1887

Abstract

High expression levels of the helper component proteinase (HC), a known virus suppressor of RNA silencing, were attained in transformed with the HC cistron of (PVA, genus ). No spontaneous silencing of the HC transgene was observed, in contrast to the PVA coat protein (CP)-encoding transgene in other transgenic lines. HC-transgenic plants were initially susceptible to PVA and were systemically infected by 14 days post-inoculation (p.i.) but, 1 to 2 weeks later, the new expanding leaves at positions +6 and +7 above the inoculated leaf showed a peculiar recovery phenotype. Leaf tips (the oldest part of the leaf) were chlorotic and contained high titres of PVA, whereas the rest of the leaf was symptomless and contained greatly reduced or non-detectable levels of viral RNA, CP and transgene mRNA. The spatial recovery phenotype suggests that RNA silencing is initiated in close proximity to meristematic tissues. Leaves at position +8 and higher were symptomless and virus-free but not completely resistant to mechanical inoculation with PVA. However, they were not infected with the virus systemically transported from the lower infected leaves, suggesting a vascular tissue-based resistance mechanism. Recovery of the HC-transgenic plants from infection with different PVA isolates was dependent on the level of sequence homology with the transgene. Methylation of the HC transgene followed recovery. These data show that the transgene mRNA for a silencing suppressor can be silenced by a presumably ‘strong’ silencing inducer (replicating homologous virus).

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2002-09-01
2020-01-19
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References

  1. Al-Kaff, N. S., Covey, S. N., Kreike, M. M., Page, A. M., Pinder, R. & Dale, P. J. ( 1998; ). Transcriptional and posttranscriptional plant gene silencing in response to a pathogen. Science 279, 2113-2115.[CrossRef]
    [Google Scholar]
  2. Anandalakshmi, R., Pruss, G. J., Ge, X., Marathe, R., Mallory, A. C., Smith, T. H. & Vance, V. B. ( 1998; ). A viral suppressor of gene silencing in plants. Proceedings of the National Academy of Sciences, USA 95, 13079-13084.[CrossRef]
    [Google Scholar]
  3. Anandalakshmi, R., Marathe, R., Ge, X., Herr, J. M.Jr, Mau, C., Mallory, A., Pruss, G., Bowman, L. & Vance, V. B. ( 2000; ). A calmodulin-related protein that suppresses posttranscriptional gene silencing in plants. Science 290, 142-144.[CrossRef]
    [Google Scholar]
  4. Andrejeva, J., Puurand, Ü., Merits, A., Rabenstein, F., Järvekülg, L. & Valkonen, J. P. T. ( 1999; ). Potyvirus helper component-proteinase and coat protein (CP) have coordinated functions in virus–host interactions and the same CP motif affects virus transmission and accumulation. Journal of General Virology 80, 1133-1139.
    [Google Scholar]
  5. Baulcombe, D. C. ( 1999; ). Fast forward genetics based on virus-induced gene silencing. Current Opinion in Plant Biology 2, 109-113.[CrossRef]
    [Google Scholar]
  6. Beclin, C., Berthome, 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, 313-317.[CrossRef]
    [Google Scholar]
  7. 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 Journal 17, 6739-6746.[CrossRef]
    [Google Scholar]
  8. Carrington, J. C., Kasschau, K. D. & Johansen, L. K. ( 2001; ). Activation and suppression of RNA silencing by plant viruses. Virology 281, 1-5.[CrossRef]
    [Google Scholar]
  9. Chicas, A. & Macino, G. ( 2001; ). Characteristics of post-transcriptional gene silencing. EMBO Reports 2, 992-996.[CrossRef]
    [Google Scholar]
  10. Citovsky, V. & Zambryski, P. ( 1999; ). Systemic transport of RNA in plants. Trends in Plant Science 5, 52-54.
    [Google Scholar]
  11. Covey, S. N., Al-Kaff, N., Langara, A. & Turner, D. S. ( 1997; ). Plants combat infection by gene silencing. Nature 38, 781-782.
    [Google Scholar]
  12. Guo, H. S. & Garcia, J. A. ( 1997; ). Delayed resistance to plum pox potyvirus mediated by a mutated RNA replicase gene: involvement of a gene-silencing mechanism. Molecular Plant–Microbe Interactions 10, 160-170.[CrossRef]
    [Google Scholar]
  13. Guo, H. S., López-Moya, J. J. & Garcı́a, J. A. ( 1999; ). Mitotic stability of infection-induced resistance to plum pox potyvirus associated with transgene silencing and DNA methylation. Molecular Plant–Microbe Interactions 12, 103-111.[CrossRef]
    [Google Scholar]
  14. Hamilton, A. J. & Baulcombe, D. C. ( 1999; ). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950-952.[CrossRef]
    [Google Scholar]
  15. Hammond, S. M., Bernstein, E., Beach, D. & Hannon, G. J. ( 2000; ). An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293-296.[CrossRef]
    [Google Scholar]
  16. Ingelbrecht, I., Van Houdt, H., Van Montagu, M. & Depicker, A. ( 1994; ). Posttranscriptional silencing of reporter transgenes in tobacco correlates with DNA methylation. Proceedings of the National Academy of Sciences, USA 91, 10502-10506.[CrossRef]
    [Google Scholar]
  17. Jones, A. L., Johansen, I. E., Bean, S. J., Bach, I. & Maule, A. J. ( 1998a; ). Specificity of the resistance to pea seed-borne mosaic potyvirus in transgenic peas expressing the viral replicase (NIb) gene. Journal of General Virology 79, 3129-3137.
