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39 Roberts, J.K.M. et al. (1992) Contribution of malate and amino acid metabolism to cytoplasmic pH regulation in hypoxic maize root tips studied using magnetic resonance spectroscopy, Plant Physiol. 98, 480487 40 Tuin, L.G. and Shelp, B.J. (1996) In situ [14C]glutamate metabolism by developing soybean cotyledons. II. The importance of glutamate decarboxylation, J. Plant Physiol. 147, 714720 41 Micallef, B.J. and Shelp, B.J. (1989) Arginine metabolism in developing soybean cotyledons. III. Utilization, Plant Physiol. 91, 170174 42 Yancey, P.H. (1994) Compatible and counteracting solutes, in Cellular and Molecular Physiology of Cell Volume Regulation (Strange, K., ed.), pp. 81109, CRC Press 43 Heber, U., Tyankova, L. and Santarius, K.K. (1971) Stabilization and inactivation of biological membranes during freezing in the presence of amino acids, Biochim. Biophys. Acta 241, 578582 44 Smirnoff, N. and Cumbes, Q.J. (1989) Hydroxyl radical scavenging activity of compatible solutes, Phytochemistry 28, 10571060 45 Turano, F.J., Kramer, G.F. and Wang, C.Y. (1997) The effect of methionine, ethylene and polyamine catabolic intermediates on polyamine accumulation in detached soybean leaves, Physiol. Plant. 101, 510518 46 Trossat, C., Rathinasabapathi, B. and Hanson, A.D. (1997) Transgenically expressed betaine aldehyde dehydrogenase efficiently catalyses oxidation of dimethylsulfoniproprionaldehyde and -aminoaldehydes, Plant Physiol. 113, 14571461 47 Baum, G. et al. (1996) Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development of plants, EMBO J. 15, 29882996 48 Kathiresan, A. et al. (1998) -Aminobutyric acid promotes stem elongation in Stellaria longipes: the role of ethylene, Plant Growth Regul. 26, 131137 49 Ramputh, A.L. and Bown, A.W. (1996) Rapid gamma-aminobutyric acid synthesis and the inhibition of the growth and development of oblique-banded leaf-roller larvae, Plant Physiol. 111, 13491352 50 Jones, R.S. and Mitchell, C.A. (1989) Calcium ion movement in growth inhibition of mechanically stressed soybean (Glycine max) seedlings, Physiol. Plant. 76, 598602 51 Ford, Y-Y., Ratcliffe, R.G. and Robins, R.J. (1996) Phytohormone-induced GABA production in transformed root cultures of Datura strammonium: an in vivo 15N-NMR study, J. Exp. Bot. 47, 811818 52 Kathiresan, A. et al. (1997) -Aminobutyric acid stimulates ethylene biosynthesis in sunflower, Plant Physiol. 115, 129135 53 Rhodes, D., Verslues, P.E. and Sharp, R.E. (1999) Role of amino acids in abiotic stress resistance, in Plant Amino Acids: Biochemistry and Biotechnology (Singh, B.K., ed.), pp. 319356, Marcel Dekker

Barry J. Shelp* and Michael D. McLean are at the Dept of Plant Agriculture, Division of Biotechnology, Bovey Bldg, University of Guelph, Guelph, Ontario, Canada N1G 2W1; Alan W. Bown is at the Dept of Biological Sciences, Brock University, St Catharines, Ontario, Canada L2S 3A1. *Author for correspondence (tel 1 519 824 4120 ext. 3089; fax 1 519 767 0755; e-mail bshelp@evbhort.uoguelph.ca).

