppressors of RNA silencing [30•]. Available evidence also shows that suppression of RNA silencing by virus-encoded proteins plays a central role in the induction of viral diseases in plants. It will be of great interest to deter-mine how much of the known roles of RNA silencing in plant viral diseases is also applicable to other organisms.
Acknowledgements
Work in the author’s laboratory was supported by grants from the National Science and Technology Board, Singapore. I apologize to the authors of many excellent PTGS-related papers that are omitted due to space constraints.
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