1887

Journal of General Virology header logo

Volume 81, Issue 3

Mutations in the coat protein gene of suppress particle assembly, heterologous encapsidation and complementation in transgenic plants of Free

Abstract

Two different motifs in the coat protein (CP) of (PPV) (RQ, D) were mutated by replacing the respective amino acids with others possessing different chemical properties. The mutated CP genes were introduced into an infectious full-length clone of PPV (p35PPV-NAT) to investigate their influence on systemic infection of transgenic wild-type PPV CP-expressing and non-transgenic plants of . All mutants failed to establish systemic infections in non-transgenic plants, but were complemented by intact CP in transgenic plants. Moreover, the CP-RQ-D mutant (carrying mutations in both the RQ and D motifs) was introduced into p35PPV-NAT engineered to express β-glucuronidase (GUS) for direct observation of systemic movement and particle assembly in leaves. GUS-staining revealed that the CP mutant (RQ-D) was restricted to initially infected cells without forming virions. Systemic movement and particle assembly were restored in CP-transgenic plants. Finally, transgenic plants were generated that expressed each of the three mutated CP genes. Homozygous T lines were selected and tested for resistance to PPV. Immunogold labelling and electron microscopy revealed that heterologous encapsidation with challenging and was suppressed in these lines. In addition, assembly mutants did not complement CP-defective p35PPV-NAT. The possible use of modified viral CP genes for the production of virus-resistant transgenic plants, thereby reducing the putative risks of heterologous encapsidation and complementation, is discussed.

  • Received:
  • Accepted:
  • Published Online:
© Microbiology Society
Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-81-3-567
2000-03-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jgv/81/3/0810567a.html?itemId=/content/journal/jgv/10.1099/0022-1317-81-3-567&mimeType=html&fmt=ahah