Virus resistance and gene silencing: killing the messenger


Peter M. Waterhouse, Neil A. Smith and Ming-Bo Wang
On occassion, virus-derived transgenes in plants can be poorly expressed and yet provide excellent virus resistance, and transgene constructs designed to supplement the expression of endogenous genes can have the effect of co-suppressing themselves and the endogenous genes. These two phenomena appear to result from the same post-transcriptional silencing mechanism, which operates by targeted-RNA degradation. Recent research into RNA-mediated virus resistance and co-suppression has provided insights into the interactions between plant viruses and their hosts, and spawned several models to explain the phenomenon.

he majority of plant-infecting viruses have RNA genomes that contain replication, movement and coat-protein genes. Initially it was thought that over-expressing one or more of these proteins in a normal or a dysfunctional state in transgenic plants would confer protection against the virus from which the transgene was derived. Although there have been some examples where this appears to be true, there are several others in which the transgene appears to have conferred resistance through its mRNA rather than by its encoded protein. This was shown first in 1992 when virus-resistant plants expressing untranslatable coat-protein mRNA were produced1. Since then there have been many examples of RNA-mediated resistance (RMVR) and they appear to share several features2: No transgene protein is required. Usually plants contain multiple transgene copies.
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Often associated with a high transcription rate but low steady state levels of transgene mRNA. Plants are either resistant to virus infection (no detectable virus replication, spread or symptoms) or initially show virus infection and symptoms, but subsequently produce new growth that is symptomless and resistant to virus infection. Usually associated with methylation of transgenes coding regions. Plants have resistance only to closely related virus strains. A few years before RNA-mediated resistance was discovered, cosuppression, a phenomenon that results in the silencing of both a transgene and its homologous endogenous gene, was described. Co-suppression was first uncovered during attempts to overexpress chalcone synthase (chs), a gene encoding a flower intermediate pigment biosynthesis enzyme, in Petunia3,4. As well as producing plants with purple flowers that are no different from

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non-transgenic plants, a small proportion produce white flowers or flowers with white and purple sectors. The chs transgenes in the white tissue cells fail not only to increase the flower pigment but also repress the floral pigment biosynthetic pathway by co-suppressing the endogenous chs gene. Although the chs mRNA level in the white tissues is dramatically reduced3, the transcription rates of the chs transgene and the endogenous chs gene in these tissues are no different from those in purple tissues5. Furthermore, many plants with entirely white or white-sectored flowers contain multiple, methylated, transgene copies6,7. These results suggest that chs cosuppression is a post-transcriptional event showing some of the characteristics found in RMVR. Pioneering work by Bill Dougherty and colleagues1,8 has provided considerable insights into RMVR that appear applicable to co-suppression and antisense suppression810. These phenomena are collectively termed post-transcriptional gene silencing (PTGS). Dougherty and colleagues were not only among the first to recognize that untranslatable coat-protein constructs give virus protection, but also identified, using isolated nuclei, the disparity between the low steady-state levels of transgene mRNAs and their high transcription rates. They proposed that transgene mRNA in virus-resistant plants, induces degradation of RNAs with the same or complementary sequence within the cytoplasm (Fig. 1). This not only explains how a virus-derived transgene can confer total protection against an RNA virus containing the same sequence, but also how a transgene derived from an endogenous gene can silence that gene in a transgenic plant. This attractive hypothesis has received much support in recent years, although the details of the mechanism, and how it is induced and maintained, remain the focus of research and debate.
Post-transcriptional gene silencing by RNA degradation

Virus transgenes (1) High transcription


Nucleus

(2) Sensing mRNA Cytoplasm RDRP (4) cRNA hybridization Virus RNA (5) Degradation
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(3) cRNA synthesis

Fig. 1. The threshold model the first model proposed to describe RNA-mediated resistance (RMVR). The model has five steps, starting with high transcription of the transgene and concluding with degradation of the RNA duplex. Abbreviations: RDRP, RNA-dependent RNA polymerase; cRNA, complementary RNA.