    [Google Scholar]
  18. Jones, A. L., Thomas, C. L. & Maule, A. J. ( 1998b; ). De novo methylation and co-suppression induced by a cytoplasmically replicating plant RNA virus. EMBO Journal 17, 6385-6393.[CrossRef]
    [Google Scholar]
  19. Jones, L., Hamilton, A. J., Voinnet, O., Thomas, C. L., Maule, A. J. & Baulcombe, D. C. ( 1999; ). RNA–DNA interactions and DNA methylation in post-transcriptional gene silencing. Plant Cell 11, 2291-2301.[CrossRef]
    [Google Scholar]
  20. Kasschau, K. D & Carrington, J. C. ( 1998; ). A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95, 461-470.[CrossRef]
    [Google Scholar]
  21. Kekarainen, T., Merits, A., Oruetxebarria, I., Rajamäki, M. & Valkonen, J. P. T. ( 1999; ). Comparison of the complete sequences of five different isolates of Potato virus A (PVA), genus Potyvirus. Archives of Virology 144, 2355-2366.[CrossRef]
    [Google Scholar]
  22. Lindbo, J. A. & 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, 725-733.[CrossRef]
    [Google Scholar]
  23. 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, 1749-1759.[CrossRef]
    [Google Scholar]
  24. Longstaff, M., Brigneti, G., Boccard, F., Chapman, S. & Baulcombe, D. C. ( 1993; ). Extreme resistance to potato virus X infection in plants expressing a modified component of the putative viral replicase. EMBO Journal 12, 379-386.
    [Google Scholar]
  25. Mallory, A. C., Ely, L., Smith, T. H., Marathe, R., Anandalakshmi, R., Fagard, M., Vaucheret, H., Pruss, G. J., 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, 571-583.[CrossRef]
    [Google Scholar]
  26. Marathe, R., Anandalakshmi, R., Smith, T. H., Pruss, G. J. & Vance, V. B. ( 2000; ). RNA viruses as inducers, suppressors and targets of post-transcriptional gene silencing. Plant Molecular Biology 43, 295-306.[CrossRef]
    [Google Scholar]
  27. Matzke, M. A., Matzke, A. J. M., Pruss, G. & Vance, V. B. ( 2001; ). RNA-based silencing strategies in plants. Current Opinion in Genetics & Development 11, 221-227.[CrossRef]
    [Google Scholar]
  28. Meins, F.Jr ( 2000; ). RNA degradation and models for post-transcriptional gene-silencing. Plant Molecular Biology 43, 261-273.[CrossRef]
    [Google Scholar]
  29. Mlotshwa, S. (2001). The helper component-proteinase of cowpea aphid-borne mosaic virus. PhD thesis, Wageningen University, Wageningen, The Netherlands.
  30. Moore, C. J., Sutherland, P. W., Forster, R. L. S., Gardner, R. C. & MacDiarmid, R. M. ( 2001; ). Dark green islands in plant virus infection are the results of posttranscriptional gene silencing. Molecular Plant–Microbe Interactions 14, 939-946.[CrossRef]
    [Google Scholar]
  31. Mueller, E., Gilbert, J. E., Davenport, G., Brigneti, G. & Baulcombe, D. C. ( 1995; ). Homology-dependent resistance: trans-genic virus resistance in plants related to homology-dependent gene silencing. Plant Journal 7, 1001-1013.[CrossRef]
    [Google Scholar]
  32. 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, 279-289.[CrossRef]
    [Google Scholar]
  33. Oparka, K. J. & Santa Cruz, S. ( 2000; ). The great escape: phloem transport and unloading of macromolecules. Annual Review of Plant Physiology and Plant Molecular Biology 51, 323-347.[CrossRef]
    [Google Scholar]
  34. Palauqui, J. C. & Vaucheret, H. ( 1998; ). Transgenes are dispensable for the RNA degradation step of cosuppression. Proceedings of the National Academy of Sciences, USA 95, 9675-9680.[CrossRef]
    [Google Scholar]
  35. Pang, S. Z., Slightom, J. L. & Gonsalves, D. ( 1993; ). Different mechanisms protect transgenic tobacco against tomato spotted wilt and impatiens necrotic spot tospoviruses. Biotechnology 11, 819-824.[CrossRef]
    [Google Scholar]
  36. Papaefthimiou, I., Hamilton, A. J., Denti, M. A., Baulcombe, D. C., Tsagris, M. & Tabler, M. ( 2001; ). Replicating potato spindle tuber viroid RNA is accompanied by short RNA fragments that are characteristic of post-transcriptional gene silencing. Nucleic Acids Research 29, 2395-2400.[CrossRef]