References

  1. Atreya, P. L., Atreya, C. D. & Pirone, T. P. (1991). Amino acid substitutions in the coat protein result in loss of insect transmissibility of a plant virus. Proceedings of the National Academy of Sciences, USA 88, 7887-7891.[CrossRef] [Google Scholar]
  2. Balázs, E. & Tepfer, M. (editors) (1997).Virus-Resistant Transgenic Plants: Potential Ecological Impact. Berlin, Heidelberg & New York: Springer Verlag.
  3. Berger, P. H., Hunt, A. G., Domier, L. L., Hellmann, G. M., Stram, Y., Thornbury, D. W. & Pirone, T. P. (1989). Expression in transgenic plants of a viral gene product that mediates insect transmission of potyviruses. Proceedings of the National Academy of Sciences, USA 86, 8402-8406.[CrossRef] [Google Scholar]
  4. Borja, M., Rubio, T., Scholthof, H. B. & Jackson, A. O. (1999). Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene. Molecular Plant-Microbe Interactions 12, 153-162.[CrossRef] [Google Scholar]
  5. Chapman, S., Kavanagh, T. & Baulcombe, D. (1992). Potato virus X as a vector for gene expression in plants. Plant Journal 2, 549-557. [Google Scholar]
  6. Daniell, H. (1993). Foreign gene expression in chloroplasts of higher plants mediated by tungsten particle bombardment. Methods in Enzymology 217, 537-557. [Google Scholar]
  7. De Block, M. & Debrouwer, D. (1992).In-situ enzyme histochemistry on plastic-embedded plant material. The development of an artefact-free β-glucuronidase assay. Plant Journal 2, 261-266.[CrossRef] [Google Scholar]
  8. Dolja, V. V., Boyko, V. P., Agranovsky, A. A. & Koonin, E. V. (1991). Phylogeny of capsid proteins of rod-shaped and filamentous RNA plant viruses: two families with distinct patterns of sequence and probably structure conservation. Virology 184, 79-86.[CrossRef] [Google Scholar]
  9. Dolja, V. V., McBride, H. J. & Carrington, J. C. (1992). Tagging of plant potyvirus replication and movement by insertion of beta-glucuronidase into the viral polyprotein. Proceedings of the National Academy of Sciences, USA 89, 10208-10212.[CrossRef] [Google Scholar]
  10. Dolja, V. V., Haldeman, R., Robertson, N. L., Dougherty, W. G. & Carrington, J. C. (1994). Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants. EMBO Journal 13, 1482-1491. [Google Scholar]
  11. Dolja, V. V., Haldeman-Cahill, R., Montgomery, A. E., Vandenbosch, K. A. & Carrington, J. C. (1995). Capsid protein determinants involved in cell-to-cell and long distance movement of tobacco etch potyvirus. Virology 206, 1007-1016.[CrossRef] [Google Scholar]
  12. Dougherty, W. G., Carrington, J. C., Cary, S. M. & Parks, T. D. (1988). Biochemical and mutational analysis of a plant virus polyprotein cleavage site.EMBO Journal 7, 1281-1287. [Google Scholar]
  13. Farinelli, L., Malnoe, P. & Collet, G. F. (1992). Heterologous encapsidation of potato virus Y strain 0 (PVY0) with the transgenic coat protein of PVY strain N (PVYN) in Solanum tuberosum cv. Bintje. Bio/Technology 10, 1020-1025.[CrossRef] [Google Scholar]
  14. Gray, D. J., Hiebert, E., Lin, C. M., Compton, M. E., McColley, D. W., Harrison, R. J. & Gaba, V. P. (1994). Simplified construction and performance of a device for particle bombardment. Plant Cell, Tissue and Organ Culture 37, 179-184.[CrossRef] [Google Scholar]
  15. Greene, A. E. & Allison, R. F. (1994). Recombination between viral RNA and transgenic plant transcripts. Science 263, 1423-1425.[CrossRef] [Google Scholar]
  16. Hobbs, H. A., Reddy, D. V. R., Rajeshwari, P. & Reddy, A. S. (1987). Use of direct antigen coating and protein A coating ELISA procedures for detection of three peanut viruses. Plant Disease 71, 747-749.[CrossRef] [Google Scholar]
  17. Holt, C. A. & Beachy, R. N. (1991).In vivo complementation of infectious transcripts from mutant tobacco mosaic virus cDNAs in transgenic plants. Virology 181, 109-117.[CrossRef] [Google Scholar]
  18. Horsch, R. B., Fry, J. E., Hoffman, N. L., Eichholz, D., Rogers, S. G. & Fraley, R. T. (1985). A simple and general method for transferring genes into plants. Science 227, 1229-1231.[CrossRef] [Google Scholar]
  19. Jacquet, C., Delecolle, B., Raccah, B., Lecoq, H., Dunez, J. & Ravelonandro, M. (1998a). Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants. Journal of General Virology 79, 1509-1517. [Google Scholar]
  20. Jacquet, C., Ravelonandro, M., Bachelier, J. & Dunez, J. (1998b). High resistance to plum pox virus (PPV) in transgenic plants containing modified and truncated forms of PPV coat protein gene. Transgenic Research 7, 29-39. [Google Scholar]
  21. Jagadish, M. N., Ward, C. W., Gough, K. H., Tulloch, P. A., Whittaker, L. A. & Shukla, D. D. (1991). Expression of potyvirus coat protein in Escherichia coli and yeast and its assembly into virus-like particles. Journal of General Virology 72, 1543-1550.[CrossRef] [Google Scholar]