PTGS is a highly specific process. Co-suppression, antisense suppression and RMVR only silence genes or viruses with a high degree of sequence homology (75%) to the transgene. This specificity and the ability of transgene mRNA to silence same-sense endogenous gene transcripts and virus genomic RNA, suggests that a plant-encoded RNA-dependent RNA polymerase (RDRP) produces small RNA molecules that are complementary to transgene mRNA; these hydridize with the target RNAs to potentiate their degradation8. The degradation could be mediated by several enzymes, such as by a dsRNAase or an endonuclease that cleaves ssRNA adjacent to a dsRNA duplex. An RDRP was identiVirus RNA Degradation fied and purified from plants in 1993, but its triggered involvement in PTGS has been hypothetical until recently. The tomato gene encodVirus RNA ing this enzyme has now been cloned11; mRNA from when its homologue in Neurospora crassa 38 transgene mRNA from is inactivated, the fungus is unable to copies 12 transgene maintain PTGS (termed quelling)12. mRNA from copies There is good evidence that co-suppres12 transgene sion operates by sequence-specific RNA copies degradation within the cytoplasm rather Resistant Recovery Susceptible Trends in Plant Science than by preventing export of the target gene mRNA from the nucleus. Plants Fig. 2. Initiation of RNA-mediated resistance (RMVR) according to the threshold model exhibiting PTGS of a GUS transgene are (Fig. 1). A threshold level of RNA containing virus sequences is required to initiate RMVR. resistant to infection by a recombinant Plants with between three and eight transgenes meet this level and show resistance. Some plants potato virus X (PVX) containing GUS with one or two transgene copies exceed this level only in conjunction with RNA from the virus, and resistance develops some time after infection. Whereas other plants with one or two transsequences within its genome, but susceptigene copies express insufficient mRNA to exceed the threshold even in conjunction with virus ble to wild-type PVX (Ref. 13). Because RNA, and show no resistance. PVX is an RNA virus with a lifecycle that is exclusively restricted to the cytoplasm,
Level of RNA, with virus sequence, in cell
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the most reasonable explanation is that the GUS sequences within the PVX genome are targeted and degraded together with the GUS transgene mRNA. The degradation rates of PTGS-target mRNA have been measured for -1-3-glucanase gn1 (GLU) RNA and class 1 chitinase (CHN) in tobacco14. The turnover rates for CHN and GLU mRNAs in silenced tissues are about five- and threefold higher, respectively, than in unsilenced tissue. Specific fragments from mRNAs or viral genomes have been identified in silenced or virus-resistant tissues, suggesting that PTGS-related RNA degradation begins with endonucleolytic cleavage at one or more sites, followed by exonucleolytic degradation1517. The degradation probably takes place exclusively in the cytoplasm because there is evidence that PTGS does not decrease full-length transcript levels in the nucleus15. Many mechanisms of mRNA degradation involve the association of RNA with ribosomes, and it might be assumed that this is the site of

