Centro Nacional de Biotecnología (C.S.I.C.), Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain
Correspondence
Juan Antonio García
jagarcia{at}cnb.uam.es
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ABSTRACT |
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Present address: Centro de Investigaciones Biológicas (C.S.I.C.), Velázquez 144, 28006 Madrid, Spain.
Present address: Institute of Molecular Agrobiology, 1 Research Link, The National University of Singapore, Singapore 117604.
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INTRODUCTION |
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Although RNA silencing in plants has been studied most extensively using transgenes, viruses can be both initiators and targets of RNA silencing (Ratcliff et al., 1997; Voinnet et al., 1999
). RNA silencing mechanisms derived from virus infection (virus-induced gene silencing, VIGS) and from transgenes are closely related and appear to share many components, but have been shown to use different pathways to generate the double-stranded RNA that triggers the process (Dalmay et al., 2000
; Béclin et al., 2002
; Morel et al., 2002
). Many data obtained in recent years support RNA silencing as an inducible host RNA surveillance mechanism used for defence against virus infection and transposon-induced abnormalities in gene expression, thus allowing the plant to recognize and destroy foreign or aberrant RNAs (for reviews, see Vance & Vaucheret, 2001
; Voinnet, 2001
; Waterhouse et al., 2001
; Vazquez Rovere et al., 2002
).
The study of this intriguing defence mechanism of plants against virus infection has led to the discovery of virus-encoded suppressors of RNA silencing (Anandalakshmi et al., 1998; Béclin et al., 1998
; Brigneti et al., 1998
; Kasschau & Carrington, 1998
). The ability of plant viruses to suppress RNA silencing is probably part of a counter-defensive strategy to overcome the silencing response (for reviews, see Carrington et al., 2001
; Baulcombe, 2002
). Silencing suppressors which have distinct modes of action have been described in diverse DNA and RNA viruses of plants (Voinnet et al., 1999
). The first viral suppressor of silencing characterized was the HCPro protein encoded in the potyviral genome, which has been shown to interfere with both transgene-induced RNA silencing (Anandalakshmi et al., 1998
; Kasschau & Carrington, 1998
) and VIGS (Anandalakshmi et al., 1998
; Brigneti et al., 1998
). HCPro acts by blocking the maintenance of RNA silencing in tissues where silencing has already been established. In contrast, the 2b protein of cucumoviruses is unable to reverse already established RNA silencing, but prevents its initiation at the growing points of the plant by inhibiting the long-range activity of the silencing signal produced during the silencing reaction (Béclin et al., 1998
; Brigneti et al., 1998
; Guo & Ding, 2002
). Other suppressors, such as the p25 protein of the potexviruses, also affect RNA silencing by preventing spreading of the silencing signal (Voinnet et al., 2000
).
RNA silencing of transgenes homologous to viral sequences has been shown to give rise to highly specific resistance against the virus from which the transgene derives (Lindbo & Dougherty, 1992; Mueller et al., 1995
). Sometimes, the transgene is constitutively silenced and the transgenic plant is immune to virus infection. However, in other cases, transgene silencing and the consequent establishment of resistance must be induced by an initial virus infection, resulting in a recovery phenotype. The recovered tissues are free of viral RNA and proteins and are resistant to superinfection by the same virus, but susceptible to infection by unrelated viruses (Lindbo et al., 1993
). Recovery from virus disease is not restricted to transgenic plants and can also be activated naturally in some virus infections. In these cases, the infected plants exhibit a response very similar to the virus-induced recovery of transgenic plants in that the upper leaves are symptom free, contain reduced levels of virus and exhibit a strong and specific virus resistance (Covey et al., 1997
; Ratcliff et al., 1997
).
We have constructed transgenic Nicotiana benthamiana plants transformed with a genome fragment of Plum pox virus (PPV), a member of the Potyvirus genus (López-Moya et al., 2000), which show a phenotype of delayed resistance mediated by RNA silencing (Guo & García, 1997
; Guo et al., 1999
). In the work described in this paper, the paradox that an infection producing an efficient silencing suppressor (HCPro) can induce RNA silencing is approached by assessing the suppression of the induced silencing and the consequent delayed virus resistance following infection with two heterologous viruses producing related and unrelated silencing suppressors.