  22. Jagadish, M. N., Huang, D. & Ward, C. W. (1993). Site-directed mutagenesis of a potyvirus coat protein and its assembly in Escherichia coli. Journal of General Virology 74, 893-896.[CrossRef] [Google Scholar]
  23. Jakab, G., Vaistij, F. E., Droz, E. & Malnoe, P. (1997). Transgenic plants expressing viral sequences create a favourable environment for recombination between viral sequences. In Virus-Resistant Transgenic Plants: Potential Ecological Impact, pp. 45-50. Edited by E. Balázs & M. Tepfer. Berlin, Heidelberg & New York: Springer Verlag.
  24. Jefferson, R. A., Kavanagh, T. A. & Bevan, M. W. (1987). GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal 6, 3901-3907. [Google Scholar]
  25. Kaplan, I. B., Shintaku, M. H., Li, Q., Zhang, L., Marsh, L. E. & Palukaitis, P. (1995). Complementation of virus movement in transgenic tobacco expressing the cucumber mosaic virus 3a gene.Virology 209, 188-199.[CrossRef] [Google Scholar]
  26. Korte, A. M., Maiss, E., Kramer, I. & Casper, R. (1995). Biosafety considerations of different plum pox potyvirus (PPV) genes used for transformation of plants. XVI International Symposium on Fruit Tree Virus Diseases. Acta Horticulturae 368, 280-284. [Google Scholar]
  27. Landsmann, J., Llewellyn, D., Dennis, E. S. & Peacock, W. J. (1988). Organ regulated expression of Parasponia andersonii haemoglobin gene in transgenic tobacco plants. Molecular and General Genetics 214, 68-73.[CrossRef] [Google Scholar]
  28. Lapidot, M., Gafney, R., Ding, B., Wolf, S., Lucas, W. J. & Beachy, R. N. (1993). A dysfunctional movement protein of Tobacco mosaic virus that partially modifies the plasmodesmata and limits virus spread in transgenic plants. Plant Journal 4, 959-970.[CrossRef] [Google Scholar]
  29. Lecoq, H., Ravelonandro, M., Wipf-Scheibel, C., Monsion, M., Raccah, B. & Dunez, J. (1993). Aphid transmission of a non-aphid-transmissible strain of zucchini yellow mosaic potyvirus from transgenic plants expressing the capsid protein of plum pox potyvirus. Molecular Plant-Microbe Interactions 6, 403-406.[CrossRef] [Google Scholar]
  30. Lecoq, H., Ravelonandro, M., Wipf-Scheibel, C., Monsion, M., Raccah, B. & Dunez, J. (1994). Significance of the heterologous encapsidation of zucchini yellow mosaic potyvirus in transgenic plants expressing plum pox potyvirus capsid protein. Bulletin OEPP 24, 555-559.[CrossRef] [Google Scholar]
  31. Lindbo, J. A. & Dougherty, W. G. (1992a). 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]
  32. Lindbo, J. A. & Dougherty, W. G. (1992b). Pathogen-derived resistance to a potyvirus: immune and resistant phenotypes in transgenic tobacco expressing altered forms of a potyvirus coat protein nucleotide sequence. Molecular Plant-Microbe Interactions 5, 144-153.[CrossRef] [Google Scholar]
  33. Maiss, E., Timpe, U., Brisske, A., Jelkmann, W., Casper, R., Himmler, G., Mattanovich, D. & Katinger, H. W. D. (1989). The complete nucleotide sequence of plum pox virus RNA. Journal of General Virology 70, 513-524.[CrossRef] [Google Scholar]
  34. Maiss, E., Timpe, U., Brisske-Rode, A., Lesemann, D.-E. & Casper, R. (1992). Infectious in vivo transcripts of a plum pox potyvirus full-length cDNA clone containing the cauliflower mosaic virus 35S RNA promoter. Journal of General Virology 73, 709-713.[CrossRef] [Google Scholar]
  35. Maiss, E., Koenig, R. & Lesemann, D.-E. (1995). Heterologous encapsidation of viruses in transgenic plants and in mixed infections. In Proceedings of the 3rd International Symposium on the Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms, pp. 129–140. Monterey, CA, USA, 14–16 November 1994.
  36. Milne, R. G. & Lesemann, D.-E. (1984). Immunosorbent electron microscopy in plant virus studies. In Methods in Virology, pp. 85-101. Edited by K. Maramorosch & H. Koprowski. Orlando, FL: Academic Press.
  37. Morozov, S. Yu., Fedorkin, O. N., Jüttner, G., Schiemann, J., Baulcombe, D. C. & Atabekov, J. G. (1997). Complementation of a potato virus X mutant mediated by bombardment of plant tissues with cloned viral movement protein genes. Journal of General Virology 78, 2077-2083. [Google Scholar]
  38. Murashige, T. & Skoog, F. (1962). A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Plant Physiology 15, 473-497.[CrossRef] [Google Scholar]
  39. Osbourn, J. K., Sarkar, S. & Wilson, T. M. (1990). Complementation of coat protein-defective TMV mutants in transgenic tobacco plants expressing TMV coat protein. Virology 179, 921-925.[CrossRef] [Google Scholar]