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on observations from RMVR. Transgenic plants have three (a) types of response to virus inoculation: susceptible, resistant and plants showing a recovery phenoAAAAA AAAAA AAAAA type. The recovery plants are iniAAAAA AAAAA AAAAA AAAAA AAAAAAAA AAAAA AAAAA tially infected systemically, but AAAAA dsRDRP AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA subsequently produce new leaves cRNA AAAAA AAAAA RDRP with progressively fewer sympcRNA toms and eventually produce virus-free leaves that are completely resistant to super-infec(b) (d) tion. Examination of the plants Ectopic pairing reveals that the resistant plants Ectopic pairing contain high numbers (three to Endogenous eight) of integrated transgene M M M M gene M M M copies whereas the susceptible and recovery plants contain one Premature termination Premature termination or two copies17. The resistance RDRP RDRP is functional at the single cell cRNA cRNA level and highly sequence-speRDRP cific, being effective against only cRNA closely related viruses1. The Trends in Plant Science threshold model (Figs 13) Fig. 3. Four models for the induction of post-transcriptional gene silencing. (a) Multicopy insertion or highly proposes that a resistant plant expressed single gene in the threshold model: the level of RNA exceeds a threshold and therefore acts as a has several transgene copies, template for RDRP. (b) Multicopy insertion. Transgenes are inserted as multicopies, ectopic pairing induces which result in large amounts of methylation that in turn leads to prematurely terminated transcription. The truncated transcripts are a template transgene mRNA being tranfor RDRP. (c) Inverted repeat transgene integration RNA with self-complementarity is produced by the transcribed. This high level of transcriptional readthrough from one transgene into another transgene inserted in the opposite orientation. The scription is sensed within the cell, duplex formed by the mRNA is a template for a dsRNA-specific RDRP. (d) Promoterless transgenes. triggering a sequence-specific Methylation or some other epigenetic modification to the endogenous gene is transferred by ectopic pairing mechanism that degrades the between methylated transgenes in an inverted repeat configuration and an endogenous gene. The modified transgene mRNA in the cytoendogenous gene produces truncated mRNA, which is a template for RDRP. Abbreviations: RDRP, RNAplasm. The model also proposes dependent RNA polymerase; dsRDRP, double strand-specific RNA-dependent RNA polymerase; cRNA, complementary RNA; M, methyl group. Green boxes with arrows represent promoters giving transcription in that the transgene mRNA in the direction of the arrows; blue boxes represent coding regions; pink boxes represent terminator sequences. recovery phenotype plants does not accumulate above the threshold required to trigger the degraPTGS-directed degradation. However, several studies using pro- dation mechanism, but will do so when combined with the RNA from tein synthesis inhibitors have shown that ongoing translation is virus infection. Plants that contain the transgene, but that are suscepnot required for this degradation18. Furthermore, dissociation of tible to the virus, produce insufficient transgene mRNA to exceed the mRNA from ribosomes using Verrucarin A does not reduce the threshold even in conjunction with virus RNA. The induction of degradation rate of CHN mRNA in tissues showing PTGS degradation following overproduction of transgene mRNA fits well (Ref. 14). These results suggest that the ribosome might not be the with many examples of co-suppression19,20. Co-suppression occurs degradation site in PTGS. efficiently when transgenes are transcribed from a strong promoter20,21, or when they are present in high copy numbers22. However, Induction of post-transcriptional gene silencing PTGS is not always associated with excessively active transgenes5,13, Clearly, not all transgenes induce PTGS because novel traits can and can be induced even with promoterless constructs5,23,24. To address be introduced using transgenesis. PTGS usually occurs in only a these anomalies, it has been suggested that an aberrant quality of the small proportion of the plants transformed with the same con- transgene mRNA, not its abundance, induces silencing8,13,25. struct. So what causes the transgene mRNA, among the thousands of other endogenous mRNAs, to be the target for degradation? Post-transcriptional induction by aberrant RNA There have been many models and variations proposed, but, with Because PTGS, whether for virus resistance or endogenous gene one exception, they all propose that RNA produced by the trans- silencing, does not always require a high level of transcription of gene is the template for RDRP, and that it is either above a certain the inducing transgenes, it has been proposed that a qualitative threshold concentration within the cell, or aberrant in some way. feature of the transgene mRNA triggers silencing8,13,25. This so-called The exception