Summary
The potato leafroll virus (PLRV) coat protein (CP) gene was directly cloned from the total RNA extracted from virus-infected plants. First strand cDNA synthesis was not necessarily specific; it was equally efficient using either random or CP-specific primers. The viral sequence encoding the coat protein was specifically amplified from the total population of cDNA molecules by polymerase chain reaction (PCR), using specific primers bordering the CP gene. The unique amplified product thus obtained was cloned blunt-end into the pT7T318U plasmid vector, and the authenticity of the cloned gene verified by sequence analysis. This cloning strategy obviates the need for virus purification. Sequence comparison of the CP gene of the Italian isolate and those of five other PLRV isolates revealed a close similarity to the three European and the Canadian isolates, and a more distant relationship with the Australian one.
Similar content being viewed by others
References
Beachy, R.N., S. Loesch-Fries & N.E. Tumer, 1990. Coal protein-mediated resistance against virus infection.Annual Review of Phytopathology 28: 451–474.
Beemster, A.B.R. & J.A. de Bokx, 1987. Survey of properties and symptoms. In: J.A. de Bokx & J.P.H. van der Want (Eds), Viruses of potato and seed potato production, PUDOC, Wageningen, pp. 84–113.
Harrison, B.D., 1984. Potato leafroll virus.CMI/AAB Description of plant viruses No. 291.
Hooft van Huijsduijnen, R.A.M., B.J.C. Cornelissen, L.C. van Loon, H.J. van Boom, M. Tromp & J.F. Bol. 1985. Virus induced synthesis of messenger RNAs for precursors of pathogenesis related proteins in tobacco.EMBO Journal 4: 2167–2171.
Keese, P., R.R. Martin, L.M. Kawchuk, P.M. Waterhouse & W.L. Gerlach, 1990. Nucleotide sequences of an Australian and a Canadian isolate of potato leafroll luteovirus and their relationships with two European isolates.Journal of General Virology 71: 719–724.
Maniatis, T., E.F. Fritsch & J. Sambrook, 1982. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory. New York, 545 pp.
Martin, R.R., P.K. Keese, M.J. Young, P.M. Waterhouse & W.L. Gerlach, 1990. Evolution and molecular Biology of luteoviruses.Annual Review of Phytopathology 28: 341–363.
Mayo, M.A., S.J. Robinson, C.A. Jolly & L. Hyman, 1989. Nucleotide sequence of potato leafroll luteovirus RNA.Journal of General Virology 70: 1037–1051.
Rosner, A., I. Ginzburg & M. Bar-Joseph. 1983. Molecular cloning of complementary DNA sequences of citrus tristeza virus (CTV) RNA.Journal of General Virology 64: 1757–1763.
Rosner, A., A. Stein & S. Levy, 1992. Transcription amplification of polymerase chain reaction products of bean yellow mosaic virus RNA extracted from gladioli corms.Annals of Applied Biology 121: 269–276.
Rowhani, A. & R. Stace-Smith, 1979. Purification and characterization of potato leafroll virus.Virology 98: 45–54.
Sanger, F., S. Nicklen & A.R. Coulson, 1977. DNA sequencing with chain-terminating inhibitors.Proceedings of the National Academy of Sciences USA 74: 5463–5467.
Tacke, E., S. Sarkar, F. Salamini & W. Rohde, 1989. Cloning of the gene for the capsid protein of potato leafroll virus.Archives of Virology 105: 153–163.
Takanami, Y. & S. Kubo, 1979. Enzyme-assisted purification of two phloem-limited plant viruses: Tobacco necrotic dwarf and potato leafroll.Journal of General Virology 44: 153–159.
Tamada, T. & B.D. Harrison, 1980. Factors affecting the detection of potato leafroll virus in foliage by enzyme-linked immunosorbent assay.Annals of Applied Biology 95: 209–219.
Vunsh, R., A. Rosner & A. Stein, 1990. The use of polymerase chain reaction (PCR) for the detection of bean yellow mosaic virus in gladiolus.annals of Applied Biology 117: 561–569.
Vunsh, R., A. Rosner & A. Stein, 1991. Detection of bean yellow mosaic virus in gladioli corms by the polymerase chain reaction.Annals of Applied Biology 119: 289–294.
Wilk, R. van der, M.J. Huisman, B.J.C. Cornelissen, H. Huttinga & R. Goldbach, 1989. Nucleotide sequence and organization of potato leafroll virus genomic RNA.FEBS Letters 245: 51–56.
Zaag, D.E. van der. 1987. Yield reduction in relation to virus infection. In: J.A. de Bokx & J.P.H. van der Want (Eds). Viruses of potato and seed potato production. PUDOC Wageningen, pp. 146–150.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Faccioli, G., Rosner, A. & Forni, M. Use of the polymerase chain reaction to clone the potato leafroll virus coat protein gene directly from the total RNA of infected plants. Potato Res 38, 211–218 (1995). https://doi.org/10.1007/BF02357934
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02357934