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METHODS |
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Transgenic plants were grown from seeds germinated in the presence of kanamycin at a concentration of 0·1 mg ml-1.
Plant regeneration.
Plants were regenerated from sterilized leaf disks by in vitro culture on solid Murashige and Skoog (MS) medium containing 6-benzylaminopurine (1 mg l-1), -naphthalene acetic acid (0·1 mg l-1) and kanamycin (0·1 mg ml-1). Regenerated shoots were rooted on MS medium without hormones. Rooted plantlets were transferred to soil and grown in a climate-controlled room at 60 % relative humidity in a 14 h light (22 °C) and 10 h dark (20 °C) cycle.
Inoculation and sampling.
Young (five-leaf stage) plants were inoculated by rubbing inocula on to three leaves dusted with Carborundum. Crude sap from Nicotiana clevelandii plants infected with PPV or N. benthamiana plants infected with CMV or TVMV (1 g in 2 ml 5 mM sodium phosphate, pH 7·2) was used as the source of inoculum for these viruses. In the co-inoculation experiments, the two extracts were mixed just before being applied to the Carborundum-dusted leaves. For sequential inoculation, the first virus was inoculated as described above and, 24 weeks later, one leaf with symptoms immediately above those first inoculated was sampled to analyse transgene expression; the second virus was then inoculated on to the following leaf (also showing symptoms). Plants were maintained in a climate-controlled room at 60 % relative humidity in a 14 h light (22 °C) and 10 h dark (20 °C) cycle. PPV accumulation was assessed at different days post-inoculation by double-antibody sandwich indirect ELISA with the REALISA kit (Durviz), and TVMV and CMV accumulation was assessed by ELISA with antibodies obtained from the ATCC. Samples consisted of single discs of 7 mm diameter collected from leaves situated above the inoculated ones.
Analysis of RNA.
Total RNA was isolated from leaf tissue by the LiCl precipitation method described by Verwoerd et al. (1989). For Northern blot analysis, total RNA (10 µg) was separated on a 1·2 % agarose gel containing 6 % formaldehyde and transferred and UV cross-linked (1200 mJ, Stratalinker; Stratagene) to a Zeta-probe membrane (Bio-Rad) (Sambrook et al., 1989
). The blot was hybridized with a 32P-labelled riboprobe specific for the PPV NIb coding sequence, synthesized by in vitro transcription with MAXIscript kit (Ambion). Methylene blue staining of the membrane after blot transfer was used to show the ribosomal RNA (rRNA) loading (Sambrook et al., 1989
). Both hybridization signals and rRNA content were quantified with a phosphorimager (model PSI-486; Molecular Dynamics) and these values were used to calculate the relative amounts of transgene mRNA in each sample.
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RESULTS |
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In order to assess the susceptibility of the VIGS established in the recovered NIbV3 plants to the suppressor activity of different viral proteins, we regenerated plantlets from asymptomatic leaf explants in vitro. Both transgene silencing and resistance to PPV were maintained in these plants in the absence of the inducer PPV infection (Guo et al., 1999 and Fig. 1
). They will subsequently be referred to as recovered plants.
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DISCUSSION |
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We used the N. benthamiana line NIbV3 (Guo & García, 1997). This line is transformed with a PPV transgene that becomes silenced after an initial PPV infection. We studied the effect of infection with a second virus in plants regenerated from the recovered tissue (recovered plants), rather than in the primarily infected and recovered plants. These recovered plants retained the silencing of the transgene and the PPV-resistance phenotype but were completely free of the infecting virus (Fig. 1B
). Thus, they allowed us to study the result of the second virus infection on juvenile tissue without possible interference of the silencing suppressor from the first infection.
Both CMV, which produces the potyvirus-unrelated silencing suppressor 2b, and the potyvirus TVMV suppressed the PPV-induced silencing of the recovered NIbV3 plants (Fig. 2). This indicated that, although the silencing of the PPV transgene is induced by an infection that produces HCPro, it is still susceptible to the suppressor activity of a presumably similarly acting potyviral protein. Two main conclusions can be drawn from this result: (i) since virus replication and thus HCPro accumulation is maintained in the primary infected leaves in the recovering NIbV3 plants, the silencing suppression activity of HCPro must not be able to act at a distance; and (ii) HCPro cannot interfere with the propagation of silencing signals, which induce transgene silencing and virus resistance in the upper leaves of the NIbV3 plants before PPV can arrive, replicate and then produce HCPro.