is a model proposed to explain the rare cases of aberrant mRNA would be distinct from normal mRNA, allowing PTGS in which no transgene mRNA is produced7. it to be recognized as a template for RDRP, thus targeting degradation of itself and other RNAs of the same sequence. The aberrant Threshold induction of post-translational gene silencing features that are suggested include: untranslatability, lack of introns, The first model1,8 for PTGS induction proposed that there is a sur- self-complementarity and transcriptional truncation or extension. veillance system within plant cells that detects mRNAs expressed Truncated RNAs might be produced in plants by several mechaabove an acceptable level, and induces the RDRP to recognize nisms. It is proposed26 that transgene protein overproduction in some these molecules as templates. This threshold model was based way causes the premature translational termination of transgene
(c) 454
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mRNA, inducing the removal of the polyA-tail, and that these aberrant RNAs form the template for RDRP. Alternatively, truncated RNAs might be produced in a similar manner to that described for the methionine biosynthetic locus (met2) in the fungus Ascobolus imersus. In the presence of met2 transgenes, the endogenous met2 locus becomes methylated over regions that are duplicated in the transgenes, and transcription is terminated at the methylated regions to produce truncated mRNA (Ref. 27). Methylation probably occurs as a consequence of ectopic pairing between homologous regions of the endogenous met2 gene and the met2 transgenes. Several observations of plant PTGS are consistent with this model (Fig. 3). A correlation between transgenes and PTGS methylation has been reported frequently9 and the presence of multiple transgenes, which is also frequently correlated with PTGS (Ref. 9), would increase the likelihood of ectopic pairing. The arrangement of transgene copies inducing PTGS appears to be important. An examination of the integration patterns of segregating chs transgenes in Petunia shows that no single-copy transgene insertion locus induces silencing and that silencing is only induced by the loci in which transgene copies are arranged as inverted repeats7,9. This organization could trigger aberrant RNA production by three possible mechanisms. The self-complementarity of repeat integrations might produce cruciform structures that are targets for methylation, resulting in a truncated transcript as described above. Second, the inverted repeat structure somehow interacts with the target endogenous gene or another active transgene, disrupting transcription through altered chromatin structure. Finally, transcriptional readthrough of an inverted-repeat arrangement produces RNA with self-complementarity (Fig. 3). Such a molecule would be capable of forming a duplex with itself. The possibility that dsRNA induces silencing has been investigated for both virus resistance and cosuppression25. Plants expressing sense mRNA of a virus-derived transgene have been crossed with plants expressing antisense mRNA of the transgene (Fig. 4). Each parent contains a single hemizygous transgene and is susceptible to the virus. All progeny that inherit both the sense and the antisense transgenes are resistant to the virus, whereas progeny inheriting the sense, antisense or neither transgene are susceptible. Similarly, GUS-expressing tissue that is super-transformed with a crippled GUS transgene that is designed to express mRNA with self-complementarity, is silenced much more efficiently than tissue that is super-transformed with sense or antisense constructs. Both results suggest that dsRNA plays an important role in PTGS. This is consistent with observations that PTGS can be induced by introducing exogenous dsRNA into nematodes (and trypanosomes, insects and planaria)28, and the discovery that some RNA viruses can be used to silence endogenous genes. Injection of dsRNA, containing sequences of endogenous genes, into nematodes silences the genes; injection with either sense or antisense RNA does not29. Plants infected with a virus containing an endogenous plant gene sequence, silence that gene30. In both these cases, no nuclear transgene transcription is required and because replicating RNA viruses have a dsRNA replicative form, dsRNA is produced. It is suggested that the PTGS, which mediates RDRP, might have an absolute requirement for a dsRNA template, and therefore recognize only mRNA that has large regions of self-complementarity, such as produced by readthough transcription of transgenes in an inverted repeat configuration25 (Fig. 3). An intriguing possibility is that the RDRP is functional in the nucleus (sequence analysis indicates it contains nuclear localization signals) and that the truncated or self-complementary RNAs cannot efficiently exit the nucleus, thereby providing abundant template for the enzyme. The small cRNAs produced by the RDRP could escape the nucleus to potentiate the degradation of cytoplasmic RNAs.