  40. Pang, S. Z., Jan, F.-J. & Gonsalves, D. (1997). Nontarget DNA sequences reduce the transgene length necessary for RNA-mediated tospovirus resistance in transgenic plants. Proceedings of the National Academy of Sciences, USA 94, 8261-8266.[CrossRef] [Google Scholar]
  41. Powell, P. A., Sanders, P. R., Tumer, N., Fraley, R. T. & Beachy, R. N. (1990). Protection against tobacco mosaic virus infection in transgenic plants requires accumulation of coat protein rather than coat protein RNA sequences. Virology 175, 124-130.[CrossRef] [Google Scholar]
  42. Prins, M. & Goldbach, R. (1996). RNA-mediated virus resistance in transgenic plants. Archives of Virology 141, 2259-2276.[CrossRef] [Google Scholar]
  43. Riedel, D., Lesemann, D.-E. & Maiss, E. (1998). Ultrastructural localization of nonstructural and coat proteins of 19 potyviruses using antisera to bacterially expressed proteins of plum pox potyvirus. Archives of Virology 143, 2133-2158.[CrossRef] [Google Scholar]
  44. Rochow, W. F. (1977). In Aphids as Virus Vectors, pp. 253–273. Edited by K. F. Harris & K. Maramorosch. New York: Academic Press.
  45. Rodrı́guez-Cerezo, E., Findlay, K., Shaw, J. G., Lomonossoff, G. P., Qiu, S. G., Linstead, P., Shanks, M. & Risco, C. (1997). The coat and cylindrical inclusion proteins of a potyvirus are associated with connections between plant cells. Virology 236, 296-306.[CrossRef] [Google Scholar]
  46. Rojas, M. R., Zerbini, F. M., Allison, R. F., Gilbertson, R. L. & Lucas, W. J. (1997). Capsid protein and helper component-proteinase function as potyvirus cell-to-cell movement proteins. Virology 237, 283-295.[CrossRef] [Google Scholar]
  47. SAS Institute (1996).SAS User’s Guide: Statistics Version 6, 12th edn. Cary, NC: SAS Institute.
  48. Schaad, M. C., Jensen, P. E. & Carrington, J. C. (1997). Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein. EMBO Journal 16, 4049-4059.[CrossRef] [Google Scholar]
  49. Scholthof, H. B., Scholthof, K.-B. G. & Jackson, A. O. (1996). Plant virus gene vectors for transient expression of foreign proteins in plants.Annual Review of Phytopathology 34, 299-323.[CrossRef] [Google Scholar]
  50. Shukla, D. D., Frenkel, M. J. & Ward, C. W. (1991). Structure and function of the potyvirus genome with special reference to the coat protein coding region. Canadian Journal of Plant Pathology 13, 178-191.[CrossRef] [Google Scholar]
  51. Silva-Rosales, L., Lindbo, J. A. & Dougherty, W. G. (1994). Analysis of transgenic tobacco plants expressing a truncated form of a potyvirus coat protein nucleotide sequence. Plant Molecular Biology 24, 929-939.[CrossRef] [Google Scholar]
  52. Timpe, U., Maiss, E., Landsmann, J. & Casper, R. (1992). ‘Coat protein mediated cross protection’ gegen das Scharka Virus. Mitteilungen der Biologischen Bundesanstalt für Land- und Forstwirtschaft 238, 212. [Google Scholar]
  53. Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, USA 76, 4350-4354.[CrossRef] [Google Scholar]
  54. Vancanneyt, G., Schmidt, R., O’Connor-Sanchez, A., Willmitzer, L. & Rocha-Sosa, M. (1990). Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Molecular and General Genetics 220, 245-250. [Google Scholar]
  55. van den Boogaart, T., Lomonossoff, G. P. & Davies, J. W. (1998). Can we explain RNA-mediated virus resistance by homology-dependent gene silencing?Molecular Plant-Microbe Interactions 11, 717-723.[CrossRef] [Google Scholar]
  56. Wassenegger, M. & Pélissier, T. (1998). A model for RNA-mediated gene silencing in higher plants. Plant Molecular Biology 37, 349-362.[CrossRef] [Google Scholar]
  57. Wen, F. & Lister, R. M. (1991). Heterologous encapsidation in mixed infections among four isolates of barley yellow dwarf virus. Journal of General Virology 72, 2217-2223.[CrossRef] [Google Scholar]
  58. White, J. L. (1999). An overview of cultivation of virus-resistant crops in the United States. In Proceedings of the 5th International Symposium of Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms. Edited by J. Schiemann & R. Casper. Braunschweig, Germany (in press).
  59. Wu, X. & Shaw, J. G. (1998). Evidence that assembly of a potyvirus begins near the 5′ terminus of the viral RNA.Journal of General Virology 79, 1525-1529. [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-81-3-567
Loading
Mutations in the coat protein gene of Plum pox virus suppress particle assembly, heterologous encapsidation and complementation in transgenic plants of Nicotiana benthamiana
J. Gen. Virol. 81, 567 (2000); https://doi.org/10.1099/0022-1317-81-3-567
/content/journal/jgv/10.1099/0022-1317-81-3-567
/content/journal/jgv/10.1099/0022-1317-81-3-567
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error