There are conflicting data on the capacity of HCPro to interfere with the systemic silencing signal. Using grafting experiments and transgenic expression of both HCPro and the silencing inducer, Mallory et al. (2001) showed that HCPro was unable to block spreading of the silencing signal. In contrast, transient expression by agroinfiltration of HCPro was shown to prevent the systemic spread of silencing of a GFP transgene induced by a homologous construct also expressed by agroinfiltration (Hamilton et al., 2002
; Pfeffer et al., 2002
). The apparent inconsistency of these experiments probably rises from the different transgenes and expression systems used (Voinnet, 2001
). It is likely that HCPro has some ability to disturb the spreading of the systemic silencing signal, either by direct interference with it or by causing a decline in its production. However, the effectiveness of the blockade of the silencing spread would depend on the quantitative balance between HCPro and the silencing signal produced in each case. In contrast with the previous approaches, in our experimental system recovery of the NIbV3 plants took place in the context of a potyvirus infection, indicating that in such a natural situation, HCPro is unable to block the systemic propagation of the silencing signal successfully in advance of the virus infection.
Interestingly, suppression of the NIb transgene silencing caused by CMV infection but not by TVMV infection restored susceptibility to PPV in the recovered NIbV3 plants (Table 1). The inability of TVMV infection to reverse the resistance to PPV is not likely to be due to inefficient silencing suppression since: (i) CMV infection facilitated PPV infection of the recovered NIbV3 plants, although it never reverted the transgene mRNA accumulation levels to those of the transgenic plant prior to the initial PPV infection (Fig. 2
); (ii) no apparent correlation between higher transgene mRNA accumulation and PPV susceptibility was observed in recovered NIbV3 plants inoculated first with CMV and then with PPV (Fig. 3
); and (iii) TVMV appears to suppress the silencing of the NIbV3 plants more efficiently than CMV. At least two possible explanations could account for our results. The first involves an inter-potyvirus cross-protection mechanism. Although no cross protection of TVMV against PPV was observed in non-transgenic PPV systemic hosts such as N. benthamiana (Table 1
) or N. clevelandii (data not shown), the infection of another potyvirus, Tobacco etch virus, has been shown to interfere with PPV replication in Nicotiana tabacum, a local host for PPV (Sáenz et al., 2002
). Thus, the possibility exists that cross protection is only apparent when PPV replication is weakened, either by an unsuitable host, such as tobacco, or by some RNA silencing activity still remaining in the TVMV-infected recovered NIbV3 plants. In this scenario, the susceptibility to Potato virus A (PVA) after Potato virus Y infection of N. benthamiana plants transformed with a constitutively silenced PVA coat protein transgene (Savenkov & Valkonen, 2001
) could be explained by a less efficient cross protection in this virus/host/transgene system.
Alternatively, the inconsistency between transgene silencing suppression and resistance breakdown in the virus-infected recovered NIbV3 plants could be revealing the existence of more than one silencing mechanism induced by the former PPV infection, which would have different susceptibility to the suppressor activities of CMV 2b and TVMV HCPro. At least two branches have been recognized in the induction pathway of RNA silencing in plants (Voinnet et al., 2000; Béclin et al., 2002
; Mlotshwa et al., 2002
). In addition, the possible existence of two separate mechanisms for silencing-related target mRNA destruction has recently been suggested (Tang et al., 2003
). Our results could be explained by assuming that both the constitutively PVA-resistant plants described by Savenkov & Valkonen (2001)
and the recovered NIbV3 plants have an RNA silencing mechanism that is suppressible by both HCPro and 2b, while a second silencing mechanism, specific for target degradation of viral RNA, is only induced by the virus-induced silencing of the recovered plants and would be efficiently suppressed by 2b but not by HCPro. We also cannot rule out the possibility that PPV infection induces non-silencing-related defence mechanisms in the recovered NIbV3 plants, which would be not active in the constitutively immune transgenic plants and which could be counteracted by CMV but not by TVMV infection. In this regard, it is important to note that CMV 2b has been shown to be involved not only in silencing-related defence mechanisms but also in virus resistance mediated by a hypersensitive response (Li et al., 1999
; Ji & Ding, 2001
).