Parent plants

X Genotype S: :A

Progeny

Genotype

S:

S:A

:A

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Fig. 4. Experiment to investigate the role of dsRNA in RNA-mediated resistance (RMVR). A plant expressing a sense virus-derived transgene was crossed with a plant expressing an antisense virusderived transgene. Both plants are hemizygous for the transgene and susceptible to virus infection. The progeny have four different genotypes. Those progeny inheriting both a sense and an antisense gene are resistant to the virus (plants depicted with white leaves). Plants of the other three genotypes are susceptible to the virus (plants depicted with grey leaves). This experiment suggests that the sense and antisense transgene mRNA form a duplex that induces RMVR. Abbreviations: S, sense allele; A, antisense allele; , no allele.

Role of transcription and methylation

Many reports suggest that transgene transcription is essential for PTGS (Refs 16,20,25). Some reports provide convincing evidence that PTGS occurs only when high levels of transgene mRNA are supplemented with developmentally regulated endogenous mRNA (Ref. 21). Furthermore, when a promoterless version of a transgene, which is known to induce RMVR with a functional promoter, is transformed into plants, none of the lines produced is resistant to the virus31. However, there are reports of promoterless constructs inducing PTGS (Refs 5,24), and in at least one of these cases there is no detectable transgene mRNA (Ref. 5), ruling out the possibility that a transgene had fortuitously inserted itself adjacent to a plant gene promoter and was transcribed. It is well known that the methylation of one gene or transgene is often copied to a homologous gene elsewhere in the genome. Therefore, it is possible that the promoterless transgenes are methylated after integration, either triggered by their repetitive structure or by their base-pair composition. Ectopic pairing with the homologous endogenous gene confers either methylation or some other epigenetic modification onto it. The epigenetically marked endogenous gene subsequently produces aberrant mRNA, which acts as a template for the RDRP (Fig. 3). This model7 also explains why the promoterless RMVR construct does not induce RMVR. In contrast with co-suppression, there is no transcriptionally active, homologous, endogenous gene in the plant that the virus-derived transgene can ectopically pair with to generate aberrant RNA. Examination of RMVR-recovery plants showed that de novo methylation of the transgenes is induced by viral infection and that this immediately precedes the onset of RMVR (Ref. 32). This suggests a causal relationship. Interestingly, only the transgene
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regions that are homologous to the virus sequence are methylated, supporting the suggestion that the methylation is directed by RNADNA interaction26. In most of the models discussed, de novo methylation plays a key role in PTGS induction by causing the premature termination of mRNA transcription. However, there is evidence that it might neither cause premature termination of transcription nor be required for PTGS induction. Studies of some transgenic plants show that a methylated, silenced neomycin phosophotransferase gene is transcribed at the same rate as its unsilenced, non-methylated counterparts, and that the transcription proceeds through the heavily methylated 3 region of the gene26,33. In addition, there are several examples of PTGS in which transgene or endogenous gene methylation is absent26. Furthermore, in Neurospora crassa, strains that are cytosine methylation-defective exhibit the same capacity for PTGS (quelling) as those capable of cytosine methylation34. Perhaps transgene methylation is an unnecessary by-product of PTGS, but indicative that the silencing process has commenced. Alternatively, methylation might be an energysaving strategy that plants use to enforce or amplify PTGS.
Maintenance and spread of post-transcriptional gene silencing

PTGS can be divided into three phases: initiation, maintenance and spread35. PTGS can be initiated by transgenes and viruses. It can also be initiated by the delivery of exogenous DNA by bombardment or agro-infiltration35, or by the grafting of unsilenced scions onto silenced rootstock36, both of these methods create localized delivery points for PTGS and, amazingly, PTGS spreads from these points into other tissues. It appears to spread by a nonmetabolic, gene-specific diffusable signal36 that is capable of travelling both between cells via plasmodesmata, and long distances via the phloem35,36, probably in source-to-sink directions. The signal is probably a nucleic acid23,35,37 that can induce PTGS in tissues expressing target transgenes, and even in tissues with elevated levels of target endogenous gene mRNAs (Ref. 23). There are at least two types of PTGS-response in target tissues. On the one hand, tissues with transgene arrangements that are incapable of spontaneous PTGS can be induced into PTGS by the signal, but when removed from the signal they cannot maintain or induce PTGS. On the other hand, unsilenced tissues that are capable of spontaneous PTGS can be induced into a silent state by the signal, and when removed from the signal can maintain and induce PTGS (Ref. 23). This spread and maintenance of PTGS appears to be facilitated by an interaction between the signal and the target gene. This has been shown by an experiment in which PTGS was induced in a previously green fluorescent protein (GFP)-expressing plant by bombardment with DNA encoding the 3 third of the GFP sequence35. These silenced plants were found to be resistant to a virus containing the other two thirds of the GFP gene sequence. Because there was no overlap between these GFPderived sequences, it is highly likely that PTGS is mediated via the intact GFP transgene.
RNA-mediated resistance is a natural process and part of an on-going battle between host and virus

