Archives of Virology

, Volume 141, Issue 8, pp 1535–1552 | Cite as

Construction and analysis of infectious transcripts from a resistance-breaking strain of tobacco vein mottling potyvirus

  • O. Nicolas
  • T. P. Pirone
  • G. M. Hellmann
Original Papers


The Burley tobacco (Nicotiana tabacum) cultivar TN 86 is “resistant” to most strains of tobacco vein mottling potyvirus (TVMV), the virus being restricted to epidermal cells of inoculated leaves. One strain, designated TVMV-S, overcomes this resistance and infects cv TN 86 systemically. To begin our investigation of the molecular basis for the resistance-breaking phenomenon, we have completed the cloning and sequencing of the TVMV-S RNA genome. The complete cDNA clone, under the control of a T7 RNA polymerase promoter, was used to produce infectious transcripts which were tested for their ability to reproduce the characteristics of TVMV-S RNA on three types of tobacco (N. tabacum cv TN 86,N. tabacum cv KY 14, andN. benthamiana). Timing of symptom appearance, symptom type, and titer of virus were identical to those of plants inoculated with TVMV-S RNA. As a step toward mapping the responsible genetic region(s) that contribute(s) to resistance-breaking by TVMV-S, the nucleotide and deduced amino acid sequences were compared to those of wild-type TVMV, a strain that does not overcome cv TN 86 resistance. Variant TVMV-S transcripts containing changes within the VPg cistron exhibited an altered pattern of infectivity on cv TN 86.