The mechanisms leading to RNA degradation through RNA silencing remain unclear at both the cellular and the plant level. Numerous models have been proposed but none fully account for the broad range of phenomena described. Further characterization of the recovery process will contribute to a better understanding of virus resistance and RNA silencing in plants and will help to design more effective and durable strategies to confer virus resistance to plants.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Baulcombe, D. (2002). Viral suppression of systemic silencing. Trends Microbiol 10, 306308.[CrossRef][Medline]
Béclin, C., Berthomé, R., Palauqui, J.-C., Tepfer, M. & Vaucheret, H. (1998). Infection of tobacco or Arabidopsis plants by CMV counteracts systemic post-transcriptional silencing of nonviral (trans)genes. Virology 252, 313317.[CrossRef][Medline]
Béclin, C., Boutet, S., Waterhouse, P. & Vaucheret, H. (2002). A branched pathway for transgene-induced RNA silencing in plants. Curr Biol 12, 684688.[CrossRef][Medline]
Brigneti, G., Voinnet, O., Li, W. X., Ji, L. H., Ding, S. W. & Baulcombe, D. C. (1998). Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J 17, 67396746.
Carrington, J. C., Kasschau, K. D. & Johansen, L. K. (2001). Activation of suppression of RNA silencing by plant viruses. Virology 281, 15.[CrossRef][Medline]
Cogoni, C. (2001). Homology-dependent gene silencing mechanisms in fungi. Annu Rev Microbiol 55, 381406.[CrossRef][Medline]
Covey, S. N., Al-Kaff, N., Lángara, A. & Turner, D. S. (1997). Plants combat infection by gene silencing. Nature 385, 781782.[CrossRef]
Dalmay, T., Hamilton, A., Rudd, S., Angell, S. & Baulcombe, D. C. (2000). An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101, 543553.[Medline]
Guo, H. S. & Ding, S. W. (2002). A viral protein inhibits the long range signaling activity of the gene silencing signal. EMBO J 21, 398407.
Guo, H. S. & García, J. A. (1997). Delayed resistance to plum pox potyvirus mediated by a mutated RNA replicase gene: involvement of a gene silencing mechanism. Mol Plant Microbe Interact 10, 160170.
Guo, H. S., Lopez-Moya, J. J. & Garcia, J. A. (1999). Mitotic stability of infection-induced resistance to plum pox potyvirus associated with transgene silencing and DNA methylation. Mol Plant Microbe Interact 12, 103111.[Medline]
Hamilton, A., Voinnet, O., Chappell, L. & Baulcombe, D. (2002). Two classes of short interfering RNA in RNA silencing. EMBO J 21, 46714679.
Hannon, G. J. (2002). RNA interference. Nature 418, 244251.[CrossRef][Medline]
Ji, L. H. & Ding, S. W. (2001). The suppressor of transgene RNA silencing encoded by Cucumber mosaic virus interferes with salicylic acid-mediated virus resistance. Mol Plant Microbe Interact 14, 715724.[Medline]
Kasschau, K. D. & Carrington, J. C. (1998). A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95, 461470.[Medline]
Li, H. W., Lucy, A. P., Guo, H. S., Li, W. X., Ji, L. H., Wong, S. M. & Ding, S. W. (1999). Strong host resistance targeted against a viral suppressor of the plant gene silencing defence mechanism. EMBO J 18, 26832691.
Lindbo, J. A. & Dougherty, W. G. (1992). Pathogen-derived resistance to a potyvirus: immune and resistant phenotypes in transgenic tobacco expressing altered forms of a potyvirus coat protein nucleotide sequence. Mol Plant Microbe Interact 5, 144153.[Medline]
Lindbo, J. A., Silva-Rosales, L., Proebsting, W. M. & Dougherty, W. G. (1993). Induction of a highly specific antiviral state in transgenic plants: implications for regulation of gene expression and virus resistance. Plant Cell 5, 17491759.