nepovirus-infected plants, the symptomless leaves exhibit the characteristics of PTGS-mediated resistance to superinfection38. Similarly, some examples of cross protection (a phenomenon recognized since 1929, in which a plant is protected from a severe virus by a previous infection with a closely related but mild strain of the virus) were shown recently to be mechanistically the same as PTGS (Ref. 39). Furthermore, co-inoculation of two different (non-recovery-inducing) viruses with a single shared homologous gene, results in one virus inducing PTGS-mediated resistance against the other39. Therefore, it is probable that successful virus infection results from a virus being able to prevent PTGS-mediated degradation of its genome by moving through the plant more rapidly than the PTGS it induces, by directly debilitating the plants PTGS response, or by a combination of both. Most plants are resistant to most viruses. Perhaps this is because upon infection, plants are able to initiate PTGS against most viruses sufficiently rapidly to prevent virus spread. Indeed, tissue-specific viruses (e.g. luteoviruses) might be restricted to cells in which they are able to inactivate PTGS. An exciting, recent discovery is that some viruses do encode proteins that debilitate the PTGS mechanism in plants. The HCPro of potyviruses41 and the 2b protein of cucumoviruses42, when expressed by a virus or as a transgene, relieve PTGS of reporter genes in plants. But the virushost interaction does not stop here. The 2b protein expressed from a recombinant tobamovirus can be recognized as an avirulence factor in some plants, initiating a hypersensitive cell-death-response, but not when expressed from its native virus43. This poses the possibility that the plant, in response to the virus possessing a mechanism for overcoming the plants PTGS-based protection (i.e. the 2b protein), uses its hypersensitive response to combat the virus. However, the strategy only works when the 2b is expressed from a heterologous virus, suggesting that the cucumovirus has a further mechanism that prevents the plant from recognizing the 2b protein as an avirulence factor. These results highlight the continual attack and counter-attack that is waged in the war between plants and their viruses.
Comments and future perspectives

RMVR and PTGS were discovered using transgenes, but there is strong evidence that they are derived from an intrinsic plant mechanism that evolved to protect the plant against virus infection. nepo-, tobra- and caulimo-viruses can induce responses in nontransgenic plants that are like the recovery phenomenon in transgene-mediated RMVR (Refs 3840). Plants infected with these viruses initially show symptoms, but subsequently produce leaves that are symptomless and contain low levels of the virus. In the
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PTGS is a powerful, specific, intracellular RNA-degradation mechanism, which has probably evolved as a defence against virus infection. In spite of intensive research over recent years, the components of the mechanism remain largely unknown, but they probably involve an RDRP and some sort of dsRNase, possibly akin to RNase III. The role of methylation remains a mystery. Is it intimately involved in the process of PTGS or is it merely a by-product? The discovery that PTGS can be fully active in cytosine methylationdeficient Neurospora suggests that it might be relatively unimportant. Nevertheless, ectopic pairing might be essential to PTGS, resulting in the production of aberrant RNA caused by an altered chromatin structure; the fact that the chromatin becomes methylated might be purely incidental. Methyltransferase-deficient plants have been generated recently, it will be interesting to see whether they can sustain PTGS, and also whether dsRNA plays a key role in PTGS and if RDRP is located in the nucleus. How PTGS is induced and operates might be elucidated by gene knock-out studies in Arabidopsis and Neurospora10. Three genetic loci that impede PTGS have been identified in Neurospora, including one that encodes an RDRP (Refs 12,44). The imminent completion of the Arabidopsis genome sequencing project, in combination with PTGS mutants in Arabidopsis, might also identify the genes involved in PTGS. Some interesting mutants have already been generated4547.

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Acknowledgements
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Sincere thanks to our colleagues, Jean Finnegan and Varsha Wesley, for stimulating discussions and critically reading this manuscript. We apologize to the authors of the many excellent PTGS-related papers we could not quote because of space restrictions.
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Peter M. Waterhouse*, Neil A. Smith and Ming-Bo Wang are at CSIRO-Plant Industry, PO Box 1600, Canberra, ACT 2601, Australia. *Author for corrspondence (tel 61 26 246 5365; fax 61 26 246 5000; e-mail peterw@pi.csiro.au).

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