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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).
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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(2) Sensing mRNA Cytoplasm RDRP (4) cRNA hybridization Virus RNA (5) Degradation
Trends in Plant Science
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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Parent plants
X Genotype S: :A
Progeny
Genotype
S:
S:A
:A
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.
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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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.
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.
References 1 Lindbo, J.A. and Dougherty, W.G. (1992) Untranslatable transcripts of the tobacco etch virus coat protein gene sequence can interfere with tobacco etch virus replication in transgenic plants and protoplasts, Virology 189, 725733 2 Baulcombe, D.C. (1996) Mechanisms of pathogen-derived resistance to viruses in transgenic plants, Plant Cell 8, 179188 3 Napoli, C. et al. (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans, Plant Cell 7, 599609 4 Van der Krol, A.R. et al. (1990) Flavonoid genes in petunia: addition of a limited number of gene copies may lead to suppression of gene expression, Plant Cell 2, 291299 5 Van Blokland, R. et al. (1994) Transgene-mediated suppression of chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover, Plant J. 6, 861877 6 Stam, M. et al. (1997) Post-transcriptional silencing of chalcone synthase in petunia by inverted transgene repeats, Plant J. 12, 6382 7 Stam, M. et al. (1998) Position-dependent methylation and transcriptional silencing in inverted T-DNA repeats: implications for posttranscriptional silencing of homologous host genes in plants, Mol. Cell. Biol. 18, 61656177 8 Dougherty, W.G. and Parks, T.D. (1995) Transgenes and gene suppression: telling us something new? Curr. Opin. Cell Biol. 7, 399405 9 Stam, M. et al. (1997) The silence of genes in transgenic plants, Ann. Bot. 79, 312 10 Vaucheret, H. et al. (1998) Transgene-induced gene silencing in plants, Plant J. 16, 651659 11 Schiebel, W. et al. (1998) Isolation of an RNA-directed RNA polymerasespecific cDNA clone from tomato, Plant Cell 10, 20872101 12 Cogoni, C. and Macino, G. (1999) Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase, Nature 399, 166169 13 English, J.J. et al. (1996) Suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes, Plant Cell 8, 787797 14 Holtorf, H. et al. (1999) Stochastic and nonstochastic post-transcriptional silencing of chitinase and -1,3-glucanase genes involves increased RNA turnover possible role for ribosome-independent RNA degradation, Plant Cell 11, 471483 15 Lee, K.Y. et al. (1997) Post-transcriptional gene silencing of ACC synthase in tomato results from cytoplasmic RNA degradation, Plant J. 12, 11271137 16 Metzlaff, M. et al. (1997) RNAmediated RNA degradation and chalcone synthase A silencing in Petunia, Cell 88, 120 17 Goodwin, J. et al. (1996) Genetic and biochemical dissection of transgenic RNA-mediated virus resistance, Plant Cell 8, 95105 18 Tanzer, M.M. et al. (1997) Characterization of post-transcriptionally suppressed transgene expression that confers resistance to tobacco etch virus infection in tobacco, Plant Cell 9, 14111423 19 De Carvalho, F. et al. (1992) Suppression of the -1,3 glucanase transgene expression in homozygous plants, EMBO J. 11, 25952602 20 Elemayan, T. and Vaucheret, H. (1996) A strongly expressed 35S-driven transgene undergoes post-transcriptional silencing in all tobacco transformants irrespective of the copy number, Plant J. 9, 787797 21 Jorgensen, R. et al. (1996) Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single copy vs. complex T-DNA sequences, Plant Mol. Biol. 31, 957973 22 Palauqui, J.C. and Vaucheret, H. (1995) Field trial analysis of nitrate reductase co-suppression: a comparative study of 38 combinations of transgene loci, Plant Mol. Biol. 29, 149159 23 Palauqui, J.C. and Vaucheret, H. (1998) Transgenes are dispensable for the RNA degradation step of cosuppression, Proc. Natl. Acad. Sci. U. S. A. 95, 96759680 24 Voinnet, O. et al. (1998) Systemic spread of sequence-specific transgene RNA degradation in plants is initiated by localized introduction of ectopic promoterless DNA, Cell 95, 177187
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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