    [Google Scholar]
  37. Rajamäki, M., Merits, A., Rabenstein, F., Andrejeva, J., Paulin, L., Kekarainen, T., Kreuze, J. F., Forster, R. L. S. & Valkonen, J. P. T. ( 1998; ). Biological, serological, and molecular differences among isolates of potato A potyvirus. Phytopathology 88, 311-321.[CrossRef]
    [Google Scholar]
  38. Ratcliff, F., Harrison, B. & Baulcombe, D. C. ( 1997; ). A similarity between viral defense and gene silencing in plants. Science 276, 1558-1560.[CrossRef]
    [Google Scholar]
  39. Ratcliff, F. G., MacFarlane, S. A. & Baulcombe, D. C. ( 1999; ). Gene silencing without DNA. RNA-mediated cross-protection between viruses. Plant Cell 11, 1207-1216.[CrossRef]
    [Google Scholar]
  40. Roberts, A. G., Santa Cruz, S., Roberts, I. M., Prior, D. A. M., Turgeon, R. & Oparka, K. J. ( 1997; ). Phloem unloading in sink leaves of Nicotiana benthamiana: comparison of a fluorescent solute with a fluorescent virus. Plant Cell 9, 1381-1396.[CrossRef]
    [Google Scholar]
  41. Savenkov, E. I. & Valkonen, J. P. T. ( 2001a; ). Potyviral helper component proteinase expressed in transgenic plants enhances titers of Potato leaf roll virus but does not alleviate its phloem-limitation. Virology 283, 285-293.[CrossRef]
    [Google Scholar]
  42. Savenkov, E. I. & Valkonen, J. P. T. ( 2001b; ). Coat protein gene-mediated resistance to Potato virus A in transgenic plants is suppressed following infection with another potyvirus. Journal of General Virology 82, 2275-2278.
    [Google Scholar]
  43. Taliansky, M. E., Ryabov, E. V. & Robinson, D. J. ( 1998; ). Two distinct mechanisms of transgenic resistance mediated by groundnut rosette virus satellite RNA sequences. Molecular Plant–Microbe Interactions 11, 367-374.[CrossRef]
    [Google Scholar]
  44. Valkonen, J. P. T., Rajam ki, M.-L. & Kekarainen, T. ( 2002; ). Mapping of viral genomic regions important for cross-protection between strains of a potyvirus. Molecular Plant–Microbe Interactions 15, 683–692.[CrossRef]
    [Google Scholar]
  45. Vance, V. & Vaucheret, H. ( 2001; ). RNA silencing in plants: defence and counterdefense. Science 292, 2277-2280.[CrossRef]
    [Google Scholar]
  46. Vance, V. B., Berger, P. H., Carrington, J. C., Hunt, A. G. & Shi, X. M. ( 1995; ). 5′ proximal potyviral sequences mediate potato virus X/potyviral synergistic disease in transgenic tobacco. Virology 206, 583-590.[CrossRef]
    [Google Scholar]
  47. van der Krol, A. R., Mur, L. A., Beld, M., Mol, J. N. M. & Stuitje, A. R. ( 1990; ). Flavonoid genes in petunia: addition of a limited number of gene copies may lead to suppression of gene expression. Plant Cell 2, 291-299.[CrossRef]
    [Google Scholar]
  48. Verwoerd, T. C., Dekker, B. M. M. & Hoekema, A. ( 1989; ). A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Research 17, 2362.[CrossRef]
    [Google Scholar]
  49. Voinnet, O. & Baulcombe, D. C. ( 1997; ). Systemic signalling in gene silencing. Nature 389, 553.[CrossRef]
    [Google Scholar]
  50. Voinnet, O., Vain, P., Angell, S. & Baulcombe, D. C. ( 1998; ). Systemic spread of sequence-specific transgene RNA degradation in plants is initiated by localized introduction of ectopic promoterless DNA. Cell 95, 177-187.[CrossRef]
    [Google Scholar]
  51. 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. Proceedings of the National Academy of Sciences, USA 96, 14147-14152.[CrossRef]
    [Google Scholar]
  52. Voinnet, O., Lederer, C. & Baulcombe, D. C. ( 2000; ). A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103, 157-167.[CrossRef]
    [Google Scholar]
  53. Wang, H., Qi, M. & Cutler, A. J. ( 1993; ). A simple method for preparing plant samples for PCR. Nucleic Acids Research 21, 4153-4154.[CrossRef]
    [Google Scholar]
  54. Waterhouse, P. M., Wang, M. B. & Lough, T. ( 2001; ). Gene silencing as an adaptive defence against viruses. Nature 411, 834-842.[CrossRef]
    [Google Scholar]
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