Nucleotide Infectious Disease Amino Acid Sequence Molecular Basis Epidermal Cell 
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  1. 1.
    Atreya PL, Atreya CD, Pirone TP (1991) Amino acid substitutions in the coat protein result in loss of insect transmissibility of a plant virus. Proc Natl Acad Sci USA 88: 7887–7891Google Scholar
  2. 2.
    Atreya CD, Atreya PL, Thornbury DW, Pirone TP (1992) Site-directed mutations in the potyvirus HC-PRO gene affect helper component activity, virus accumulation, and symptom expression in infected tobacco plants. Virology 191: 106–111Google Scholar
  3. 3.
    Atreya PL, Lopezmoya JJ, Chu MH, Atreya CD, Pirone TP (1995) Mutational analysis of the coat protein N-terminal amino acids involved in potyvirus transmission by aphids. J Gen Virol 76: 265–270Google Scholar
  4. 4.
    Atreya CD, Pirone TP (1993) Mutational analysis of the helper component-proteinase gene of a potyvirus: effect of amino acid substitutions, deletions and gene replacement on virulence and aphid transmissibility. Proc Natl Acad Sci USA 90: 11919–11923Google Scholar
  5. 5.
    Atreya CD, Raccah B, Pirone TP (1990) A point mutation in the coat protein abolishes aphid transmissibility of a potyvirus. Virology 178: 161–165Google Scholar
  6. 6.
    Carrington JC, Dougherty WG (1987) Small nuclear inclusion protein encoded by a plant potyvirus genome is a protease. J Virol 61: 2540–2548Google Scholar
  7. 7.
    Carrington JC, Freed DD, Oh CS (1990) Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete processing. EMBO J 9: 1347–1353Google Scholar
  8. 8.
    Carrington JC, Freed DD, Sanders TC (1989) Autocatalytic processing of the potyvirus helper component proteinase inEscherichia coli and in vitro. J Virol 63: 4459–4463Google Scholar
  9. 9.
    Carrington JC, Herndon KL (1992) Characterization of the potyviral HC-Pro autoproteolytic cleavage site. Virology 187: 308–315Google Scholar
  10. 10.
    Domier LL, Franklin KM, Hunt AG, Rhoads RE, Shaw JG (1989) Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus. Proc Natl Acad Sci USA 86: 3509–3513Google Scholar
  11. 11.
    Domier LL, Franklin KM, Shahabuddin M, Hellmann GM, Overmeyer JH, Hiremath ST, Siaw MFE, Lomonossoff GP, Shaw JG, Rhoads RE (1986) The nucleotide sequence of tobacco vein mottling virus RNA. Nucleic Acids Res 14: 5417–5430Google Scholar
  12. 12.
    Dougherty WG, Parks TD (1991) Post-translational processing of the tobacco etch virus 49-kDa small nuclear inclusion polyprotein: identification of an internal cleavage site and delimitation of VPg and proteinase domains. Virology 183: 449–456Google Scholar
  13. 13.
    Frenkel MJ, Ward CW, Shukla DD (1989) The use of 3′ non-coding nucleotide sequences in the taxonomy of potyviruses: application to watermelon mosaic virus 2 and soybean mosaic virus-N. J Gen Virol 70: 2775–2783Google Scholar
  14. 14.
    Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85: 8998–9002Google Scholar
  15. 15.
    Gallie DR, Sleat DE, Watts JW, Turner PC, Wilson TMA (1987) A comparison of eukaryotic viral 5′-leader sequences as enhancers of mRNA expression in vivo. Nucleic Acids Res 15: 8693–8711Google Scholar
  16. 16.
    Garcia JA, Martin MT, Cervera MT, Riechmann JL (1992) Proteolytic processing of the plum pox potyvirus polyprotein by the NIa protease at a novel cleavage site. Virology 188: 697–703Google Scholar
  17. 17.
    Gibb KS, Hellmann GM, Pirone TP (1989) Nature of resistance of a tobacco cultivar to tobacco vein mottling virus. Mol Plant Microbe Interact 2: 332–339Google Scholar
  18. 18.
    George DG, Barker WC, Hunt LT (1990) Mutation data matrix and its uses. Methods Enzymol 183: 333–351Google Scholar
  19. 19.
    Gubler U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25: 263–269Google Scholar
  20. 20.
    Hellmann GM, Shaw JG, Lesnaw JA, Chu L-Y, Pirone TP, Rhoads RE (1980) Cell-free translation of tobacco vein mottling virus RNA. Virology 106: 207–216Google Scholar
  21. 21.
    Hellmann GM, Shaw JG, Rhoads RE (1988) In vitro analysis of tobacco vein mottling virus NIa cistron: evidence for a virus-encoded protease. Virology 163: 554–562Google Scholar
  22. 22.
    Jayaram C, Hill JH, Miller WA (1992) Complete nucleotide sequences of two soybean mosaic virus strains differentiated by response of soybean containing the rsv resistance gene. J Gen Virol 73: 2067–2077Google Scholar
  23. 23.
    Kavanagh T, Goulden M, Santa Cruz S, Chapman S, Barker I, Baulcombe D (1992) Molecular analysis of a resistance-breaking strain of potato virus X. Virology 189: 609–617Google Scholar
  24. 24.
    Laliberté J-F, Nicolas O, Chatel H, Lazure C, Morosoli R (1992) Release of a 22-kDa protein derived from the amino-terminal domain of the 49-kDa NIa of turnip mosaic potyvirus inEscherichia coli. Virology 190: 510–514Google Scholar
  25. 25.
    Lütcke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GA (1987) Selection of AUG initiation codons differs in plants and animals. EMBO J 6: 43–48Google Scholar
  26. 26.
    Matthews REF (1991) Plant Virology, 3rd edn. Academic Press, San DiegoGoogle Scholar
  27. 27.
    Mavankal G, Rhoads RE (1991) In vitro cleavage at or near the N-terminus of the helper component protein in the tobacco vein mottling virus polyprotein. Virology 185: 721–731Google Scholar
  28. 28.
    Miller RD (1987) TN 86. A Burley tobacco resistant to TVMV, TEV and PVY. UNiversity of Tennessee, Agriculture Experimental Station, Knoxville, TN, Bull No. 657Google Scholar
  29. 29.
    Murphy JF, Rhoads RE, Hunt AG, Shaw JG (1990) The VPg of tobacco etch virus RNA is the 49-kDa proteinase or the N-terminal 24-kDa part of the proteinase. Virology 178: 285–288Google Scholar
  30. 30.
    Murphy JF, Rychlik W, Rhoads RE, Hunt AG, Shaw JG (1991) A tyrosine residue in the small nuclear inclusion protein of tobacco vein mottling virus links the VPg to the viral RNA. J Virol 65: 511–513Google Scholar
  31. 31.
    Padgett HS, Beachy RN (1993) Analysis of a tobacco mosaic virus strain capable of overcoming N gene-mediated resistance. Plant Cell 5: 577–586Google Scholar
  32. 32.
    Reddick BB, Miller RD, Gooding GV (1991) Resistance inN. tabacum cultivar TN 86 to naturally occurring tobacco viruses. Tobacco Sci Dec: 96–99Google Scholar
  33. 33.
    Rodriguez-Cerezo E, Gamble Klein P, Shaw JG (1991) A determinant of disease symptom severity is located in the 3′-terminal noncoding region of the RNA of a plant virus. Proc Natl Acad Sci USA 88: 9863–9867Google Scholar
  34. 34.
    Rodriguez-Cerezo E, Shaw JG (1991) Two newly detected nonstructural viral proteins in potyvirus-infected cells. Virology 185: 572–579Google Scholar
  35. 35.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  36. 36.
    Shukla DD, Ward CW (1988) Amino acid sequence homology of coat proteins as a basis for identification and classification of the potyvirus group. J Gen Virol 69: 2703–2710Google Scholar
  37. 37.
    Shukla DD, Ward CW, Brunt AA (1994) The Potyviridae. CAB International, Wallingford, Oxon, pp 74–112Google Scholar
  38. 38.
    Verchot JM, Carrington JC (1995) Debilitation of plant potyvirus infectivity by P1 proteinase-inactivating mutations and restoration by second-site modifications. J Virol 69: 1582–1590Google Scholar
  39. 39.
    Verchot JM, Koonin EW, Carrington JC (1991) The 35-kDa protein from the N-terminus of the potyviral polyprotein functions as a third virus-encoded proteinase. Virology 185: 527–535Google Scholar
  40. 40.
    Ward CW, McKern NM, Frenkel MJ, Shukla DD (1992) Sequence data as a major criterion for potyvirus classification. In: Barnett OW (ed) Potyvirus taxonomy. Springer, Wien New York, pp 283–297 (Arch Virol [Suppl] 5)Google Scholar
  41. 41.
    Weber H, Schultze S, Pfitzner AJP (1993) Two amino acid substitutions in the tomato mosaic virus 30-kilodalton movement protein confer the ability to overcome theTm-2 2 resistance gene in the tomato. J Virol 67: 6432–6438Google Scholar

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • O. Nicolas
    • 1
  • T. P. Pirone
    • 2
  • G. M. Hellmann
    • 1
  1. 1.Biotechnology ResearchBowman Gray Technical Center, R. J. ReynoldsWinston-Salem
  2. 2.Plant Pathology DepartmentUniversity of KentuckyLexingtonUSA

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