López-Moya, J. J., Fernández-Fernández, M. R., Cambra, M. & García, J. A. (2000). Biotechnological aspects of plum pox virus. J Biotechnol 76, 121136.[CrossRef][Medline]
Mallory, A. C., Ely, L., Smith, T. H., Marathe, R., Anandalakshmi, R., Fagard, M., Vaucheret, H., Pruss, G., Bowman, L. & Vance, V. B. (2001). HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal. Plant Cell 13, 571583.
Mitter, N., Sulistyowati, E., Graham, M. W. & Dietzgen, R. G. (2001). Suppression of gene silencing: a threat to virus-resistant transgenic plants? Trends Plant Sci 6, 246247.[CrossRef][Medline]
Mlotshwa, S., Voinnet, O., Mette, M. F., Matzke, M., Vaucheret, H., Ding, S. W., Pruss, G. & Vance, V. B. (2002). RNA silencing and the mobile silencing signal. Plant Cell S289S301.
Morel, J. B., Godon, C., Mourrain, P., Béclin, C., Boutet, S., Feuerbach, F., Proux, F. & Vaucheret, H. (2002). Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance. Plant Cell 14, 629639.
Mueller, E., Gilbert, J., Davenport, G., Brigneti, G. & Baulcombe, D. C. (1995). Homology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing. Plant J 7, 10011013.[CrossRef]
Napoli, C., Lemieux, C. & Jorgensen, R. (1990). Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2, 279289.
Pfeffer, S., Dunoyer, P., Heim, F., Richards, K. E., Jonard, G. & Ziegler-Graff, V. (2002). P0 of beet western yellows virus is a suppressor of posttranscriptional gene silencing. J Virol 76, 68156824.
Ratcliff, F., Harrison, B. D. & Baulcombe, D. C. (1997). A similarity between viral defense and gene silencing in plants. Science 276, 15581560.
Sáenz, P., Salvador, B., Simón-Mateo, C., Kasschau, K. D., Carrington, J. C. & García, J. A. (2002). Host-specific involvement of the HC protein in the long-distance movement of potyviruses. J Virol 76, 19221931.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Savenkov, E. I. & Valkonen, J. P. T. (2001). Coat protein gene-mediated resistance to Potato virus A in transgenic plants is suppressed following infection with another potyvirus. J Gen Virol 82, 22752278.
Smith, C. J., Watson, C. F., Bird, C. R., Ray, J., Schuch, W. & Grierson, D. (1990). Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants. Mol Gen Genet 224, 477481.[Medline]
Tang, G., Reinhart, B. J., Bartel, D. P. & Zamore, P. D. (2003). A biochemical framework for RNA silencing in plants. Genes Dev 17, 4963.
van der Krol, A. R., Mur, L. A., Beld, M., Mol, J. N. & Stuitje, A. R. (1990). Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2, 291299.
Vance, V. & Vaucheret, H. (2001). RNA silencing in plants defense and counterdefense. Science 292, 22772280.
Vazquez Rovere, C., del Vas, M. & Hopp, H. E. (2002). RNA-mediated virus resistance. Curr Opin Biotechnol 13, 167172.[CrossRef][Medline]
Verwoerd, T. C., Dekker, B. M. M. & Hoekema, A. (1989). A small scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17, 2362.[Medline]
Voinnet, O. (2001). RNA silencing as a plant immune system against viruses. Trends Genet 17, 449459.[CrossRef][Medline]
Voinnet, O., Pinto, Y. M. & Baulcombe, D. C. (1999). Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci U S A 96, 1414714152.
Voinnet, O., Lederer, C. & Baulcombe, D. C. (2000). A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103, 157167.[Medline]
Waterhouse, P. M., Smith, N. A. & Wang, M. B. (1999). Virus resistance and gene silencing: killing the messenger. Trends Plant Sci 4, 452457.[CrossRef][Medline]
Waterhouse, P. M., Wang, M. B. & Lough, T. (2001). Gene silencing as an adaptive defence against viruses. Nature 411, 834842.[CrossRef][Medline]
Zamore, P. D. (2002). Ancient pathways programmed by small RNAs. Science 296, 12651269.
Received 1 April 2003;
accepted 24 May 2003.